Abstracts 1230-1630

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1. PART

1230

1231

Abstracts

Hualin ('. Yip, Thomas Forsttmher aml Paul 1,'~Lehmaml. Case Western Rescrvc University, Department of Pathology, School of Medicine, Clcvehmd Ohio, 44106 One of the major objectivcs of wlccine devclopmcnt is to establish immunization protocols that permit the selective induction of either Th I or Th2 type immunity, as required, for the immune system's successful encounter of different infectious agents, ltere we report thai intraperitoneal (i.p.) injection of antigen m incomplete Frcund's adjuvant (IFA), a protocol thought to induce tolerance, actually induces vigorous and pure T helper 2 (Th2) type immunity. In contrasl, immunization with complete Freund's adjuvant (CFA) results in unipolar Thl type immunity. These adjuwmts override the genetic bias of the host with IFA inducing Th2 immunity in Thl biassed mouse strains and CFA triggering Thl immunity in Th2 biassed strains. The data suggcst that adjuvants can reliably guide the T cell response ahmg the Th I or Th2 differentiation pathway.

either E, coli (but not calf thymus) DNA, oligonuclcotides (ONs) that contain an unmethylatcd CpG motif, or a combination of LPS and IFN3,. Furthermore, following these treatments, the populations acquired the ability to transfer moderate-to-severe E A E with 100% incidence. IL-12 p40 production was induced in these cultures by each of these treatments and the ability of the microbial products to confer a Thl phenotype as well as cnccphalitogenic capacity was abrogated by the addition of a neutralizing antibody to IL-12 to the cultures. Wc conclude that the induction of IL-12 production in response to bacterial pathogens and their prodncls can unmask the cneephalitogenie potential of bystander MBP-reactive T cells, precipitating autoimmune demyelination. Our results have implications with respect to the therapeutic uses of IL-12, IL-12 antagonisls, and bacterial DNA vaccines.

Adjuvant guided Thl or Th2 differentiation.

Differential Expression of the Beta-2-Adrenergic Receptor (I]2AR) on Activated Thl and Th2 Cells Provides a Mechanism for Selective Modulation of Thl Cell Cytokine Production. DS Ramer-Quinn. RA Bake1; and VM Sanders Loyola Univ Med Ctr, Maywood, IL We propose that a functional dichotomy exists between the ability of activated Th l/Th2 cells to respond to a ligand that binds to the [3eAR. Resting Thl/Th2 chines differentially express the [3eAR. However, activation of lymphocytes can modulate the level of expression of a variety of cell surface molecules. Therefore, wc measured expression of the [3,AR on murine Th cell subsets either before or after cell activation with anti-CD3 mAb. Radioligand binding shows that the lAzAR is detectable on both resting and activated Thl cells. The number of binding sites increases, with no change in affinity, on activated Thl cells. In contrast, detectable levels of the [3~AR on resting Th2 chines arc absent and arc not induced at any time examined following activation. However, it is possible that stimulation of a low level of the [3eAR, not detected by radioligand binding, could alter Th2 cell function. Therefore, Th l/Th2 cells were activated via anti-CD3 mAb and cxposed to a [3:AR agonist ul increasing times after cell activation. Cytokine levels were mcasurcd 24 hours after cell activation. IL-2 and IFN-y production by Thl cclls is inhibited and enhanced respcctively, while IL-4 and IL-5 production by Th2 cells is not modulated. Furthermore, the physiological ligand, norepincphrinc, also induccd a significant decrcase in IL-2 production by binding to the ~3eAR on Thl cells. These results suggcst that a dctcctablc level of [3eAR expression is limitcd to Thl cells and may providc a mechanism by which Thl ccll cytokine production can be directly modulated by endogenous ligands such as norepinephrine. Supported by NIH Gm~u AI 3Z~20 and the

Schweppe Foundati
S301

2

Activation of Pathogenic Th I T cells by Microbial Products is Mediated by IL-12. BM Segal, DM Klinman and EM Shevach. Ll, NIAII), NIH and FDA, Bethcsda, MD Experimental autoimmunc encephalomyelitis (EAE) is a dcmyelinating disease of the central nervous system mediated by CD4+ Thl T cells reactive with myelin basic protein (MBP). BI0.S mice are resistant to induction of EAE despite their expression of the permissive H-2 ~ haplotype. We have shown that the resistance of BI0.S mice to EAE is secondary to an antigen-specific defect in the generation of MBP-reaetive Thl cells even though the frequency of MBP-reaefive T cells in I310.S mice is similar to thc susceptible SJL strain; this deficiency can be overcome by exposure of BI(t.S T cells to IL-12 in vitro (J. Exp. Mcd. 184: 771-5). As multiple sclerosis and other human autoimmune diseases frequently present/exacerbate in the setting of infectious disease, we have evaluated the effects of microbial products with known cytokinc modulating properties to convert BI0.S MBP-reactive cells into Thl populations which are capable of transferring EAE. Primed BI0.S MBP-reactivc cells acquired the ability to produce IFNy following exposure in vitro for 96h to antigen and

1233

T Cell Activatiun by Liposumes and Polarization of Thl Type Cytukine Response. Santa Sehra, Lalita Chugh and S.K Gangal, Centre for Biochemical Technology, Delhi 110007, India. Th2 responsc induced by an allcrgen is involved in pathogensis of allergic disorders. We have observed that the Th2 response of the allergen can be changed to Thl type whcn allergen is entrappcd in liposomes. Liposomes entrapped allergen (LEA) showcd 10 timcs increasc in proliferation of spleenic cells as compared to that of free allergen (FA) in in-vitro stimulation studies. Furthcr invivo and in-vitro studies with FA and LEA indicated that there was substantial increase in IL-2, IFN-3, and IgG 1 and decrease in IL-4 and IgE levels in Balb/C mice injected with the same concentration of FA. In-vitro and in-vivo studies were conducted using Balb/C mice and allergen from Artemisia Scoparia pollcn. The results indicated that when allergen entrapped in liposomes was injected in-vivo it stimulated preferentially Thl cells thereby showing increased Thl type of cytokine response resulting in decrease IgE levels and increascd lgG levels. Reversal of T cell response from Th2 to Thl and increase in 1FN-~,/IL-4 ratio on injection of LEA, provides liposomes as an unique delivery system with both immunoadjuvant and immunomodulation properitics fl~r use in immunothcrapy of allergic disorders.

1234

Highly Stimulated T Cells Expressing Intracellular IFN~/ or TNFo~ Enhance Expression of ICAM-I on Cultured Endothelial Cells. DD Smith and HB Lindsl~% University of Kansas Medical Center, Kansas City, KS Resting or highly stimulatcd (PMA, 10 ng/ml + ionomycin, 1 ug/ml for 3 to 4 h), nylon wool purificd T cells from normal blood donors were allowed to adhere to monolayers of HUVECs. Monolayers were unstimulated or previously stimulated for 24 h with either interferon y (IFN) (100 U/ml) or tumor necrosis factor-c~ (TNF) (10 U/ml). After one h at 37 ~ non-adherent cells werc washed off and co-culture permitted for 24 h. Expression of intraccllular adhesion molecule-1 (ICAM-I) by these monolayers was characterized by cellular ELISA. Similarly stimulated T cells were assayed for intracellular expression of the cytokincs IFN and TNF by means of flow cytometry. Unstimulated T cells variably enhanced ICAM-1 expression on HUVECs (A,5 o from 0.404 to 1.013) over base line (A,~5. from 0.164 to 0.885) depending upon the stimulation of the monolayer, while stimulated T cells elicited presumab}y maximal ICAM-I production (A~,sI~ flom 1.106 to 1.277) in all instanecs regardless of stimulation of the monolaycr (two experiments). Highly stimulated T cells also displayed cnhanecd intraccllular expression of IFN over background (23.0% versus 0.6%) whilc TNF production was enhanccd less significantly (6.8% vcrsus I).9%) (average of 2 experiments). Thus, highly stimulated T cells may activate H U V E C s via a paraerine pathway due to thc production of proinflammatory cytokines. (Supported, in part, by thc Carey and Ewms Arthritis Funds, Kansas University Endowment Association)

S302

1235

1236

1237

Abstracts

Recombinant soluble TCR (sTCR) protect T cells from suppression: Requirement for aggregated, pentameric, disulfide-linked oq5 hetrodimers of sTCR. V Paliwal, W Ptak, M Szczepanik. E BraswelI, P W Askenase, Yale Univ. School of Medicine New Haven, CT; Jagiellonian Univ, Krakow, Poland, and U Conn, Storrs, CT Recombinant a13 TCR were released enzymatically from the surface of thymoma cells transfected with a and 13TCR cDNA and expressing a membrane PI-linkage, enabling PI-PLC enzymatic release of sTCR. In vitro treatment of contact sensitivity (CS) effector T cells with these sTCR protected the cells from active T cell suppression. The CS-protective activity bound to and eluted from anti-TCRa and 13 mAb columns. Upon heat treatment at 62~ C for 30 min, sTCR formed an aggregate corresponding to a pentamer according to HPLC (m.w. 516 kDa) and analytical ultracentrifugation (m.w. 518). HPLC purified sTCR pentamer was protective while monomer (104 kDa) was not. Aggregated biotinylated sTCR bound to peritoneal exudate macrophages. These results suggesting that the pentameric form of a[3 sTCR protected CS effector T cells from suppression via binding to putative receptors on macrophages. IL-12 reverses established tolerance of contact sensitivity (CS). H Ushio, RF Tsuji, M Szczepanik, K Kawamoto, H Matsuda and P W Askenase, Yale Univ Sch Med New Haven, CT. CS are in vivo examples of Th-1 responses and specific tolerance can be induced by high dose hapten i.v.. We evaluated tolerance by measuring proliferation, and IFN-~/ production in vitro of CS T cells, stimulated by haptenlinked APC, and in vivo CS. Responses of T cells to hapten-APC, became absent in mice tolerized with hapten i.e., paralling impaired in vivo CS. Addition of IL-12 restored proliferative and IFN-3, responses of tolerized CS-effector T cells, and IL-12 also restored established tolerance in vivo. We examined if IL-12 reversed tolerance via enhanced production of IFN--/, by directly injecting IFN-~/ into tolerized mice, and found that IFN--/ partially mimicked IL-12 in restoring impaired CS. We also attempted to block established tolerance with neutralizing mAb against IL-4, IL-10 TGF-13, but none restored established tolerance. Thus, these results demonstrated that IL-12 reversal of established tolerance may have been partially due to IFN-3,, but was not due to antagonizing effects against Th-2 cytokines, such as IL-4, IL-10, nor opposing TGF-!3. Prostaglandin E2 promotes the maturation of naive human CD4 T cells into effector cells producing high levels of anti-inflammatory cytokines and low levels of pro-inflammatory cytokines. C.E. Demeure, L.P. Yang, C. Desjardins, and G. Delespesse. Centre de Recherche Louis-Charles Simard, Hopital Notre-Dame, Montreal University, Quebec, Canada. The potent immunomodulator PGE2 was reported to inhibit preferentially the production of Thl cytokines. We investigated the effects of PGE2 present at priming of human neonatal naive CD4 T cells on their development into cytokine-producing effectors. We report that T cells primed with anti-CD3 and CD32-B7 transfected L cells (CD32-B7L) in the presence of PGE2 produced higher quantities of the anti-inflammatory cytokines IL-4, IL-10 and IL-13, and lower amounts of the pro-inflammatory cytokines IFN-~, TNF-a, and LT at restimulation with anti-CD3 and CD32-B7L without PGE2. Only inactive TGF-13 was produced, which was not affected by PGE2. This anti-inflammatory cytokine profile can be defined as Th2-1ike. CD28 and CD40L expression was normal. PGE2 inhibited IL-2, IFN-~/ and TNF-a but not IL-4 or IL-13 release in the primary culture, showing that increased IL-4 production capacity was IL-4-independent. Inhibition of IFN-'7 production was independent of IL-2 or IL-10 production, and very high production induced at restimulation by PMA+IONO was also inhibited. We show also that PGE2-induced inhibition of TNF-a was IL-10-dependent. We conclude that PGE2 at priming favors the development

J ALLERGY CLIN IMMUNOL JANUARY 1997

of cells producing mostly anti-inflammatory cytokines, and suggest that this may be one mechanism by which PGE2 and analogs exert beneficial anti-inflammatory effects in transplantation.

1238

Nitric Oxide Modulates IFN-~, Responsiveness of T Lymphocytes by Regulating the Expression of IFN-y Receptor oL and [~ chains. A. Allione, L. Rigamonti, P. Bernabei, G. Forni, F. Novelli. Dept. of Clinical and Biological Sciences, University of Turin, Italy. Nitric oxide (NO) is a pleiotropic molecule involved in neuro-trasmission, vascular homeostasis and the effector functions of the immune system. It is derived from the oxidation of L-arginine by NO-synthase, an enzyme that exists in both a costitutive and an inducible form (cNOS and iNOS). Previous data showed that IFN-3, induces the apoptosis or proliferation of neoplastic T cells in function of the greater or lesser membrane expression of its receptor (IFN-~/R) in response to as yet undefined "environmental factors". In this study we show that serum deprivation led to a parallel increase of NO production and IFN-',/R and 13chain expression in malignant T cell lines, correlating with apoptosis induced by serum deprivation and increase of production of endogenous NO. Also the presence of sodium nitroprusside (SNP), a NO donor, increased IFN-,/R c~ and 13 chain expression and apoptosis. The regulation of IFN-~,R by NO can influence the responsiveness of these cells to IFN--,/. In fact addition of IFN-3, to the cells cultured in the presence of SNP increased apoptosis, similar to that obtained by the addition of tFN-"t in the cultures assessed in the absence of serum. These findings identify NO as one of the "environmental factors" governing the response ofT cells to IFN-~/. They also indicate that the effects of "specific" molecules, such as cytokines, can be modulated by "nonspecific" molecules, such as NO, through regulation of their receptor.

1239

Induction of the High-Affinity Conformation of o~4131 Integrin on Human T cells by Vasoactive Intestinal Peptide (VIP). RR Pankhaniya, TA Yednock, PF Dazin and EJ GoetzL University of California Medical Center, Howard Hughes Medical Institute and Athena Neurosciences, Inc., San Francisco, CA Human T lymphoblastoma cells of the Tsup-1 cultured line express a mean of 9• type II VIP receptors (VIPR2) with a mean Kd(_+S.D.) of 6.0 +_ 1.8nM, but no VIPRls. Tsup-1 cells respond to nM-txM concentrations of VIP with increases in [cAMP]i and [Ca++]i , chemotaxis, enhanced production of matrix metalloproteinases-2 and -9, and decreased generation of ILs-2, 4, and 10. As VIP enhanced adherence of Tsup-1 cells to fibronectin and neutralizing monoclonal antibodies to ol.4 and 13~ diminished this increment by up to 70%, the capacity of VIP to activate integrins was examined directly using the 15/7 monoclonal antibody specific for a high affinity conformation of a413~-Suspensions of 1 • 106 Tsup-t cells in 0.2 ml of medium were incubated for 2 hrs at 37~ with 10 -'~ to 10-6M VIP alone or after 15rain at 37~ C with 10-160nM PMA or 0.05-2.51xg/ml of fibronectin III complexes. Increases in binding of 15/7, assessed by fluorescence flow cytometry, were maximal with a combination of 10nM PMA and 1• 7M VIP (range of 23-45% increase in mean population fluorescence) and of 0.031xg/ml fibronectin III complexes and l x l 0 6M VIP (10-17% increase in mean population fluorescence) compared to VIP, PMA or fibronectin III complexes alone, that had no detectable effect. VIP enhancement of expression of an immunochemically-determined high affinity state of a413t by the T cells thus requires two signals, one from a priming stimulus and the other through VIPR2. (Supported by NIH grants U01 AI 34570 and AI 29912.)

J ALLERGYCLIN IMMUNOL

Abstracts

$303

VOLUME 99, NUMBER 1, PART 2

1240

Type 1 and type 2 CD8 + CTL are activated by different exogenous protein antigens in CFA. HL Ma, Y Ke, Q Li, JA Kapp. Department of Pathology and Winship Cancer Center, Emory University School of Medicine, Atlanta, GA Wc have been studying the mechanisms by which exogenous protein antigens actiw~tc CD8 + T cells. Previously, we have shown that chicken owdbumin (OVA) primed C D 8 ' , MHC class l-restricted cytotoxic T lymphocytcs (CTL) prccursors in vivo when administered with complete Freund's adjuvant (CFA). tlere, we report that CD8 +, insulin-specific, MHC class I-restricted CTL precursors can be primed by injection of beef insulin in CFA. Primed splenic CTL precursors from C57BL/6 mice were stimulated in vitro with EL4 thymoma transfected with OVA cDNA (E.G7-OVA) or human insulin cDNA (EL4-1NS), respectively. Long-term CTL lines wcrc established by weekly rcstimulation with irradiated stimulators, syngeneic filler cclls and exogenous IL-2. The resulting CTL were antigen specific sincc OVA-CTI, recognize only E.G7OVA but not EL4-1NS, and vice versa. Both CTL werc CD4 CDB', oq3"l'cR ' CD3 ~ but thc OVA-CTL expresscd the CT-1 epitopc whereas INS-CTL did not. In addition, upon antigen stimulation, the OVA-CTL produced IFN-y and TNF-~ whilc the INS-CTL produccd IL-4, IL-5, IL-10, GM-CSF, low Icvels of IFN-y and no TNF-~. None of these CTL lines produced Ik-2. Thus, under same immunization protocols and culture conditions, thc OVA-CTL developed into thc type I whereas the INS-CTL devclopcd into thc type 2 CTL, respectively. We conclude thai the exogenous antigcn plays an important role in determining the phenotype of CD8 ~ CTL. Supportcd by the grant CA70372 from Nltl.

1241

Recombinant soluble all3 TCR induce IL-12 in antigenpresenting cells tAPC). K Kawamoto, I'" Paliwal, R Ramabhadran, M S:c:epanik, RF T~'uji, & P W Askenase. Univ of Osaka Pref, Osaka, Japan; Yale Univ Sch of Med, New Haven, CT, USA Contact sensitivity (CS) is a Thl mediated, haptenMHC-specitic cutaneous immune response. Soluble recombinant oq3 TCR (sTCR) obtained as Pl-linkcd chimeric molecules cnzymatically released from T cell surfaces, have CS regulatory activity ill viva. We tested in vitro effects of sTCR on Ag-induced IFN-y production by CS T cells stimulated by hapten-conjugated APC. When CS T cells were inhibited via T cell suppressive factor, and IFN-y production was reduced, then sTCR blocked the suppression and enhanced IFN-y production. Reversal of suppression also was obtained when CS-cffcctor cells were clutured with APC pre-pulsed with sTCR. This APC effect of sTCR was mediated by IL-12 production, and blocked by anti-IL12. Further, FACS showed direct binding of sTCR to APC, and ELISA showcd increased production of ll.-12 by APC stimulated with rceombinant sTCR. Thus, reversal of suppression by sTCR was mediated via 1L-12 stimulatcd APC aftcr direct surface binding of sTCR, resulting in enhanced production of IFN-y by the CS-effcctor T cclls.

1242

Factors Regulating IL-12 Production From Human Purified Monocytes and P B M C s . . l o h n F.McDver and Robert A. Seder Laboratory of Clinical Investigation, NIAID, Bethesda, MD. Interleukin 12 (IL-12) ha becn shown to be a critical mediator in the generation of an effective Th 1 immune response to a varicty of intraccllular pathogens. While the requirements regulating IL-12 induction are not clearly defined, there is good evidence to supporl a role for CD4+ T cell derived soluble and costimulatory factors such as IFNg, and CD4(IL respcctively. Efforts were focused on the respcctive roles of IFNg and/r CD4OL/CD40 stimulation in the presence or absence of a panel of microbial antigens on IL-12 induction from fresh human monocytes and PBMCs. In the abscncc of antigenic stimulation, we were unable to detect IL-12, IL-I(I r TNFa protein from highly purified fresh human monocytes in response to CD4(I stimulation. By contrast, monocytcs stimulated with a SAC produced a small amount of IL-12 p7ll protein, which was strikingly enhanced by addition of IFNg to the cultures but

not by soluble CD40 stimulation. However, cells stimulated in the presence of both IFNg and soluble CD40 had a marked augmentation in their production of IL-12. Purified monocytcs stimulated with live M. tuberculosis (H37Ra) or Heat-killed Listeria Mono{ytogenes (HKLM) produced no detectable IL-12 p7{}. Addition of IFNg but not soluble CD40 induced a small amount of IL-12 p7(I protein in response to either stimulus, while addition of both IFNg and soluble CD4(} caused a marked increase in IL-12 production. These results suggest that activated CD4+ T cells through IFNg and CD4(IL/CD4I} stimulati~m are required for the induction of an optimal IL-12 in rcsponse to various microbial antigenic stimuli.

1243

Anti-class II MHC monnclonal antibodies inhibit the stimulation of ~ T cells by Daudi cells. John Faven, Jui-Han Huang, and Mark 71vkocinski. Institute of Pathology, Case Western Reserve Univcrsity, Clevcland, Ohio Daudi cells arc potent inducers of a polyclonal y8 T cell expansion which is dependent on hsp6l) expressed on the Daudi cells. We investigated how the expression of class II MHC by Daudi cells affects y8 T cell stimulation. 11)~' normal human PBMNC were cultured with 3 x 1()5 irradiated Daudi cells, and the expansion of y8 T cells was assessed using FITC-conjugated yB-I antibody and FACS analysis. Daudi-stimnlated y~ T cells increased both in pcrccntagc (25.4% versus 1.7% unstimulated control) and absolute numbcr (4 • 10~ versus 0.1 • 105) after a 10-day co-culture. In contrast to thc findings of other invcstigators (J. lmmunol. 150:2046, 3/93), prc-eoating the Daudi cells with any one of several anti-class I1 monochmal antibodies (mAb) substantially inhibited -y8 T cell induction (L243: 82% inhibition, 9.3FIll: 94%, I-1C4: 94%, isotype control 0%). Fab fragments of these mAb, however, were not inhibitory, suggesting a possible effect by the bivalent intact mAb on the cellular physiology of the stimulatory Daudi cells. Effccts of anti-class II mAb on several indices of B cell actiwltion, including homotypic aggregation, will be presented. The stimulation of 78 T cells by Daudi cells can be substantially altered by cellular changes induced by anti-class I1 mAb.

$304

1244

1245

Abstracts

Antigen presentation by T-helper cells: clonotypic specificity guides selective acquisition and presentation of antigen. PYArnold, MD Mannie. East Carolina University School of Medicine, Greenville, NC. Under certain circumstances, rat T cells express class II MHC/peptide complexes and serve as antigen presenting cells (APCs) for other class II MHC-restricted T cells. However, it is not known if specific T cell antigen recognition is required for T cell acquisition and presentation of antigen. We have shown that myelin basic protein (MBP)specific Lewis rat R1 T cells cultured with antigen and irradiated syngeneic splenocytes (irrSPL) in the presence of tolerogenic monoclonal antibodies (mAbs) express high levels of surface class II MHC molecules. R1 T cells purified from this primary culture presented MBP in a secondary assay to another T cell line, the R2, inducing high levels of proliferation. In these studies, we investigated transfer of specific (MBP) and unrelated (conalbumin) antigens from APCs to T cells through use of antigenpulsed, extensively washed SPL. R1 T cells cultured with MBP- and conalbumin-pulsed irrSPL efficiently acquired both antigens and presented them to the appropriate T cell responders in a secondary assay, suggesting a physical transfer of antigen from the APC to T cell surface. However, when splenic APC were pulsed separately with MBP or conalbumin, R1 T cells acquired and presented MBP but did not efficiently acquire and present conalbumin. Addition of anti-class II I-A monoclonal antibody OX6 to the primary culture inhibited transfer of antigen from splenic APC to R1 T cells. Lastly, no transfer of antigen occurred when allogeneic SPL were used as APC. In conclusion, R1 T cell acquisition of antigen requires specific antigen recognition, whereby an initial cognate interaction of the T cell receptor (TCR) with the appropriate antigen/self-MHC complex is necessary for efficient acquisition of antigen from APC. The specificity of T cell antigen presentation, like that described for B cell APCs, may be critical to maintaining the homeostasis of the immune system. This research was supported by a grant from the National Multiple Sclerosis Society.

Activated B cells Are Effective APC for Induction of Primary CD4 Responses In Vivo. SM Bradley, PR Rogers, DD Duncan, ME Malo, SL Swain, LM Bradley. The Scripps Research Institute, La Jolla, CA. Although dendritic cells (DC) are highly effective APC for naive CD4 cells in vivo, the function of B cells in this regard is controversial. We showed previously that activated B cells present Ag to naive CD4 cells in vitro. Here we evaluated their capacity to function as APC in vivo. Highly purified, resting B cells were activated with ionomycin and/or PMA, pulsed with KLH, and injected iv into syngeneic recipients. Primary CD4 responses were measured 4-5 days later by cytokine production in vitro to KLH. KLH-pulsed B cells induced predominantly IL-2 responses of similar magnitude and kinetics to those elicited by KLH in CFA. Comparable responses were also induced when pulsed DC or the B cell line, LB-1, were used for priming. Responses to KLH-pulsed B cells were not elicited in recipients that were depleted of naive T cells by thymectomy, and were thus solely due to priming of naive CD4 ceils. Unlike KLH in CFA, KLH-pulsed B cells failed to elicit humoral responses. This indicated that KLH was presented as peptides and induced a CD4 response in the absence of B cell priming. Transfer of Ag to host APC was excluded since in vitro restimulation of CD4 cells from F1 recipients was restricted to the parental MHC haplotype used for immunization. B cells activated in vivo by priming with the NP hapten conjugated to an unrelated carrier, CGG, in CFA were found to induce primary CD4 responses to KLH following iv injection of soluble NP-KLH. Our results indicate that naive CD4 cells are not limited to use of DC as primary APC, and may respond to B cells which become activated by Ag in vivo.

J ALLERGY CLIN IMMUNOL JANUARY 1997

1246

Induction of Human Cytotoxic CD4+ T Cells: Role of CD80 and CD86 Expression on Antigen Presenting Cells. Thomas Wendland, Davide Mauri, Karin Frutig, Elisabeth Frei, Karl Brander attd Werner J. Pichler Institute of Immunology and Allergology, Inselspital Bern, CH-3010 Bern Aim of the study: We previously investigated the role of T cells as antigen presenting cells (APC) and found that T cells as APC can induce cytotoxic CD4+ T cells. Blocking experiments suggested that the level of CD80 adhaesion molecule expression on T cells determines the induction of CD4+CTL. To further investigate the presumed nessessity for CD80 costimulation in the primary induction of CD4+ cytotoxicity and to analyse the mechanism of CD4+ T-cell mediated cytotoxicity, we: a) examined other classical (monocytes, dendritic cells, EBV-transformed B-cell lines [B-LCL]) and non-classical APC's (Keratinocytes, activated T cells) upon their ability to induce cytotoxic CD4+ T cells, b) generated stable mouse fibroblast transfectants bearing human MHCII and expressing different levels of either human CD80 or CD86 and analysed their capacity to induce cytotoxic CD4+ T cells, c) examined the inhibitory effects of drugs (Concanamycin A and Brefeldin A) on CD4+ mediated cytotoxicity and measured Fas-Ligand expression by competitive RT-PCR on cytotoxic and noncytotoxic CD4+ T cells. Results and Conclusion: a) Only APC's (monocyte derived dendritic cells, but not e.g. B-LCL) with levels of CD80 expression comparable to T cells were able to induce CD4+ cytotoxicity. b) The level of CD80/CD86 expression on transfected fibroblasts, together with the ratio of cell numbers (APC's vs CD4+ T cells) determines the development of CD4+ cytotoxicity. c) CD4+ cytotoxicity induced in our system is mainly Perforin and not Fas-Ligand mediated.

1247

Anti-HIV Activity of CD8+ Cells Can Be Enhanced by Antigen Presenting Cells Bearing B7. Edward Barker, K. Bossart, S. Fujimura, and J.A. Levy. Dept. Medicine, UCSF, San Francisco, CA 94143. A subset of CDS+ T-cells which express CD28, a membrane receptor for the B7 molecule found on antigenpresenting cells (APC), is primarily responsible for noncytotoxic suppression of HIV replication in CD4+ cells from people infected with HIV. Triggering the CD28 molecule on CD8+ cells from HIV-infected people with anti-CD28 antibodies during stimulation through the CD3 complex increases their ability to suppress HIV replication. This enhancement of CD8+ cell antiviral activity is due to an increased production of IL-2 and expression of its receptor. We now demonstrate that exposure of CD8+ cells from HIV-infected individuals with APC bearing B7 increases the ability of the CD8+ cells to suppress HIV replication. The effect of the APC on CD8+ cell antiviral activity appears to be related to enhanced production of IL-2 by CD8 + cells, since interfering with the ability of IL-2 to bind to its receptor, during stimulation in presence of macrophages, abrogated the ability of CD8+ cells to suppress HIV replication. Blocking the B7 molecule on macrophages with the anti-CTLA-Ig fusion protein in the absence of exogenous IL-2 abrogated the ability of CD8+ cells to suppress HIV replication. Furthermore, this effect of blocking B7 is reversed by triggering CD28 with antiCD28 antibody. In this regard, a recent study (AIDS Res. and Hum. Retro. 12:885) has shown that expression of B7 on the surface of macrophages is significantly decreased in AIDS patients when compared with healthy infected individuals. Based on these findings and the observations described above, triggering CD28 during stimulation of CD8+ cells could provide benefit to HIV-infected individuals during disease progression.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1248

Distinct class I MHC receptors on class 11 MHC-restricted T cells co-activate or inhibit immune responses to low doses of antigen. D M Davis, 0 Mandelboim, H T Revbum, M Valds-G6mez, E G Sheu, L Pazmany and J L Strominger. Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA. Class II MHC-restricted T cells expressing class I HLA-C allotype specific co-activating receptors, previously found only on Natural Killer (NK) cells, have been cloned (TANK cells). In addition, T cell clones that express inhibitory NK receptors or those that express both types of receptor have becn obtained. The response of these clones to superantigen or antigen presented by class II MHC molecules on cells also expressing appropriate class I MHC molecules is either augmented or suppressed, depending on the type of NK receptors present. In particular, the amplification of the T cell response mediated by coactiw~ting NK receptors is especially evident at low doses of antigen and is maintained for up to nine days. Such enhancement may amount to as much as a 10 fold increase in the proliferative response of T cells to superantigen. These data coupled with other recent publications examining T cells with inhibitory NK receptors infer that NK receptors do not alter the nature of T cell responses per se, but rather alter the strength of stimulus that is required to elicit a proliferative response and may also determine the duration of T cell-mediated immune responses in viw).

1249

Biologically Active Covalently-Linked Recombinant c~1-[31 Peptide Binding Domain of MHC II Molecules. Subha Arimilli, lrina Astafieva, Prabha Mukku, Kiara Bechta, John Mumm attd Bishwajit Nag Anergen, hw., 301 Penobscot Drive, Redwood City, CA 94063 Major histocompatibility (MHC) class II molecules are cell surface heterodimeric (ab) glycoproteins that display processed antigens to T cell receptors of CD4 positive T cells. Both ed and [31 domains are involved in creating the peptide binding groove of the MHC class II molecules. In this report we describe the E. coil expression of covalentlylinked c~1-131 sub-structure of HLA-DR2 (DRB5*0101) using a 12 amino acid linker [(GGGS)~]. The expressed protein was purified by SI(10 size-exclusion followed by High-Q anion exchange chromatography using salt step gradient. Purified and refolded protein was recognized by heterodimerie specific L243 monoclonal antibody. Like the native DR2 heterodimer, the covalently-linked cd-[31 can specifically bind MBP(83-102)Y83 antigenic peptide demonstrated by biotin-MBP and earboxyfluorcscein-MBP peptides. The specificity of the peptide binding was demonstrated using MBP(1-14) peptide that has been shown to have no binding affinity with native DR2. The T cell recognition of complexes of cd-[31 and MBP(83-102)Y83 peptidc was demonstrated by a two fold increase in g-IFN secretion in a virally transformed T cell clone which led to the induction of time dependent antigen-specific apoptosis in 20% of the total cell population. These results provide the first evidence that soluble recombinant covalentlylinked c~1-[31 domains of human MHC class I1 molecules can bind antigenic peptide, and complexes of c,l-~l can recognize TCRs to induce antigen-specific T cell response in vitro. Since the CD4 is known to interact directly with the [32 domain of MHC class lI molecules, these results reported here with the cd-[31 domain suggest that the interaction of CD4 molecules with MHC class II molecules may not be required for the induction of T cell apoptosis.

1250

T-ceil clonal survival prolonged by self- and non-seifpeptide partial agonists. S Matsushita, Y Nis'himura. Div. Immunogenetics, Kumamoto Univ. Grad. Sch. Med. Sci., Kumamoto, Japan. Recognition of certain peptide analogs by T cells results in T-cell activation without proliferation (partial agonism) and leads to functional activation, anergy, lymphokine production or survival and differentiation of thymocytes (positive selection). In addition to one-residue substitution of wild-type peptides, ram-self-reactive T-cell clones can be fully or partially actiwLted by minimally homologous selfpeptides, or vice versa; i.e., self-reactive T cells are acti-

Abstracts

S305

vated by non-self-peptides. However, the roles of peptide partial agonists in the survival of mature T cells are unclear. In this study, we tested whether non-self Bacillus Calmette-Gu&in (BCG)-reactive human CD4 + T-cell clones can be partially activated by analog or minimally homologous self-peptides to prolong their life span. We found that (a) stimulation of T cells with a non-self analog or a minimally homologous self-peptide fragment can prolong the T-cell survival, in vitro; (b) this prolongation is associated with the up-regulation of Bcl-x~, without proliferation; and (c) these peptide-clone combinations are capable of inducing lymphokine secretion. Thus, peptide partial agonism may play a role in the survival of not only thymocytes but also mature T cells, in the absence of wild-type ligands.

1251

T Cell Proliferation and Cytokine Transcription Are Independently Regulated in Response to Peptides Altered by a Single Amino Acid Substitution at Anchor Position 1. DK Newton-Nash, KL Kennedy, D Ante and NK Garlie, The Blood Research Institute, Milwaukee, WI The T cell receptor (TCR) recognizes antigen as a peptide bound to molecules encoded within the major histocompatibility complex (MHC). The peptide binds to a groove in the MHC molecule which accommodates a limited set of amino acid side chains at anchor positions within the peptide. The functional significance of amino acid substitution at peptide anchor positions is not clear. Previous work (Wu, S., et al. 1996, J. Immunol. 156:3815) demonstrated that effects on T cell recognition of amino acid substitution at a peptide anchor position are independent of peptide-binding affinity or SDS stability of resulting peptide/MHC complex. In this study, we demonstrate that conservative amino acid substitutions at anchor position 1 of influenza A matrix protein peptide (MP19 3n) can induce cytokine gene transcription independent of thymidine incorporation by T cells. Two DRl-restricted, MP~93~specific Th0 clones (MP10.4 and MP10.8) were cultured with native peptide (PLKAEIAQRLEDV) and peptide substituted at anchor position 1 (position 20) with alanine (MPI~ 3~A20) or tryptophan (MPI, 31W20). MP10.4 incorporated thymidine in response to both native and variant peptides. MP10.8 incorporated thymidine in response to stimulation with native peptide and MPjg_~W20, but not MP~,,_31A20. RT-PCR analysis of cytokine mRNA demonstrated that IL-4 and IFN-y transcription correlated with thymidine incorporation for both MP10.4 and MPI0.8. In contrast, TGFBI transcription but not thymidine incorporation was observed upon culture of MP10.8 with MP,)_3jA20. These results indicate that peptide variants can induce different signaling events and that thymidine incorporation and cytokine gene transcription may be independently regulated.

$306

1252

Abstracts

J ALLERGY CLFN FMMUNOL JANUARY 1997

apparent selectivity of the other predominant TCR functional pocket, thus suggesting a remarkable degree of receptor plasticity. This ability of the TCR/MHC/peptide complex to undergo conformational changes provides a conceptual framework for reconciling the apparent paradox of the extreme selectivity of the TCR and yet its remarkable cross-reactivity with different MHC/peptide complexes.

Combinatorial Peptide Libraries allow identification of crossreactive exogenous and endogenous ligands for human class II restricted T cells. Bernhard HemmeF, Marco

VergellF, Burkhard Fleckenstein "~, Guenther Jung~, Henry McFarland I, Karl-Heinz Wiesmueller~, Roland Martin z. 11 Neuroimmunology Branch, NINDS, National Institutes of Health,Building 10, Room 5B-16, 10 Center DR MSC 1400, Bethesda, MD 20892-1400, USA 2Institut fuer Organische Chemie, Universitaet Tuebingen, 72076 Tuebingen, Germany -~Naturwissenschaftliches und Medizinisches Institut an der Universitaet Tuebingen, 72762 Reutlingen, Germany CD4+ class II restricted T cells are likely to be involved in the pathogenesis of most human autoimmune diseases. Molecular mimicry between foreign and self ligands has been discussed as an important autoimmune mechanism. In this report we introduce combinatorial peptide libraries to identify crossreactive ligands for these T cells. The T cell recognition of a CD4+ T cell clone (TCC) specific for MBP (86-96) was completely dissected by the response to a set of 220 ll-mer peptide sublibraries. Based on the prediction by the libraries, peptide ligands derived from protein sequences of self and microbial proteins were identified and shown to be stimulatory for the T cell clone. These data demonstrate that (1) in at least some class II restricted T ceils TCR recognition is highly degenerate, (2) that for these TCC combinatorial peptide libraries are useful to define TCR recognition, and (3) that combinatorial peptide libraries provide a powerful tool to clarify the spectrum of stimulatory ligands for a specific TCC. 1253

a TCR [3 chain interacts with the H-2Kb/VSV8 complex at the area flanking the amino terminus of the peptide.

Goyarts EC, Vegh Zs, Kalergis A, Thomson C, Nathenson SG. Albert Einstein College of Medicine, Dept Micro & Immunol, Bronx, NY TCRs with single amino acid changes in the CDR3 loop of the [3 chain were expressed with the wt c~chain in a TCR deficient cell line to probe questions concerning TCR interactions with class 1 MHC/peptide complex. Of the seven positions scanned, 96-102, four positions inactivated TCR recognition, demonstrating that these sites were critical for recognition. Four mutants which maintained TCR recognition permitted further mapping on the H-2Kt'/ VSV8 complex. The recognition patterns of TCR mutants $96T and E101A were identical, and two others, $96A and G97A, were different from the wt TCR transfectant. The latter two restored recognition of the K ~" point mutants, R62Q and E166K, and enhanced sensitivity for peptide variants at P1 position. These compensatory mutations suggest that this particular TCR[3 chain assumes an orientation over the N-terminus of the peptide and interacts with both the peptide and the MHC class I resiudes. 1254

Complementary mutations in an antigenic peptide allow for cross-reactivity of autoreactive T cell clones. LJAusubel, CK Kwan, A Sette, V Kuchroo, DA Hailer. Brigham and Women's Hospital and Harvard Medical School, Boston, MA and Cytel Corp, San Diego, CA T ceils recognize antigen by formation of a trimolecular complex in which the T-cell receptor (TCR) recognizes a specific peptide antigen within the groove of a major histocompatibility complex (MHC) molecule. It has generally been assumed that T cell recognition of two distinct MHC/antigen complexes is due to similarities in the threedimensional structure of the complexes. Here we report results of experiments examining the cross-reactivity of T-cell receptors (TCRs) recognizing a myelin basic protein peptide and several of its analogs in the context of MHC. We demonstrate that single conservative amino acid substitutions of the antigenic peptide at the predominant TCR contact residues at positions 91 and 93 totally abrogate reactivity of specific T cell clones. Yet, when a conservative substitution is made at position 91 concomitant with a substitution at position 93, the T cell clones regain reactivity equivalent with that of the original stimulating peptide. Thus, the exact nature of the amino acid side chains engaging one TCR functional pocket may change the

1255

Immunization with peptide variants as strategy to learn about the TCR structure and function. A M Kalergis, T

Ono, T P Dilorenzo, F Wang, E C Goyarts, S Vegh, H E Horig and S Nathenson. Department of Microbiology and Immunology. Albert Einstein College of Medicine, Bronx, New York The highly diverse heterodimeric ~[3TCR of T CD8+ cells, recognizes antigens as short peptides bound to the MHC class I molecules. Three different approaches are being followed by our group to study the TCR-antigen interaction: 1) site directed mutagenesis of the TCR; 2) mice immunization with variants of a known antigenic peptide; 3) TCR photo-cross-linking by a modified antigenic peptide. Mutations on the CDR3 loop of the [3 chain of one specific TCR recognizing a VSV nucleocapsid 8 mer peptide bound to Kb, showed that the [3CDR3 loop interacts with both MHC and peptide residues. The analysis of those interactions allows the prediction of a major orientation for this specific TCR, in which the [3chain interacts preferentially with residues flanking the amino terminus of the peptide bound to Kb. To further analyze the role played by the peptide in the TCR/antigen interaction, we immunized B6 mice with the VSV-nucleocapsid peptide and single amino acid mutated peptides. Thus, a CD8+ T cell mediated immune response is triggered, and the effect that peptide single amino acid substitutions have on the structure of the elicited TCRs can be reflected in V[3 segment usage on the CD8+ population recognizing that peptide. Preliminary data show that the T CD8+ cells recognizing the wild type VSV peptide use predominately V[313 and V[38. However, the substitution Arg(P1)Lys dramatically reduces the usage of V[313 by specific CD8+ cells, whereas increases V[38. The decrease on V[313 usage was even more striking after immunization with Gln(P6)Lys, where again V[38 increased. On the other hand, the peptide Val(P4)Tyr significantly increases V[313 and V[311. These results show that by making discrete changes on the antigenic peptide used for the immunization, the repertoire of triggered TCRs can be manipulated. The observed changes in the V[3 gene segment usage as result of single replacement in the peptide, suggest that, besides CDR3, CDR1 and 2 could also interact with the peptide. Furthermore, considering that single replacements at the N, as well at the C terminus of the peptide, affect the V[3 gene segment usage, it is probable that in a peptide-specific T cell population, some TCR [3-chains interact with the N-terminus (case of the T cell clone studied by site directed mutagenesis), while others with the C terminus of the peptide. These studies are being extended by cloning individually the TCRs recognizing each peptide variant, looking for motifs at the amino acid sequence of their CDR3 loops, and refined by the TCR cross-linking approach. We have synthesized a VSV 8 mer derived photoreactive peptide, able to bind K b, which is being currently used to immunize mice.

Abstracts

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1256

Understanding the Mechanism of T Lymphocyte Anergy. Debra Bloom, Bethany Moore, and C. G, Fathman. Stanfl~rd University, Stanford, CA A THI cell can be rendered unresponsive to antigen stimulation by triggering the TcR (signal one) in the absence of costimulatory signaling (signal two). This anergic state is defined as the lack of proliferation due to the inability to produce [L-2. The intracellular pathways which lead to T cell anergy are not well understood. Howcver, two points are clcar. First, the induction of anergy can be circumvented by cyclohexamide which suggests that new protein synthesis is required. Second, the Ras signaling pathway appcars to be blocked aflcr 24 hours of ancrgy induction. To further understand the mechanism(s) of anergy, wc have analyzed its kinetics in murine T lymphocytes. Our data demonstrate that the unresponsive phenotype is not present until 3-4 hours after giving T cell chines signal one without signal two. Furthermore, we show that the Ras pathway is blocked even at this early stage of unresponsiveness, as measured by ERK-2 phosphorylation levels. Thcse data support the hypothesis that Ras inactivation results in the anergic phenotype and suggests that a protein may be synthesized in anergic cells which dysregulutes Ras.

1257

T Cell Anergy Induced by Tetanus Toxoid-Derived Native Peptides is Accompanied by Th2 Cytokine Production. EK. Chou, 1. Robey, C.N. Woody, H. O~wr, A.A. Vandenhark and M.P. Davey, Dept. of Veterans Affairs Medical Center and Oregon Health Sciences University, Portland, OR Peptide-induced T cell ancrgy, using high concentrations of native peptide ligand, has been well characterized for relatively few antigens. Tetanus Toxoid (TT)-specific, CD4+ human T cell lines wcre generated from 2 donors by 4 cycles (cycle time = 7 days) of stimulation with TT, autologous APC and rlL2 (5 ng/ml). Using a panel of IT-derived peptidcs in standard T cell proliferation assays, the T cell lines from donors I and 2 reacted predominately to peptidcs T2 (amino acids 616-631) and T3 (amino acids 640-651), respectively. Each T cell line could be rendered unresponsive to its dominant epitope in a dose dependent manner (1C5,~=0.113 and 1.7 gg/ml for T2-donor 1 and T3-donor 2 lines, respectively) by culture for 2 additional cycles with peptkte, APC and rlL2. Anergy induction was not associated with a decrease in T-cell viability, nor could it be prevented or revcrscd by 50 ng/ml of rlL-2 or co-culture with intact TT. The TT-specific line from donor 1, as well as 3 T-cell chmcs established from this line, produced INF-y and negligible levels of IL-4 or -10 as detected by ELISA when maintained with intact TT. A significant increase in IL-4 and -10 production, and sustained INF-y production, occurred following 1 cyclc of anergy induction with T2. The cultured cells from donor 2 produced INF-y and low levels of IL-4 in response to TT. Following anergy induction with T3, INF-y production remained stable and there was a significant increase in IL-4. These data are consistent with previous reports of anergy induction by nativc peptides and show that this process may be accompanicd by a shift from a Thl cytokine pattern to a mixed profile containing both Thl and Th2 cytokincs.

1258

The role of protein kinase C-beta in T-lymphocyte activation and tolerance induction. JT Snyder, II. OJ Finn. University of Pittsburgh School of Medicine, Pittsburgh, PA Whether a T cell becomes activated or tolerized on encounter with antigen is probably determined by differences in intracellular signal transduction. Thc major pathway for T cell receptor signalling involves activation of protein kinase C (PKC). We hypothesizc that PKC has a role in activation and tolerance induction in mature, peripheral T cells, and in positive and negative selection during T cell development. To test our hypothesis, we produced transgenic mice in which PKC expression can be temporally regulated in T cells. Two specific promoters were used for this purpose. The lck proximal promoter is

S307

active predominantly in thymocytes. Expression of PKC from this promoter enables examination of the role of PKC in positive and negative selection of T-lymphocytes in the thymus. The lck distal promoter is active predominantly in mature, peripheral T cells. Expression of PKC from this promoter enables an examination of its role in activation or tolerance induction in mature T cells. Founder mice carrying the PKC-beta isoform driven by each of these promoters have been generated, with germline transmission to FI progeny. The mice carrying PKC-beta driven by the lck distal promoter develop a Iymphoproliferative disease which becomes increasingly severe with age. All lymphoid organs in these mice become greatly enlarged with age. The mice carrying PKC-beta driven by the lck proximal promoter appear to have a relatively normal distribution of thymocyte subsets except for a slight skewing towards the double positive subset. These results suggest that PKC-beta plays a significant role in activation of mature T cells in vivo, and in positive and negative selection during T cell development.

1259

Giant-l, a gene induced in anergic T lymphocytes. Korthiiuer U, Menon RS, Manoli DS and Bluestone JA. Ben May Institute for Cancer Research and Committee on Immunology, University of Chicago, 5841 S. Maryland Ave MC 1089, Chicago, IL 60637 This project is aiming at the identification, isolation and characterization of genes that are differentially expressed in anergic versus responsive murine T lymphocytes. To achieve this goal we employed differential display RT-PCR on responsive versus anergized murine Thl clones. Giant-1 was found to represent a 4 kb mRNA that was upregulated in anergic T cell after anergy was induced using either immobilized anti-CD3 mAb in the absence of costimulation or antigen on chemically fixed antigen presenting cells (APC). Giant-I was only marginally induced upon normal activation with antigen presented by live APCs. mRNA expression was hmg-tived, evident as late as seven days after stimulation in anergic but not in control Thl clones. Finally, Giant-I seems to be the only member of a multigene family or alternatively spliced products displaying this anergy-associated mRNA expression pattern. Giant-1 mRNA expression is clearly not restricted to cells of hematopoietic origin. We observed a strong signal from lung or heart tissue. In addition, high levels of constitutive expression of giant-1 were observed in fibroblast cell lines indicating a potentially important role in fibroblast function. Scquence analysis of a 2.5 kb partial cDNA clone 1AI revealed only homology to expressed sequence tags (EST). Current studies are underway to obtain a full length clone for sequencing and transfection studies in order to finally examine whether giant-1 plays a functional role in the maintenance of anergy.

S308

Abstracts

1260

Cytokine regulation of activation-induced cell death in the CD4 T helper subsets. Lisa V. Tsui, Xiaohong Zhang, Susan L. Swain. Trudeau Institute, Saranac Lake, New York The factors that regulate activation-induced cell death (AICD) in the CD4 T helper (Th) cells are not well defined. Previously, we have shown that Th2 polarized effector cells, generated from TCR transgenic mice, undergo AICD between days 4 and 7 after restimulation with peptide antigen. We found that IL-2 and TGFI3 together synergize to nearly completely block apoptosis in Th2 effectors. Th 1 effector cells undergo a more rapid cell death mediated by Fas:FasL interaction with maximal apoptosis occurring between 1 and 2 days after restimulation with antigen. IL-2 and TGFI3 together also block this cell death and prolong cell expansion. Other cytokines such as IL-3, IL-7, IL-10 or IL-15 did not block apoptosis in Thl cells. IL-4 showed a slight block in cell death 1 day after restimulation. However, Th2 effectors generated from IL-4 knockout mice underwent AICD much earlier compared to wildtype controls with a kinetics similar to that of AICD in Thl effectors. These results suggest a role for IL-4 in the regulation of cell death in the CD4 T helper subsets perhaps at the level of their differentiation to a state where they are resistant to short term death. The expression of cell death genes is currently being investigated. We have found that the expression level of bcl-2 and bcl-xL do not differ in the Thl and Th2 subsets.

1261

IL-2-induced Cell-mediated Lysis by Thl Clones: A Fasbased Pathway Involving LFA-1 and Protein Tyrosine Kinase Fyn. David W.. Lancki. U of Chicago Ag-specific CD4 + murine Thl clones can lyse nucleated target cells bearing relevant Ag. Thl clones can also be induced to lyse target cells that lack relevant Ag in the presence of IL-2. Two cell-mediated cytolytic pathways have been identified in Ag-specific T cells, based on short-term lytic assays, a perforin-based pathway and a Fas-based pathway. The perforin pathway is expressed by CD8 + CTL clones and by some CD4 + Th2 clones but not by CD4 § Thl clones. The role of the Fas pathway in IL-2-induced lysis by Ag specific CD4 + Thl clones derived from normal mice, was tested using target cells derived from normal (C57BL/6) or mutant (B6.Mrl.lpr) mice that lack the Fas molecule. Thl cells lysed target cells in the presence of IL-2, but only when the target cells express Fas. Thl cell-mediated lysis induced by IL-2 is inhibited by a mAb against Fas. Lysis was also inhibited by mAb against LFA-1 or ICAM-1. Collectively our results indicate cellmediated lysis by Thl clones induced by IL-2 as well as by Ag, mAb against CD3, Thy-1, or Ly-6C, or phorbol ester alone or with calcium ionophore requires expression of Fas on the target cells. Thl clones derived from mice that lack expression of normal Fyn protein lyse target cells and respond to Ag stimulation normally, but responses induced by stimulation through the GPI-linked molecules Thy-1 and Ly-6C are defective. While proliferative response of Thl clones derived from Fyn mutant mice to IL-2 is normal, lysis induced by IL-2 is defective as compared to that of clones derived from normal mice. The basis for this defect is not clear. However, changes in cell adhesion may be involved. LFA-l-mediated homotypic adhesions are induced by IL-2 plus IL-12, and it appears that these and other adhesion properties of T cell clones derived from Fyn mutant mice are altered.

1262

Peptide Gly-Am-Phe-PO4 inhibitors inactivate GrA, GrK and perforin-mediated lysis. SA Fraser, U Winkler, DS Jackson, C Kam, J Powers, D Hudig. University of Nevada, Reno; Reno, Nevada Cytotoxic lymphocyte granules contain two trypsin-like granzymes (Gr), GrA and GrK. The role of these granzymes in perforin-mediated lysis was investigated using peptide based phosphonate inhibitors containing amidinophenyl glycine (AmPhGly, which is structurally similar to Arg) in the P1 position. The inhibitors' selectivity for GrA or GrK is conferred by varying the amino acids in the P2 and P3 positions. A panel of the peptide-based phospho-

J ALLERGY CLIN IMMUNOL JANUARY 1997

nate inhibitors were synthesized and screened for their effect on GrA, GrK and lytic activities. The efficacy of each inhibitor was measured in two ways. First, we determined the second order inhibition rate constants (kob,a/[I]) with purified rat GrA and GrK to assess the selectivity of the inhibitors. Secondly, we pretreated granule extracts of RNK-16 cells with 0.1 mM inhibitor and assayed for hemolytic perforin activity. Benzyloxycarbonyl (Cbz)-AlaGlyAmPhe-P(O)(OPh)2 was a good inhibitor of GrA (1270 M ~s 1). 3,3-Diphenylpropionoyl Pro-GlyAmPheP(O)(OPh)2 was a good inhibitor of GrK (405 M-Is ~). These inhibitors also inactivated lysis: Cbz-Ala-GlyAmPheP(O)(OPh)2 (73% of control lytic units). Each selective inhibitor had lesser reactivity with the other tryptase. These data indicate that peptide phosphonate inhibitors can select between the 2 granzymes and suggest that even greater selectivity can be achieved using peptides than are optimally matched to each granzyme. The data also indicate that a tryptase is involved in perforin lysis. Furthermore the key tryptase(s) can be effectively inactivated in the presence of granule proteins that may include their natural substrates. Finally, we believe tryptase granzymes are molecule to target for immunosuppression. (NIH R01 CA38942, RO GM42212)

1263

Effect of perforin on DTH induced by CD8 T subsets. Li Li and Tim R. Mosmann. Dept. of MMI, Univ. of Alberta, Edmonton, AB, Canada, T6G 2H7 The short-term cytotoxic activity of CD8 T cells is mediated by perforin and Fas pathways. In vitro cytotoxieity of allo-reactive Tcl and Tc2 cells (CD8 T cells producing Thl and Th2 cytokines respectively) is mainly through the perforin-mediated pathway, and both Tcl and Tc2 cells induced adoptively transferred footpad DTH in mice bearing the target MHC molecules. However the effect of the CTL activity of these cells on DTH is not known. The generation of perforin-deficient mice provides a good system to address this question. Using the adoptively transferred DTH model, and Tc cells generated from both normal and perforin-deficient mice, we tested the effects of perforin-dependent cytotoxicity on CD8 T cell-mediated DTH. As expected, Tc cells from perforin-deficient mice showed no killing to Fas- target cells in a short-term cytotoxic assay. However perforin-deficient Tcl and Tc2 cells were able to induce footpad DTH, although in some cases the magnitude of the reaction was significant lower than that induced by normal Tc cells. These results suggested that perforin-mediated cytotoxicity of CD8 T cells may enhance the extent of DTH responses, but might not be a critical factor for the reaction. When testing the cytokine levels in the DTH footpads, we found that normal and perforin-deficient Tcl and Tc2 cells all retained their in vitro cytokine patterns in vivo, and Tcl or Tc2 cells from normal and perforin-deficient mice produced similar levels of the corresponding cytokines, suggesting that these cells may be activated in vivo similarly. Furthermore similar levels of inflammatory cytokines, IL-6 and TNF, were detected in footpads injected with either perforin-deficient or normal Tc cells, again suggesting a similar DTH induced by these cells.

J ALLERGYCLIN IMMUNOL

Abstracts

$309

VOLUME 99, NUMBER 1, PART 2

1264

Fas signaling causes GI arrest by a p53-independent pathway. Tao Dao, R Hingorani, J Huleatt, N Littlewood, IN Crispe, lmmunobiology Section, Yale University Medical School, New Haven, CT The induction of apoptosis is intimately linked to cell cycle events and occurs at distinctive phases of the cell cycle. In T lymphocytes, a major physiological mechanism of apoptosis is death following the engagement of Fas (CD95) by its ligand (FasL), but the relationship between Fas signaling and cell cycle control has remained largely uncharacterised. The present study shows that Fas-signaling induces apoptosis and cell cycle arrest in the G0/GI phase in both immature and mature T cells. Biochemical changes of p21 ~iv-~ induction and pRb dephosphorylation, that are characteristic of G1 arrest, may hold cells in the susceptible phase of the cycle for Fas induced killing. Furthermore, in contrast to the G1 arrest induced by DNA damage, this process is p53-independent.

1265

Expansion of C D l l b {Mac-1)-positive T cells in perforin/ Fas ligand double-deficient mice. J. Spielman, R.K. Lee and E.R. Podack. University of Miami, Miami, FL. Mice deficient in both perforin and Fas-ligand cytolytic pathways were generated by crossing perforin knockout mice with gld mice lacking functional Fas-ligand. We have previously demonstrated that these mice lack both the perforin and Fas-ligand short-term lytic pathways, but are capable of slow lysis of susceptible targets via TNF. Doubly-deficient (DKO) mice derived from both C3H and C57Bl/6gld backgrounds and maintained under barrier conditions developed splenomegaly, and increased numbers of Mac-t+ adherent cells in both spleen and lymph nodes. All DKO mice screened to date (29/29) developed pancreatitis and died at 12 - 16 weeks. Female DKO mice developed infiltration of the uterus and failed to reproduce. Analysis of cells infiltrating the pancreas of 10-12-week-old DKO mice revealed thc presence of large numbers of Mac-l+, Thy-l+ CD3+ cells which were only present in small numbers in control gM mice expressing a single copy of the perforin gene. These cells were also present in lower numbers in the spleen and lymph nodes of DKO mice. These data suggest a role for perforin in the regulation of an unusual subset of Mac-I +, Thy-I + CD3+ cells.

1266

A low molecular weight component of RNK-16 granules facilitates lysis of purified perforin. U. Winkler, S. A. Fraser and D. Hudig. Department of Microbiology, MS 320, University of Nevada, Reno NV 89557 Perforin is a pore-forming protein found in the granules of cytotoxic lymphocytes and natural killer cells. These granules also contain proteoglycans, a number of serine proteases (granzymes) and other proteins. We hypothesize that granzymes in conjunction with a yet unidentified component of these granules play a key role in the perforin system of lysis. In support of this hypothesis, we show here that by supplementing perforin with a low molecular weight component from the granules we were able to increase the lytic activity by approximately 200% as compared to nonsupplemented control. Purified perforin was obtained using a two step column chromatography procedure. The first step involves separating perforin from the proteoglycan and granzymes using an immobilized metal affinity chromatography procedure (IMAC) where copper was the ligand. Perforin was further purified using hydrophobic interaction chromatography. At this stage there was a substantial loss of lytic activity which coincided with the loss of granzymc activity. We found that supplementing perforin with granzymes was not enough to restore lysis, while restoration with a hydrophobic fraction did. Microgram quantities of the perforin facilitating material were isolated from the fraction that was not bound to the 1MAC column during pcrforin preparation. This material was further purified on a MonoQ anion exchange column. It eluted at approximately 0.65M NaCI from the MonoQ column and has a molecular weight of approximately of 15 kD. We believe that this component has a significant role in the perforin system of lysis. (NIH R01 CA38942) TM

TM

1267

A Two Hit Model of CTL Killing in vivo. YNakamoto, L G Guidotti, F V Chisari. The Scripps Research Institute, La Jolla, CA/USA Cytotoxic T lymphocytes (CTL) kill their target cells either by Fas ligand-Fas interactions or by releasing perforin/granzymes. Most of the evidence for this concept has been produced using easily lysable target cells in vitro. In this study we asked if activation of each of these death pathways is sufficient to kill primary hepatocytes and to cause liver disease in a living animal. Equal numbers of BrdU-labelled, CD8-positive, Ld restricted, HBsAg specific CTL clones derived from wild type (wt), Fas-ligand-deficient (gld), perforin gene knockout (pko) and interferon gamma knockout (gko) mice were injected into hepatitis B virus (HBV) transgenic mice. CTL entry into the liver and induction of hepatocellular apoptosis were monitored immunohistochemically and by TUNEL assay, while the severity of the liver disease was followed by serial analysis of serum alanine aminotransferase (sALT) activity. As shown below, all CTL clones displayed comparable cytolytic activity in vitro, measured as 5~Cr release from HBsAg + P815 target cells.

CTL

Fas L

Pe~orin

IFNy

wt gld pko gko

+ + +

+ +

+ + +

+

%51Cr Release

Apoptosis

Peak sALT

89 87 32 86

64 7 1 67

618 95 76 475

In contrast, the ability of the CTL clones to induce hepatocellular apoptosis and to cause liver disease were drastically different from one another in vivo. Specifically, the wt and the gko CTL clones induced apoptosis in 64-67 hepatocytes per 100 high power fields and caused serum ALT activity to increase from 50 (baseline) to 475-618 units per liter. In contrast, the gld and pko CTL clones caused virtually no apoptosis or liver disease in the recipients. These results demonstrate that the requirements for CTL killing are much more stringent in vivo than in vitro. Furthermore, they suggest a two hit model in which Fas ligand- and perforin-dependent death pathways must be activated simultaneously for CTL to be cytopathic in the liver, thereby enhancing CTL specificity and minimizing bystander killing in this vital organ.

1268

Molecular Principles of Aberrant CTL Function. B Kessler, JC Cerottini, IF Luescher, Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Switzerland. We tested for antigen recognition and TCR-ligand binding 12 peptide derivative variants on seven H-2Ka-re stricted CTL clones specific for a bifunctional photoreactire derivative of the Plasmodium berghei circumsporozoite peptide 252-260. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to K d molecules and photoactivation of the orthogonal group to TCR. In most cases (>80%) cytotoxicity (sICR release) and TCR-ligand binding (TCR photoaffinity labeling) differed by less than 5-fold. Exceptions included: i) partial agonists (8 cases) for which recognition was more efficient than binding, ii) antagonist (2 cases), which were not recognized and inhibited the recognition of the wild type conjugate, iii) heteroclitic agonists (2 cases) and one partial agonist that activated only Fas (CD95), but not perforin mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding. Remarkably, CD8 dependent clones clearly inclined more to TCR antagonism than CD8 dependent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 can directly interfere with CTL function.

$310

1269

1270

1271

Abstracts

Dexamethasone- and Activation- Induced Apoptosis is Mediated by a Conserved Death Protease Pathway Downstream of Bcl-2. Richard K. Lee and Eckhard R. Podack. Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33136 Activation-induced cell death (AICD) of lymphocytes by CD3 signalling is mediated by Fas and Fas ligand and is not blocked by overexpression of bcl-2. In contrast, dexamethasone-induced DNA degradation and apoptosis is independent of Fas and Fas ligand and is susceptible to inhibition by bcl-2. This dichotomy in apoptosis signalling and regulation by two disparate triggers has led to speculation that AICD and steroid-induced apoptosis utilize different cell death pathways. Dexamethasone readily induces apoptosis in 2B4 (a thymic hybridoma) that is blocked by protease inhibitors selective for members of the interleukin 113 converting enzyme (ICE) and CPP32 protease family. Likewise bcl-2 overexpression blocks dexamethasone-induced death of 2B4. 2B4 readily undergo anti-CD3 triggered AICD that is also blocked by inhibitors of the ICEand CPP32-1ike proteases but is insensitive to bcl-2 overexpression. Thus, glucocorticoid- and activation-induced apoptosis are mediated by a conserved death pathway regulated at the level of signal integration and activation of ICE- and CPP32-1ike proteases by upstream molecules such as bcl-2. Recombinant Mouse Bcl-2~.203~: Two Domains Connected by a Long Protease-Sensitive Linker. DM Segal, CM Zacharchuk, BA Vance, NIH:National Cancer Institute: Experimental Immunology Branch, Bethesda, MD Bcl-2 is a cytoplasmic integral membrane protein with potent anti-apoptotic activity, but whose mechanism of action is poorly understood. The purpose of this study was to obtain large amounts of soluble Bcl-2 protein for structural and functional studies. Mouse Bcl-2r (missing the C-terminal hydrophobic tail) was produced in bacterial inclusion bodies, solubilized in guanidine and refolded by dialysis. The resulting protein was monomeric in non-denaturing solution and was active in protecting mouse T hybridoma cells from glucoeorticoid induced apoptosis. Refolded Bcl-2<] 203) showed no tendency to homodimerize by gel filtration or analytical ultracentrifugation. Limited proteolysis experiments identified a region between the BH3 and BH4 homology domains of Bcl-2cj_ 203) that was extremely susceptible to digestion by several common proteases, but not by a cell extract known to contain CPP-32-1ike (ICE-family) protease activity. The protease-sensitive sites were located within a 50 residue stretch that contained most of the non-conserved and proline residues of Bcl-2tt 203). Trypsin-cleaved Bcl-2(j_203~ eluted in the same position as the undigested protein on gel filtration in non-denaturing solution, indicating that the two portions of the molecule connected by the proteasesensitive region associate stably and noncovalently. The solution properties of Bcl-2~_2o3) suggest that it consists of two non-covalently associated domains connected by a long protease-sensitive linker and that its structure is similar to that of Bcl-XL, which has been determined by X-ray and NMR analysis. Apoptotic signaling in T cells: the role of ICE-family proteases. SA Memon, MB Moreno, CM Zacharchuk. NIH, Bethesda, MD The mouse T cell hybridoma 2B4 dies by apoptosis in response to dexamethasone (Dex) and anti-TCR ligation, mediated through the interaction of Fas and Fas ligand. We have shown previously that overexpression of Bcl-2 blocks cell death induced by Dex but not through Fas. This suggested that there are distinct apoptotic signaling pathways with differential sensitivity to Bcl-2. We have now examined the role of ICE-family proteases (caspases) in apoptotic signaling using caspase inhibitors. Transient expression of CrmA, a potent inhibitor of ICE, selectively blocked Fas-mediated apoptosis in 2B4 cells. In contrast, p35, a more general caspase inhibitor, interfered with both Dex- and anti-Fas-induced apoptosis. Similarly, AcDEVD-CHO, a potent peptide inhibitor of CPP32, blocked

J ALLERGY CLIN IMMUNOL JANUARY 1997

both apoptotic stimuli whereas Ac-YVAD-CHO had no effect. These data support a model for distinct apoptosis signaling pathways; Dex- or drug-induced killing uses a Bcl-2 sensitive, CrmA-resistant CPP32-1ike protease path while Fas-mediated apoptosis uses a Bc[-2 resistant, CrmAsensitive ICE-like protease and CPP32-1ike protease pathway,

1272

E2/CD99 molecule mediates apoptosis of CD4+CD8+CD3i"t human thymocytes. G. Bernard, M. de Matteis, J.P. Breittmayer, P. Trampont, A. Senik and A. Bernard Unit6 INSERM 343 Hopital de l'Archet-NiceFrance E2/CD99 is a 32 kD transmembrane molecule which does not belong to any known family of protein. It appears to regulate adhesion properties of T-cells as previously reported, in particular, the induction of homotypic adhesion in CD4 § CD8 + thymocytes. We report here that E2/CD99 also mediates apoptosis of jurkat cells and a subpopulation of thymocytes. Apoptotic cells display characteristic morphological features. However E2/CD99 induced apoptosis is not followed by detectable DNA fragmentation, but includes early mitochondrial alterations and phosphatidylserine exposure at the outer leaflet of the plasma membrane. This effect is restricted to double positive (DP) thymocytes carrying an intermediate density of CD3 and including all CD69 + cells. It occurs even when apoptosis induced by the Fas pathway is blocked by a mAb or soluble Fas molecule. It is however dependent upon interleukin-ll3-converting enzyme (ICE)-type proteases since it is blocked by a specific ICE inhibitor. Thus E2/CD99 mediates apoptosis at a critical stage of thymocyte differentiation i.e., when positive selection is known to occur.

1273

Negative selection and apoptosis of CD30-transgenic thymocytes independent of CD28. T. Nguyen, R. Adkins, J. Spielman, F. Rhoderick and E.R. Podack. Dept. of Microbiology and Immunology, Univ. of Miami School of Medicine, Miami, F1. Potential autoreactive T-cells, the TCR of which have a high affinity for self antigen presented by MHC of thymic APC are eliminated by apoptosis in the thymus through a process known as negative selection. However, TCR stimulus alone is not sufficient to mediate negative selection. The process requires the interactions of other thymocyte surface receptors with their ligands on thymic APC. The abundance of CD30 mRNA in the thymus and the presence of CD30 expression seen in immunohistochemistry of thymic sections suggest a role of CD30 in thymocyte development. Here we investigate the potential role of CD30 as costimulator for negative selection. In mouse thymocyte cultures, strong deletion of double positive thymocytes requires both the cross-linking of CD3 and CD28 receptors. The addition of CD30-CD30 ligand interaction enhances this anti-CD3/B7 - induced cell death by 10%. CD30 involvement in thymocyte death is also demonstrated in vivo through the administration of CD30-Ig and anti-CD3 anti-bodies. Blocking of CD30-CD30 ligand signaling by CD30-Ig protects a small population (10%) of double positive thymocytes from anti-CD3 - induced deletion. Recognizing the likehood that CD30 expression is transient on thymocytes destined to commit suicide, we created CD30 transgenic mice, in which CD30 is expressed constitutively in only double positive thymocytes through the lck proximal promoter. CD30 transgenic, double positive thymocytes die by stimulation with anti-CD3 and CD30 ligand, bypassing the CD28 signal required by normal thymocytes. Our results consistently point to CD30 as a costimulator during negative selection of thymocytes.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1274

Peripheral Tolerance: The Fate of Fetal Antigen-Specific T cells during Pregnancy. Shi-Ping Jiang and Melanie S. Vacchio. Food and Drug Administration, CBER, Division of Hematologic Products, Bethesda, MD While thymic negative selection of self-reactive T cells has been shown to occur, not every serf antigen is expressed in the thymus. Therefore, the primary encounter between some self-reactive T cells and autoantigens may occur in the periphery, resulting in either activation, clonal deletion or anergy. Pregnancy provides an excellent model to address the fate of these self-reactive T cells, Fetal antigens are expressed at physiologically relevant levels, and will never have been encountered in the thymus during T cell development. The fate of T cells specific for an antigen on male fetuses was evaluated in transgenic mice that express a T cell receptor (TCR) specific for H-Y + D/'. On day 7 and day 14 of pregnancy, and 5 days postpartum, lymphoid tissues were analyzed for changes in cell number, expression of the clonotypic TCR and other T cell markers. Numbers of both total splenic T cells, and clonotypeexpressing T cells from H-2 ~', H-Y specific TCR transgenic mice decreased steadily during pregnancy. This increase was not observed in H-2a TCR transgenic mice that cannot recognize H-Y indicating an antigen specific response to H-Y. Interestingly, both the spleen and lymph nodes of H-2t" TCR transgenic mice continued to have decreased numbers of H-Y specific T cells when evaluated postpartum. This long-term decrease in fetal specific T cells argues against a transient downregulation of thc TCR, and supports clonal deletion as a mechanism for thc elimination of fetal reactive T cells.

1275

Jacalin-Indueed Apoptosis in Cultured PBLs from HIV+ Subjects is Reduced by Nitric Oxide Synthase Inhibitors. ML Quesada, S Saavedra, A M del Llano, JA Lavergne,. School of Medicine, UPR and Veterans Adm. Hospital, San Juan, P.R. Jacalin (JAC), a lectin mitogenic for T lymphocytes has been reported to block in vRro HIV infection of human CD4+ lymphocytes (Favero et al., 1993). The proliferative responses and the apoptosis (Ao) induction of lymphocytes cultured with this lectin were studied in fifty samples from HIV+ patients and compared to those of ten uninfected subjects. These effects were assessed by flow cytometry and compared to those of PWM- and PHA-stimulated PBLs. Among uninfected subjects, PHA stimulation resulted in the highest proliferativc response followed by PWM and JAC, while no mitogen induced Ao. In contrast, HIV+ patients showed decreased proliferation responses with the three mitogens when compared to normal values, and their unstimulated PBLs exhibited low percent values of spontaneous Ao (21.6 • 10.4) which increased by stimulation with PWM (25.4 + 13), JAC (29.1 + 12.8) and PHA (34.2 • 15.5). PBLs were also cultured in the presence of N-Monomethyl-L-arginine (L-NMMA) and 7-Nitroindazolc (7-NIl, two nitric oxide synthase inhibitors, resulting in a significant inhibition of Ao. The percent of inhibition with L-NMMA in JAC stimulated PBLs was highest (67.3 • 15.6; 36.7 • 19.3 with 7-NIl, followed by PHAstimulated PBLs (6131 -+ 13.4) and PWM-stimulated (42.4 • 20.4). These results indicate that JAC is a potent inducer of apoptosis in cultured PBLs from HIV+ patients. This phenomenon is reduced by I-NMMA and 7-NI, supporting the role of NO in apoptosis induction in this model. Supported by RCMI-RR03/151.

1276

Very low oxygen conditions distinguish between oxygen dependent and independent apoptosis. J F Ton'es-Roca, D R Greenwald, J Brown, 1 L Weissman. L A Herzenbetg, L A Herzenbe N and P D Katsikis. Departments of Genetics, Radiation Oncology and Pathology, Stanford University School of Medicine, Stanford, CA. Supported by grants AI-07290 and NlH CA 42509-11. Many of the stimuli that induce apoptosis arc also known to elicit oxidative stress. This has led to the proposal of oxidative stress as a mediator of apoptosis. However, recently several investigators showed that anaerobic culture does not inhibit several forms of apoptosis. Using low

Abstracts

S311

oxygen chambers with <20 ppm of oxygen, we have confirmed that some forms of apoptosis such as Fasinduced death of Jurkat T cells are not affected by very low oxygen conditions. In contrast, anaerobic culture completely inhibits dexamethasone-induced apoptosis of immature mouse thymocytes. Although spontaneous apoptosis is increased in anaerobia (18% in aerobia vs 30% in anaerobia) dexamethasone did not have any effect above background death under low oxygen conditions (50% in aerobia vs 30% in anaerobia). Consistent with these results, inhibitors of two important intracelIular oxyradical generating centers, mitochondrial respiratory complex I (rotenone) and cytochrome p 450 (metyrapone), were able to efficiently inhibit glucocorticoid-induced thymocyte death, while not affecting Fas-induced death. Rotenone and metyrapone, inhibited 75% and 78% respectively of dexamethasone-induced apoptosis. In conclusion low oxygen conditions and inhibitors of oxyradical generating centers distinguish between oxygen dependent and independent apoptosis. The level at which oxygen is involved in the oxygen dependent apoptosis is currently being investigated.

1277

Suppressed lectin-induced proliferation of T lymphocytes in simulated microgravity is restored by direct activation of PKC. D Coopd'-" and NR Pellisl' ~. LThe University of Texas, Graduate School of Biomedical Sciences: 2University Space Research Association; and ~The Biotechnology Program, NASA Johnson Space Center, Houston, Texas Several factors associated with spaceflight may be responsible for the immunosuppression observed in astronauts during and following spaceflight. We utilized Rotating Wall Vessel (RWV) bioreactors that simulate aspects of microgravity for cell cultures to assess the role of microgravity alone on polyclonal activation of lymphocytes. Previously we showed that PHA responsiveness was dramatically diminished in RWV culture as measured by [3H]-thymidine incorporation (>90%) and that T cells but not accessory monocytes are responsible for this impairment. In the current study, addition of exogenous IL-2 (10-100 U/ml) to these cultures did not restore PHA responsiveness in the RWV. However, activation of PBMC or column purified T cells with PMA and ionomycin is unaffected in RWV culture, indicating that signaling mechanisms downstream of PKC activation and calcium flux are unaffected by simulated microgravity. Furthermore, submitogenic doses of PMA alone, but not ionomycin alone (at any concentration), can restore PHA responsiveness in the RWV. Thus, a defect upstream of PKC activation and not calcium flux is most likely responsible for the suppression of polyclonal activation observed in simulated microgravity. This research was supported by NRA-94-OLMSA-02.

S312

Abstracts

1278

The Effect of Peptide Affinity for MHC Class II on T-Cell Deletion. Daniel A. Peterson & Emil R. Unanue. Washington University, St. Louis, MO, USA. We investigated the effects of peptide affinity for the MHC class II molecule [-A k on the process of T-cell deletion using mice transgenic for the 3A9 TCR. This receptor recognizes the hen egg white lysozyme epitope 48-61 presented in the context of I-A k. The aspartic acid at position 52 of the 48-61 peptide is critical for the binding affinity. The alanine substituted 48-61 (A52 48-61) peptide, with 150 fold lower binding affinity, will stimulate both the 3A9 hybridoma and T-cells from the TCR transgenic. A recently developed anti-clonotypic monoclonal antibody demonstrated that 70-80% of single positive CD4 cell are clonotype positive in both thymus and peripheral lymphoid tissues. We injected 25 nM of the high affinity 48-61 peptide or low affinity A52 48-61 intraperitoneally every 24 hours for 3 days, and then harvested the thymi lymph nodes and spleen. We observed a near 4 fold reduction of the percent CD4 single positive cells in the 3A9 thymus after injection with 48-61 compared to the saline injected mice (ie 41% to 11%). We also observed a 3-4 fold reduction of CD4 cells in the periphery (26% to 7%), both in the spleen and lymph nodes. A52 48-61 failed to delete CD4 SP (41% to 36%) in the thymus. In the periphery however, the deletion of the CD4 cells was of similar magnitude as the wildtype peptide (26% to 10%). Thus a low affinity peptide can fail to delete in the thymus, and yet delete peripheral T-cells. This may reflect a differential role for peptide affinity in alternative forms of tolerance.

1279

Elevated expression of a soluble form of Fas by human hepatocytes. CK. Fox, OM. Martinez, SM. Krams. Transplant Immunobiology Lab, Stanford University School of Medicine, Stanford, CA 94305 Fas (Apo-1/CD95), a member of the TNFR family, has been shown to mediate apoptosis when engaged by its ligand or by anti-Fas antibody. A secreted, soluble form of Fas (sFas) can be expressed by activated human PBMC and some tumor cell lines through alternative splicing of RNA. It is thought that sFas may play a regulatory role in the immune system by inhibition of apoptosis through competitive binding of Fas ligand. Human liver is one of the few tissues in the human body which constitutively expresses cell surface Fas, and thus, is uniquely sensitive to Fas mediated apoptosis. To determine whether sFas is also expressed in liver, we examined liver tissue, hepatocytes and hepatoma cell lines. Lysates of liver tissue and isolated hepatocytes showed elevated levels of sFas when assayed by an ELISA specific for the soluble form of Fas. Additionally, at least one form of alternatively spliced Fas was demonstrated by RT-PCR of hepatocytes isolated from both diseased and normal liver tissue, and of hepatoma cell lines. A comparison of the relative levels of the Fas alternative transcript and the cell surface Fas transcript showed a higher ratio of sFas to cell surface Fas in hepatocytes than in activated PBMC. Interestingly, the source of elevated levels of sFas in liver appears to be hepatocytes rather than liver infiltrating mononuclear cells. Functional studies are ongoing to confirm that hepatocyte derived sFas can modulate apoptosis. High levels of sFas produced by hepatocytes may influence the immune response within the liver. Further, these observations raise the possibility that dysregulation of the expression of sFas may be important in liver disease.

1280

Determination of optimal stimulation conditions for inducing cytokine producing cells among the peripheral blood lymphocytes. J.F. Yu, X.F. Yang, D. Sehy, C. Shih, Z. Chen and E. Huang. PharMingen, San Diego, CA. PMA (phorbol 12-myristate 13-acetate) and calcium ionophore A23187 are commonly used as stimulators for activation of lymphocytes. In this report we elucidated the optimal concentrations of these two reagents in the induction of cytokines (i.e., IL-2, IL-4, IFN--/ and TNF-c0 production in human peripheral blood lymphocytes (PBL).

J ALLERGY CLIN IMMUNOL JANUARY 1997

The PMA and A23187-induced intracellular cytokine production, in the presence of monensin (a protein transport inhibitor), were evaluated by using flow cytometric analysis. Our studies showed that: (1) PMA and calcium ionophore must be used together (rather than individually) for induction of cytokine producing cells in human PBL. Under the optimal concentration of A23187 (1 Ixg/ml), the effect of PMA in cytokine production reached plateau at 16 ng/ml. (2) Under the optimal concentration of PMA (50 ng/ml), the effect of A23187 in cytokine production reached plateau at 480 ng/ml. When whole blood was used as cell source, at least 1 ug/ml of A23187 was needed for the detection of cytokine producing cells. (3) Kinetically, under the optimal concentration of both PMA and A23187, the cytokine producing cells were detectable as early as 2 hr and reached to a plateau at 4-6 hr. While IL-4 producing ceils decreased significantly when the stimulation time was prolonged, no significant changes in IL-2, IFN3, and TNFc~ producing cells were detected. (4) PMA significantly down regulated cell surface CD4 expression at a concentration as low as 1 ng/ml, but it had less effect in CD3 and CD8 expression. Our data suggested it is important to determine optimal concentration of PMA and calcium ionophore to activate PBL for the detection of cytokine producing cells dependent on different target cells (PBL or whole blood). It also suggested that PMA and calcium ionophore may not be the suitable reagents for activation of PBL, when cell surface markers are needed for the cytokine producing cell analysis.

1281

Mouse IL-12 Detection with a Two-site Immunometric ELISA Specific for the p70 Heterodimer. P.C. Leverone and L. A. Beausang; Endogen, Inc. Woburn, MA Spon: J. Mier Biologically active Interleukin-12 (IL-12) is a heterodimeric cytokine of 70kDa (p70) made up by two covalently linked glycosylated chains of approximately 40 (p40) and 35 (p35) kDa. The biologically inert sub-unit of IL-12, p40, is often present 10 to 100 fold higher than the heterodimer in vivo and in vitro, intensifying the need for a p70 specific detection method. We have developed a sensitive two site immunometric (sandwich) ELISA specific for the detection of mouse IL-12 p70 in serum and tissue culture supernatants. The range of the assay is 26pg/ml to 2500pg/ml with a lower limit of detection of less than 5pg/ml. The mean recovery of recombinant p70 in serum is 96.5% (n=22). Natural IL-12 was produced in vivo by injecting BALB/c mice interperitoneally with 50ug of E. coli lipopolysaccharide per mouse. Serum samples collected every 30 minutes for 6 hours yielded p70 and p40 as high as 0.45ng/ml and 12ng/ml respectively. The p40 was detected using a sandwich ELISA composed of p40 specific monoclonal antibodies C15.1 and C15.6. In vitro samples were derived from mouse macrophage cell line J774A.1 and mouse peritoneal exudate cells (PEC) co-stimulated with rmINF~/and either S. enteriditis LPS or formafinized S. aureus. The J774A.1 cells produced nanogram levels of p40 and no p70. The PEC cells produced p70 levels as high as 10ng/ml and p40 levels as high as 4ng/ml. Reported levels of p40 and p70 were neutralized by a polyclonal antibody specific for mouse IL-12. Recombinant mouse IL-12 p40 homodimer, rmIFN~/, human IL-12 p40 and p70 did not cross react or interfere with the p70 ELISA. The specificity of the Endogen mouse IL-12 p70 ELISA, supported by cross-reactivity data and comparison with the p40 ELISA, make it a useful research tool for those interested in studying biologically active mouse IL-12.

Abstracts

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1282

Performance of a Human IL-12 ELISA Specific for the p70 Heterodimer. .IM MeCabe, KS P~harski, LA Beausang; Endogen, Inc. Woburn, MA Spon: J. Micr Interleukin-12 (IL-12), composed of a 35 kDa chain (p35) and a 41) kDa chain (p4/)), is mainly produced by dendritic cells, macrophages and neutrophils and has multiple effects on T cells and NK cells. A two-site ELISA for the detection of human 1L-12 utilizing 2 monoclonal antibodies was optimized for testing tissue culture supernatams, sera and plasma. The range of the assay is 15 to 600 pg/ml with a lower limit of detection of 5 pg/ml. The mean recovery of natural IL- 12 in sera and EDTA plasma is 82 _+ 15% and 88 _+ 6% respectively. A dose of 100 ng/ml of the p40 sub-unit does not cross react with or interfere in the IL-12 ELISA. The dose responses of stimulated whole blood, peripheral blood lymphocyte and THP-1 supernatants were neutralized 95% by an IL-12 specific polyclonal antibody. The p40 and p35 subunits by themselves have no IL-12 activity, but p40 is secreted in excess of IL-12 and may be antagonistic. Stimulated whole blood supernatants (Dr. G. Szabo, U. of MA. Medical Center, Worcester, MA) were tested in Endogen's p70 spccific ELISA and Endogen's total p40 and p70 EL1SA. The results indicate that p4(/and p71) production are regulated separately. Stimulation of whole blood with staphylococcus enterotoxin B (SEB) and IFN~ induces significant IL-12 production, but LPS induces minimal IL-12 production. The same whole blood supernatant samples contain p40 produced in response to both LPS and SEB/IFN'y at similar high levels. Our results indicate that Endogen's ELISA is a useful tool in the study of IL-12.

1283

Effects of Anti-B7 Monoclonal Antibodies on Cytokine Messenger RNA Expression Detected by Quantitative RTPCR. JE Woodward, AL Bayer, KD Chavin, P Baliga. Medical University of South Carolina, Charleston, SC, and The Johns Hopkins Hospital, Baltimore, MD. The role of B7-1 and B7-2 in T cell activation remains ill-defined. Others have previously described differential

t t

( t (

1 ( (

TN F-c~

;

T T

6-actin

~

~

~,

In vitro administration of anti-B7-t and anti-BT-2 mAbs appears to lead to a shift away from the TH1 cytokines with the combination having a greater immunosuppressive effect than either agent alone. These mAbs acted in a global manner by altering mRNA expression of cytokines produced by antigen presenting cells as well as T lymphocytes.

$313

1284

Differential Effects of Monensin Versus Brefeldin A on Intracellular Cytokine Staining. Jeanne 34. Elia, Elton B. Chu, David N. Ernst and Charles C-Y. Shih. PharMingen, San Diego, CA 92121. Monensin and brefeldin A are protein transport inhibitors that are commonly used in intracellular cytokine staining to enhance the detection of cytokine-producing cells. These two inhibitors were compared in mouse splenocytes using different activation times and two different activators: immobilized anti-CD3 (anti-CD3i)+soluble anti-CD28 (anti-CD28s), and PMA+ ionomycin. Cells were incubated (4 or 18 hr) in culture with one of the 2 activators in the presence of an optimal concentration of monensin (2 txM) or brefeldin A (1 txg/ml), and were stained by immunofluorescence for intracellular cytokines and analyzed by flow cytometry. Dramatic differences were observed in cells from cultures that were activated for 18 hours in the presence of monensin or brefeldin A. Brefeldin A allowed the detection of an increased frequency of TNF~-producing cells when compared with monensin, for both activators. Similarly, brefeldin permitted the detection of increased frequencies of IL-2 producing cells compared to monensin when cells were stimulated with antiCD3i+anti-CD28s. Comparable frequencies of IL-2 + cells were detected when PMA+ionomycin was used. In contrast, short term activation cultures (4 hrs) yielded comparable amounts of IL-2 + and TNFc~ ~ cells for both activators using either brefeldin or monensin. Brefeldin and monensin were also compared in restimulation cultures, SEB-primed splenocytes (4 days) or activated CD4 + cells were restimulated with PMA+ionomycin for 5 hours in the presence of monensin or brefeldin. In the restimulation cultures, monensin was comparable to, or increased the detectable numbers of cytokine-producing cells over that observed with brefeldin for all the cytokines measured (IL-2,-3,-4,-5,-10,GM-CSF,1FN-~/) except for TNF~, whereas brefeldin gave noticeably higher detectable frequencies of TNFa ~ cells than monensin. Cell viabilities were similar using either brefeldin or monensin even when the cell populations were cultured up to 24 hours. These data suggest that the effectiveness of either monensin or brefeldin can be time, activator and cytokine dependent. These factors need to bc considered when carrying out intracellular cytokine staining.

1285

An Enzyme-Linked Immunosorbant Assay (ELISA) for the Measurement of Human Interleukin-8 (hIL-8) in Serum, Plasma and Tissue Culture Fluids. MD Sullivan, T Liponis, P Durda, J Pritchard. Genzyme Diagnostics, Cambridge, MA An ELISA using an antibody sandwich format has been developed to quantitate hlL-8 in serum and plasma samples and in tissue culture fluids. The Predicta IL-8 ELISA kit (Genzyme Diagnostics) has a standard curve range of 512 pg/mL to 8 pg/mL calibrated against an amino acidanalyzed mass standard. The calculated sensitivity is 1 pg/mL. The assay uses one diluent for the measurement of hlL-8 in the above matrices. Mean recovery of recombinant and natural IL-8 was determined to be 100% in serum; 88% in EDTA, citrated or heparinized plasma; and 94% in tissue culture fluid. A linear correlation of expected vs measured values ranged from 1.000 to 0.993 for diluted serum, plasma and tissue culture fluid preparations. Intraassay precision ranged from 5 to 6%. Inter-assay precision was 10% for serum and plasma preparations and 15% for tissue culture fluid preparations. Serum and plasma samples from apparently healthy donors were evaluated for an expected normal range. A median value of 42 pg/mL was calculated from 48 serum samples (ranging from 2 to 2390 pNmL). A median value of 0 pg/mL was calculated from 50 plasma samples (ranging from 0 to 3.3 pg/mL). Serum samples (n = 25) with positive reactivity for rheumatoid factor or anti-nuclear antibodies registered values below 30 pg/mL. A panel of cytokines and human serum proteins was evaluated for interference and cross-reactivity to ascertain the assay's specificity for hlL-8. The Predicta IL-8 ELISA kit was shnwn to be a highly sensitive, specific, reliable and accurate system for the measurement of human IL-8 in serum, plasma and tissue culture fluids.

$314

1286

1287

Abstracts

ELISA Systems for the Rapid Quantitation of Total Human ILl2 and Human ILl2 p70. Jeremy E. Schonhorn, Harmesh K. Sharma, and John Pritchard. Genzyme Corporation Interleukin-12 (IL-12) is a potent immune modulating cytokine that contributes to the regulation of the TH1 response and hematopoeitic progenitor homeostasis. Biologically active hiLl2 (p70) is secreted as a heterodimer consisting of two disulfide-linked subunits of 35 kDa and 40 kDa. Biologically inactive monomer (p40) and its homodimer (p40)2 are secreted in 5 to 90fold excess of IL12p70. All three forms, p70, p40, and p402 specifically bind to ILl2 receptors. While p70 provokes the biological response, p40 and its variants are considered regulatory molecules by being antagonists both in vitro and in vivo. Described herein are two ELISA systems based on the antibody "sandwich" principle designed to work in tandem to measure both forms of hiLl2 in serum, plasma, or culture fluid. One system measures both hlL12p40 and hlL12p70 (total), the other system specifically measures hlL12p70. The total hiLl2 and hlL12p70 assays have standard curve ranges of 25 to 1600 pg/mL and 8 to 512 pg/mL, respectively. The normal range for hlL-12 determined in the total hiLl2 assay is 138 _+ 92 pg/mL for serum, and 169 _+ 80 pg/mL for plasma (n = 49 samples). Calculated sensitivities in the total and hlL12p70 assays are 4 pg/mL and 1 pg/mL, respectively (n = 4 assays). Neither assay crossreacts with other cytokines. In both assays inter and intraassay variances are <15% (n = 12 assays) and < 10% (n = 20 replicates/sample), respectively. Both assays accurately recover 80100% of spiked hlL12p70 in appropriate matrices. Accuracy of recovery was confirmed by measurement of hiLl2 in linearly-diluted spiked samples. Together, both assays rapidly and precisely measure hlL-12 content in samples comparable to current bioassay methods.

Development of Sandwich ELISA for Measurement of Rat IL-2. Qi Guan, David. N. Ernst, Jeanne M. Elia, Fariba Hosseini, Daine Mochizuki, Jason Mirabile, Elton B. Chu and Charles C-Y Shih. PharMingen, 10975 Torreyana Road, San Diego, CA 92121-1111, USA An Enzyme-linked Immunosorbent Assay (ELISA) capable of specifically and quantitatively measuring rat IL-2 protein levels was developed. This assay was found to sensitively measure rat IL-2 protein levels and was developed as an alternative to the current bioassay procedure used for measuring rat IL-2 protein. The sandwich ELISA consists of the purified IgG fraction of a polyclonal rabbit anti-rat IL-2 antiserum for the capture antibody. Insect cell-expressed, recombinant rat IL-2 protein is used as the standard with biotinylated A38-3 (mouse IgGl,k anti-rat IL-2) antibody as the detector antibody. The ELISA can then be developed with streptavidin-horse radish peroxidase or streptavidin-alkaline phosphatase and their respective substrates. This rat IL-2 ELISA was found to be capable of measuring E.coli- and baculovirus-expressed recombinant rat IL-2 as well as native rat IL-2. Supernatants containing native rat IL-2 were generated by either stimulating CD4 § spleen cells with plate-bound mouse anti-rat CD3 and soluble mouse anti-rat CD28 antibodies or PMA/Ionomysin, and stimulating unseparated rat spleen cells with Con A. Supernatants containing native rat IL-2 reduced bioactivity after immunoabsorbed by the polyclonal rabbit anti-rat IL-2 antibody or A38-3. Because the CTLL-2 proliferation bioassay measures biologically active rat IL-2, not rat IL-4, the results obtained in rat IL-2 ELISA were compared to those determined in bioassay. Good correlation between the levels of native rat IL-2 proteins measured by the ELISA and the bioassay was found. This ELISA pair did not crossreact with other rat cytokines including rat IL-4, IL-6, IL-10, GM-CSF, IFN-g, TNF-a, MCP-1, and did not crossreact with mouse IL-2 or human IL-2. The sensitivity of this rat IL-2 ELISA was -> 10 pg/ml of rat IL-2.

J ALLERGY CLIN IMMUNOI_ JANUARY 1997

1288

Characterization of Mouse Anti-human IL-12 Antibodies Through Epitope Mapping. Kristin Murray, Mark Stahl, Kathy Murphy, and Victor Van Cleave, Pre-Clinical Biology, Genetics Institute, Inc., Andover, MA 01810. We report here the characterization by epitope mapping and immunological reactivity of several mouse anti-human IL-12 monoclonal antibodies (mAbs). This panel of mouse mAb reagents includes Cll.5.14, and Cll.79.15, (D'Andrea et. al., J. Exp. Med. 176:1387, 1992) 12hl/8.2.1, and 12h4/1.2.8. Cll.5.14, Cll.79.15, and 12hl/8.2.1 are specific for the p40 or heavy chain of IL-12 while 12h4/1.2.8 is specific for the p35 or light chain subunit of IL-12. We have previously described the sensitivity and specificity of Cll.5.14 and biotinylated 12h4/1.2.8 when used in a sandwich ELISA format (Murray et. al., 9th Int Cong. Imm. Abs#130, 1995). Utilizing the FLITRIX technology (Lu et. al., Bio/Technology 13:366, 1995), we have determined that the p40 specific mAbs recognize sequences between amino acids (aa) 229 and 233 (Cll.5.14), aa between 71 and 76 (Cll.79.15), and aa 215 and 219 (12hl/8.2.1). The p35 specific mAb recognizes the sequence between aa 134 and 139 (12h4/1.2.8). Further studies to characterize the neutralizing capacity of these four mAbs is underway using PHA-stimulated lymphocytes and IL-12. Finally, noting the structural similarities between the p40 subunit and human growth hormone receptor, we have now constructed computer models which localize the epitopes for Cll.5.14 and 12hl/8.2.1 to spatially distinct surface exposed regions of the molecule. Similar models were prepared to examine the epitope recognized by 12h4/8.2.1 using the homology between p35 and human growth hormone.

1289

Dual activation signals for CD4-p561ck mediated T cell migration. T. Ryan, W. Cruikshank, H. Danis, P. Sell, and D. Center. Pulmonary Center, Boston University School of Medicine. Boston, MA 02118 The initiation of CD4 antibody induced T lymphocyte chemotactic activity requires the physical coupling of CD4 with the src family tyosine kinase p561ck. Migration is however independent of the catalytic activity of lck. Murine T cell hybridomas expressing chimeric CD4-1ck were used to identify the domains which regulate T cell motility. Chimera containing either the src homology (SH)3 domain or the N-terminus alone mediated an antibody induced response. We then investigated the involvement of factors which might bind to these domains, the first of which was the lipid kinase phosphatidylinositol 3'kinase(PI3K). Treatment of chimeric cell lines with wortmannin, a PI3K inhibitor, significantly reduced CD4 antibody induced T cell migration in lines containing SH3 chimera, while the N-chimera were uneffected. PI3K in vitro kinase assays of antibody treated cells demonstrated similar results. We propose that there are at least two distinct and separable pathways which mediate T lymphocyte migration. The first an SH3 associated, wortmannin sensitive PI3K pathway and the second an N-terminus associated wortmannin insensitive pathway.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1290

Superantigen-Activated T Lymphocytes Adhered to Human Endothelial Cells. T Krakauer. USAMRIID, Fort Detrick, Frederick, MD 21702. Adhesion of leukocytes to endothelial cells (EC) precedes extravasation and is a critical step in the establishment of an inflammatory response. The superantigens, staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin 1 (TSST), are potent stimulators ofT cells and are capable of inducing toxic shock. A sensitive ELISA was used to study the changes in adhesion of superantigenactivated human peripheral blood mononuclear cells (PBMC) to human umbilical vein EC. A statistically significant increase in adhesion of both CD4' and CD8 + T lymphocytes were observed 20 or 44 hours after SEB or TSST treatment of PBMC. There was no change in the adhesion of monocytes (detected with OKMI) to EC. When IL-I, a proinflammatory cytokine known to increase adhesion of leukocytes to EC, was added to EC, an additional small increase in adhesion to IL-l-activated EC was observed with superantigcn-activated CD4 + and CD8 ~ cells. Thus, both SEB and TSST-1 induced T cell adhesion to EC and this might partly be responsible for toxic shock induced by these bacterial proteins.

1291

Acquisition of Endothelial Cell Surface Determinants by Extravasating T Cells. RI Brezinschek, PE Llpsky, HL Wisbev, N Oppenheimer-Marks. UT Southwestern Medical School, Dallas, TX During the development of inflammation, endothelial integrity is challenged by local cellular events occurring both intra- and extra-vascularly. The transendothelial migration of leukocytes and elaboration of soluble mediators both impact endothelial activity. Although the etiology of certain vasculopathies is not known, in these disorders endothelial cells (EC) appear to be persistently acted upon by pernicious stimuli which antagonize endothelial integrift. Inasmuch as certain chronic inflammatory disorders may have a vasculitic component, we examined whether endothelial damage results from interactions with extravasating T lymphocytes. Monolayers of HUVEC were formed on the surface of hydrated collagen gels and then exposed to the proinflammatory cytokine, TNFc~ (4 hours, 2(IOU/ ml). Subsequently, the EC were incubated with purified CD4+ T cells that had been previously cultured in the absence or presence of phorbol dibutyrate (PDB), to activate protein kinase C, or immobilized CD3 mAb, to crosslink the CD3/TCR complex. Examination of migrating [ymphocytes by flow cytometry demonstrated the appearance of EC determinants, including CD31, CD49b, CD54, CD61 and CD62E on the surfaces of activated, but not resting, adherent and migrating T cells. Inhibition of protein synthesis demonstrated that this was not due to dc novo synthesis of these molecules by the T cells. Studies using the lipophilic membranc dye. DiO16, revealed that the acquisition of endothelial determinants by aetNated T cells resulted from the exchange of plasma membrane between EC and adherent and migrating T cells. T cells that did not bind to or migrate through endothelium did not acquire EC receptors. These results suggest that during cell-cell interactions, when both T cells and EC are activated, both cell types undergo remarkable phenotypic changes which may have marked effects on the progression of inflammatory disease.

1292

Monocyte Migration Across a Synovial Fibroblast Barrier Involves CD18, VLA-4 and VLA-5 Integrins. X Z Shang, BJ Lang, A C lssekutz. Dalhousie Univ., Halifax, NS Canada In arthritis, monocytes migrate through vascular endothelium and then in the synovium. We investigated the molecular mechanisms required for monocytc migration through a barrier of human synovial fibroblasts (HSF) grown on microporous filters and compared the mechanisms with monocyte migration through human umbilical vein endothelium (HUVE). Little monocyte migration occurred spontaneously (6-7%) through either HSF or HUVE barriers. Migration markedly increased to 27-35% of added monocytes through either cell barrier, when a C5a chemotactic gradient was present. Monocyte migration

Abstracts

S315

across unstimulated HUVE was partially inhibited (50%) by mAb to CD18 ([32 integrin) and completely blocked by anti-CDl8 plus anti-a 4 (VLA-4) mAbs. In marked contrast, migration across HSF was not inhibited by mAb to CDI8 and was only partially inhibited (33%) by mAb to CDI8 plus mAb to VLA-4. This monocyte CD18 and VLA-4 independent migration across HSF barriers was completely inhibited by adding mAb to % of VLA-5. The inhibitory effect of mAbs to the VLA-4 and VLA-5 [3~ integrins required treatment of the monocytes, but not the HSF and was only observed when the function of CD11/ CDI8 on monocytes was also blocked by a mAb. Treatment of HSF with 1L-la (5 hrs) upregulated their VCAM-I expression and slightly increased the spontaneous monocyte transfibroblast migration. However, there was no qualitative difference in the mechanisms utilized for migration in response to C5~ through unactivated or IL-lc~ activated HSF. These results demonstrate that: (a) CD18independent mechanisms can totally substitute for the CD18 mechanisms in mediating monocyte migration through an HSF barrier, and (b) unlike transendothelium migration, where VLA-4 is the alternate mechanism to CDt 1/CD18, migration through HSF can also be mediated by VLA-5 alone. Thus multiple [31 integrins may mediate monocyte migration through connective tissue. (Supported by MRC Canada.)

1293

Systemic expression and activation of adhesion molecules on leukocytes and endothelial cells is not sufficient for leukocyte emigration in vivo. B E Schleiffenbaum, B Odermatt, J Fehr, RA Sperb. Hematology, University Hospital Zurich, Switzerland In keeping with the multiple-step model of leukocyteendothelial cell interaction, stimulation of endothelium by various cytokines or endotoxin (LPS) in vitroleads to Lselectin/integrin-mediated neutrophil adhesion followed by neutrophil transmigration through the endothelial monolayer. The intraperitoneal injection of 10 p,g LPS leads to a systemic inflammatory reaction in an animal mouse model with generalized activation of both endothelial cells and neutrophils (PMN). Increases in PMN adhesion molecule Mac-1 expression accompanied by a slight reduction in the expression of L-selectin as measured by FACS begin at 15 rain post injection, followed by the up-regulation of endothelial adhesion molecules ICAM-I, VCAM-1, and Eselectin (ca. 4h) as demonstrated by immunohistology. However, no intravascular endothelial adhesion or tissue emigration of neutrophils can be observed in any of the organs examined. Moreover, the in vivo emigration of PMN at sites of a local inflammatory reaction (foot pad, locally injected with 0.1 ~g LPS) is markedly reduced when the mice are systematically inflamed, although the neutrophils respond fully to a rechallenge with LPS (50 ng/ml) ex vivo. In contrast to human PMN and endothelial cell interactions in vitro, up-regulation of PMN and endothelial adhesion molecules is not sufficient to induce PMN adhesion and emigration through endothelium in vivo. The reduced emigration of PMN in mice systemically challenged by LPS at local inflammatory sites argues for an inhibitory principle which is not present in vitro, but active in vivo, and cannot be explained by the loss of L-selectin or PMN deactivation towards LPS.

$316

Abstracts

J ALLERGYCLIN IMMUNOL JANUARY 1997

1294

1295

Lymphocyte Adhesion to Murine High Endothelial Venule Cell Lines: Evidence for a novel ct4-integrin ligand. J.M. Cook-Mills and K.S. Tudor. Dept. Path. & Lab. Med,, Univ. of Cincinnati, Cincinnati, OH. High endothelial venules (HEV) regulate transendothelial migration of lymphocytes from the blood into lymph nodes. We compared B and T cell adhesion to the murine HEV cell lines mHEVa and mHEVc which were derived from axillary and cervical lymph nodes, respectively. When 1x 10~' lymphocytes/well were plated on confluent mHEV cell monolayers, significantly more B cells adhered to the mHEVa cell lines (8.1_+0.2 x 105 cells/well) than adhered to the mHEVc cell lines (6.0-+0.2 • 105 cells/well). By contrast, mHEVa and mHEVc cells bound similar numbers of T cells (2.7-+0.2 • 105 and 2.2___0.2 x 105 cells/well respectively). The greater B cell adhesion to mHEVa than mHEVc cells is not due to a higher number of mHEVa binding sites because mHEV monolayer binding sites were only 50% saturated in all experiments. B and T cell adhesion to the mHEV cell lines was mediated by c~4 integrins since anti-a4 completely blocked adhesion. Importantly, there were differences in mechanisms of a4-mediated lymphocyte adhesion to the mHEVa versus mHEVc cells. Antibodies against the a4-1igand VCAM completely blocked B and T cell adhesion to the mHEVc cells whereas it significantly but incompletely blocked adhesion (75% inhibition) of B and T cells to the mHEVa cells. In addition, although the mHEVa cells expressed the 3 and 7 domain forms of VCAM and the mHEVc cells expressed only the 7 domain form, the differences in B cell adhesion were not due to the different forms of VCAM because there was no effect on adhesion when expression of the 3 domain form was inhibited. Moreover, the level of 7 domain VCAM-mediated B cell adhesion to the mHEVa and mHEVc cells was the same, indicating that the greater B cell adhesion to the mHEVa cells was not VCAM-mediated. Differences in B cell adhesion were not mediated by c~4137 or MAdCAM-1. Therefore, the greater B cell adhesion to the mHEVa cells than to the mHEVc cells suggests that B cells are capable of adhering to mHEVa cell lines through a novel ligand on the mHEVa cells that binds c~4-integrin on the B cells. (Supported in part by NIH A134585) Gender-Specific Differences In T Cell Homing May Increase Disease Severity In Female Mice. B Richardson, R Williams, and R Yung. U of MI, Ann Arbor MI. The reason lupus is more severe in women is unknown, but a recent report suggests a role for gender-specific differences in adhesion molecule expression. Injection of procainamide (Pca)-treated D10 cells, a conalbumin reactive T cell line, causes a lupus-like disease in syngeneic (AKR) recipients. We used this model to compare lymphocyte trafficking in male and female mice. Female and male AKR mice received 6 i.v. injections of Pea-treated ceils. Both developed autoimmune renal and lung disease, but only females developed liver disease. Females also developed greater titers of anti-ss- and ds-DNA antibodies. Pea-treated or untreated D10 cells were then labelled with 5~Cr, injected into male or female AKR mice, and 51Cr and organ weight measured in the liver, spleen, lungs, lymph nodes, brain, heart, skeletal muscle, skin, thymus and salivary glands. 1% of the ~lCr remained at 24 hours. Of this, the liver retained 83%, the spleen 3-7%, and lung 3%. No differences were observed between treated and untreated cells, and liver and lung homing was identical in males and females. However, twice as many cells accumulated in female spleens relative to males (p<0.01). This was confirmed using CMFDA-labelled cells, which demonstrated a 7-fold greater number of CMFDA-labelled CD4+ cells homing to the spleen in females relative to males (p<0.02). There was no difference in splenic weight between groups. To determine the significance of splenic homing, splenectomized female AKR mice received 6 injections of Pea-treated D10 ceils. Splenectomized mice developed no anti-DNA antibodies, liver or renal disease. Oophorectomy decreased splenic homing, liver disease and autoantibody titers to levels of males. These results demonstrate a crucial role for splenic homing in this model, and

suggest that gender-specific differences in lymphocyte trafficking to lymphoid tissues may contribute to severity of autoantibody production, liver and renal disease.

1296

Significance of the size and chemical composition of biomaterial particles on the deposition pattern and the host response in mice. VJ Tomazic, K Merrill and TH Umbreit, US Food and Drug Administration, Rockville, MD. Biomaterial particles, either generated by wear or introduced as such, may migrate into various tissues and organs and lead to the activation of the host's inflammatory and immune responses. This study evaluated the relevance of size and chemical composition on the pattern of particle distribution in the host tissues. Adult female B6C3F1 mice were injected intraperitoneally with polymethylmethacrylate (PMMA) particles (1.4p~ and 6.4~ in diameter) and polystyrene (PS) particles (1.2~, 5.2~ and 12.51x in diameter) and sacrificed 1, 7 and 24 days later. Macroscopic examination of peritoneum revealed accumulations of PS particles (colored) in the adipose tissues adjacent to the spleen, pancreas and caudal to the stomach. Distribution of PS particles appeared similar regardless the particle size. The PMMA particles, which were not colored, could not be observed in this manner. Peritoneal exudate cells (PEC) were collected and the percentage of particle-containing phagocytic cells was determined microscopically. Intensive phagocytosis of particles by PEC's was observed on day 1, diminishing by day 7 after injection, with exception of the largest PS particles (12.51x), which could not be engulfed by peritoneal macrophages. Histological examination of the spleen, lymph nodes and the adjacent adipose tissues revealed a marked difference in the deposit patterns of the two polymers used. PS particles were mainly accumulated in the white adipose tissues adjacent to spleen and pancreas gland, some in the splenic capsula, and very few in the splenic tissue. In contrast, mice injected with PMMA had enlarged and activated spleens with marked deposits of particles in the red pulp. These results indicate that PS and PMMA particles induce different patterns of particle distribution and the intensities of the host response.

1297

High Cell Density Delays Human Neutrophil Apoptosis in Culture. BAM Walker, RH Boone, C Rocchini, SA Ahmad. MacDonald Wing Research Laboratories, Vancouver, B.C. Canada Experiments were performed to determine if neutrophil cell density delayed apoptosis in vitro. Purified human neutrophils were cultured in 96-well tissue culture plates (0.32 cm2/well) in RPMI 1640 with 10% blood group AB serum, penicillin (5U/ml) and streptomycin (51xg/ml) at varying densities and assessed for apoptosis after 16 hrs of culture by morphology on Wright's stained cytospins. Increasing cell density resulted in a logarithmic decline in the percentage of apoptotic neutrophils when cells were cultured at cell densities between 103 to 3• ~' cells per well compared to media alone. The percentage of neutrophils showing apoptotic features at 16 hrs at cell densities of 103 and l0 s' neutrophils/well were 82-+3% and 22_+6% respectively. The transfer of culture supernatants from cells of different densities failed to demonstrate a significant effect on fresh neutrophils cultured at 104 cells/well compared to media alone. The addition of GMCSF (10 nM) reduced apoptosis as 16 hr to levels comparable to those seen at high neutrophil density (n=3). These studies demonstrate that neutrophil density has a profound effect on the appearance of apoptosis. Neutrophil conditioned medium cannot reproduce this effect. We postulate that the delay in apoptosis is due to cell-cell contact.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1298

Detection of B7-1 (CD80) in synovial fluid of arthritis patients. RS McHugh, R Gilmartin, W Ratnoff, K Sell, and P Seh,araj. Emory University School of Medicine, Atlanta, GA Many cell adhesion molecules, including ICAM-1 and VCAM-1, have recently been found to exist as soluble forms in many bodily fluids. Serum from patients with various maladies, such as malignancies, allografls and arthritis, have higher quantities of soluble adhesion molecules. To this day, a soluble fl~rm of the adhesion/costimulatory molecule B7-1 has not been reported in biological fluids. Recently, investigators reported that infiltrating immune cells in the synovium of RA patients have increased expression of B7-1 and B7-2. Cells found in the lining of the synovium also have increased B7 antigen expression as compared to synovium of OA patients. Using a sandwich ELISA developed to detect B7-1, wc found that a solube form of B7-1 was present in the synovial fluids of many arthritis patients. Not all patients, however, had detectable B7-1 present in the synovium, which may be a refelction of the state of inflammation. In addition, there seemed to be no difference in the concentration of soluble B7-1 found in RA versus OA synovial fluids. Therefore, the presence of soluble B7-1 does not correlate with an increase in B7-1 expressing cells. The presence of soluble B7-1 was confirmed by immunoprecipitation using PSRM-3 (anti-B7-1) coupled Sepharose beads. At this time, the mechanism for the production of soluble B7-1 is unknown, as well as its significance in the synovial fluid. It is possible that soluble B7-1 may be inw)lved in the modulation of T cell activation locally. Further characterization is necessary to determine function and presence of soluble B7 in other bodily lluids of patients with various diseases.

1299

Changes in cell adhesion molecules early in picornavirus infection. MJ Maurer, JW Peterson, JD Manzon, and NJ Biglev. Wright State University, Dayton, OH. Splenocyte cultures from 9 ICR Swiss mice, resistant to the pathogenesis of the D variant of encephalomyocarditis virus (EMCV), produce high amounts of interferon-y (IFN-3,) and no interleukin 10 (IL-10) at 12 hr post infection (pi) while splenocytes from infected diseasesusceptible ds produce appreciable amounts of IL-10 in contrast to IFN-y at 12 hours pi. Disease development was prevented in the susceptible gs by enhancing IFN-y production and inhibiting IL-10. At 24 hours pi, CD4+ T cells were markedly depleted in the spleens of infected ds but not in virus-infected splenocyte cultures of either gender. The working hypothesis of this study is that IFN-y and IL-10 affect the pathogenesis of the D variant of EMCV in modulating lymphocyte trafficking through affecting expression of cell adhesion molecules on both lymphoid and epithelial cells. We sought differences in expression of ICAM-I, LFA-I, VCAM-I and VLA-4 by spleen cells during the first 20 hr pi using flow cytometry. No differences in expression of ICAM-1 and LFA-1, and only slight increases in the % of VCAM-l-labeled, spleen cells were seen in either infected d or <2 mice compared with values seen on sptenocytcs from sham-infected mice. In comparisons with the values obtained for sham-infected mice, VLA-4 expression on splenocytes from infected c~s at 9 hr pi was increased. Marked decreases in VLA-4-1abeled splenocytes from infected 9s occurred at 9 hr pi and could be accounted for by the decreased percentages in VLA-4 expression by y~,T cells, c~[3Tcells and B cells. At 12 hr pi, splenocytes from infected 7es showed greater increases in VLA-4 expression (19.6 _-. 7.69b) versus a 5 _+ 5.6% increase in c~s. By 20 hr pi, decreased percentages of VLA-4-1abeled cells were seen in splenocytes from both genders (~12% J, for cos & and -17% ~, for 9s). These early fluctuations in VLA-4 expression by splenocytes in infected mice may contribute to pathogenesis via lymphocyte trafficking and interaction with VCAM-I, the ligand for VLA-4 & a receptor for EMCV-D on cardiac endothelial cells (Huber. SA. J. Virol. 68:3453, 1994l.

Abstracts

S317

1300

Analysis of CD40 Expression in the Upper and Lower Respiratory Tract. M.J. Yellin, A. Szema, J. Samson, H. Rodriguez, E. DiMango and M. Szabolcs, Columbia University. NY, NY. Interactions between CD40 and its counter-receptor, CD40L, play important roles in humoral and cellular immune responses. While CD40 is expressed on many different cells in vitro, the cellular distribution of CD40 expression in vivo in humans is not completely known. In this report we studied in situ CD40 expression in human upper (URT) and lower (LRT) respiratory tract by staining frozen sections of nasal turbinates, nasal polyps and normal lung with anti-CD40 mAb G28.5 or control mAb. Large and small vessel endothelial cells of the URT and LRT express CD40. Epithelial cells (EpC) lining minor salivary glands in the URT express CD40 particularly when infiltrated by inflammatory mononuclear cells, which also express CD40. In the LRT of normal lung, CD40 is strongly expressed on bronchiolar EpC and alveolar macrophages, while alveolar lining cells weakly express CD40. The functional significance of CD40 expression on respiratory EpC has not been previously reported. Therefore we investigated functional consequences of CD4() ligation on pulmonary EpC utilizing a lung carcinoma line (A549) and a SV40 transformed tracheal EpC line (IHAEo). Both cell lines constitutively express CD40 as determined by FACS analysis. CD40L + Jurkat DI.I cells induce ICAM-1 upregulation on A549 cells. In the presence of IFN-y, DI.1 cells also induce VCAM-I expression and augment ICAM-I expression on A549 cells. These effects are specifically inhibited by anti-CD40L mAb 5C8. DI.1 cells, but not control cells, also induce 1HAEo cells to secrete IL-8. Together, these studies suggest that interactions between CD40L + cells and CD40 + parenchymal target cells may play roles in immune mediated respiratory tract diseases.

1301

Extremely Low Frequency Electric Fields Promote Metabolic Resonance and Cell Extension During Neutrophil Migration. H.R. Pet~ and A.L. Kindzelskii. Wayne State University, Detroit MI Previous studies indicate that migrating human neutrophils have a phase-locked metabolism/signal transduction/ receptor apparatus. We now report that application of extremely low frequency (ELF) electric fields from 10~V/m to 10 4 - 1 0 s V/m which are frequency- and phasematched with endogenous metabolic oscillations leads to greatly exaggerated cell extension for locomotion and metabolic (NAD(P)H) resonance. Migrating cell length grows from 10 ~m in the absence of a resonant field to about 40 ~m in its presence. In contrast, cells stop locomotion and become spherical when exposed to phasemismatched fields. Although cellular effects were independent of electrode type, buffer, and cell orientation, they were sensitive to temporal constraints (phase and pulse length) and cell surface charge (as judged by parallel reductions in surface charge and field sensitivity). Microfilament extension sites, as judged by fluorescent cytochalasin binding, were highly dependent on field orientation and pulsed DC versus AC modes. We suggest an electromechanical coupling model wherein applied electric fields acting on cell surface charges and polymerization forces at the cytosolic membrane face act together to overcome surface/cortical tension of neutrophils. Thus, electric fields promote net cytoskeletal assembly and drive heightened biochemical (metabolite and signaling) amplitudes (metabolic resonance). These results provide fundamental insights in cell locomotion and a candidate explanation of ELF health effects.

$318

1302

Abstracts

Generation of a supernatant fluid by stimulated lymphocytes that causes selective migration of lymphocytes through endothelial monolayers. E Wilson and MA Jutila Veterinary Molecular Biology, Montana State University, Bozeman MT 59717 In this abstract, we describe the generation and functional activity of a unique, unidentified factor found in the supernatant fluid of activated bovine peripheral blood mononuclear cells. When stimulated by this supernatant fluid, the migration of bovine ~/8 T cells across 3 vm pore filters, as well as endothelial monolayers, is dramatically and selectively increased. The supernatant fluid has been shown to be highly chemokinetic, but not chemotactic, for bovine -/8 T cells. Furthermore, the active component does not bind heparin and is very heat stabile, retaining full activity after boiling for 5 minutes. The activity of this supernatant fluid is not restricted to bovine cells and causes a similar, selective migration of some human lymphocyte subsets. The basic functional and biochemical nature of this supernatant fluid distinguishes the active factor(s) from any known lymphocyte migration altering cytokine, including all known chemokines. Funded by NRI 96-02218.

1303

The Addition of Ipratropium Bromide to Continuous AIhuterol and Steroids in the Treatment of Acute Severe Asthma. JL Brauckmann MD, JA Pongracic MD Children's Memorial Hospital, Chicago, IL A recent study has suggested that the addition of ipratropium bromide (IB) to high dose albuterol in the Emergency Department (ED) treatment of asthma is more effective than albuterol alone and may reduce the need for hospitalization. The purpose of this study was to further assess the role for IB by evaluating the efficacy of adding IB to high dose albuterol and steroids to treat children hospitalized with a severe asthma exacerbation. Twenty three children who required continuous nebulized albuterol (10 mg/hr) and Solumedrol (2 mg/kg or 60 mg Q6 x 24 hrs then 1 mg/kg Q6) were admitted to the Asthma Care Unit (ACU) at Children's Memorial Hospital and were enrolled in the randomized, double blind, placebo controlled trial. Both groups received the same treatment outlined above. The 11 patients in group 1 also received 500 meg IB in saline via nebulizer every 4 hours for 24 hours while the 12 patients in Group 2 received nebulized saline at these times. Peak expiratory flow rates (PEFR) were measured in the ED, on admission to the ACU, and before and after the study treatments. As patients improved, albuterol was decreased to 5 mg/hr continuously and then to 2.5 mg every 2 hours. The two groups were comparable on presentation to the ED with an average PEFR of 63% in Group l and 60% in Group 2. There was no significant difference between groups for mean percent change in PEFR from admission to 24 hours (p = .61) or in time required to reduce albuterol to every 2 hours (p = .50). In conclusion, our study does not support an increased benefit for the addition of IB to high dose albuterol and steroids for the hospital management of acute severe asthma.

1304

Fluticasone Propionate (FP), Triamcinolone Acetonide (TAA) and Flunisolide (FLN) Have Similar Effects On The HPA Axis. W Storms,* WC Howland, CA Sorkness, C LaForce, M Goldstein, WR Lincourt, PR Rogenes, A E Schaberg, L Edwards, And The FP HPA Axis Clinical Study Group, * Colorado Springs, Colorado. Two studies were conducted to compare the effects of commonly prescribed doses of FP, TAA and FLN on the HPA axis using the 6-hour cosyntropin (synthetic ACTH) infusion method, a sensitive test of the ability to mobilize adrenal reserve. 264 adult subjects with mild asthma were enrolled in two separate double-blind, placebo (PL) controlled studies comparing the effect of FP powder via Diskhaler TM Inhaler (100, 250 or 500 mcg bid), TAA (300 or 500 meg bid), or FLN (500 meg bid) on the HPA axis. Oral prednisone 10 rag/day (PRED) qd in the AM was used as a positive control in study 1. Plasma cortisol was measured at baseline and following 28 days of therapy using HPLC

J ALLERGY CLIN IMMUNOL JANUARY 1997

analysis of serial plasma samples collected at 0, 1, 2, 4, 6, 8, 10 and 12 hrs during the infusion. Response to the infusion was assessed by mean 12-hour AUC, mean peak cortisol concentration (PK), and number of subjects who failed to achieve a normal response (defined as a peak of at least 18 mcg/dl). There were no significant differences in response to the infusion between the FP vs TAA vs PL treatment groups in study 1 or the FP vs FLN vs PL treatment groups in study 2. In study 1 only PRED was associated with a significant (p -< 0.05) decline in AUC and PK cortisol. Three (11%) PRED subjects failed to have a normal response compared with 1 (4%) subject in both the TAA 300 mcg and the FLN 500 mcg group and none in the placebo, FP 100 meg, FP 250 mcg, FP 500 meg, and TAA 500 mcg groups. We conclude that doses of FP, TAA and FLN commonly used to treat asthma are comparable to placebo in effects on adrenal response to a 6-hr cosyntropin infusion and significantly less than effects observed with PRED 10 nag/day for 4 weeks. 1305

Pranlukast (Ultair TM) is effective in improving asthma: Results of a 12-week, multicenter, dose-range study WJ Calhoun, SC Weisberg, I Faiferman, P W Stober on behalf of the Ultair Study Group. University of Pittsburgh, Pittsburgh, PA, University of Minneapolis, Minneapolis, MN, SmithKline Beecham, Collegeville, PA. The efficacy and safety of oral pranlukast (Ultair TM, SB 205312, ONO-1078) were evaluated in a multicenter, double-blind, placebo-controlled, parallel group, dose-ranging study in 521 patients with asthma (FEV I 50-85% predicted) who were receiving only prn albuterol. About 80% of these patients also had co-morbid allergic rhinitis. All doses of pranlukast (75, 150, 300, and 450 nag bid) produced clinically and statistically significant improvements in asthma symptoms beginning at week 1 (p <: 0.05) when compared to placebo. The reduction in asthma symptom scores was progressive and by week 12 had reached 31% in the 300 mg group (p < 0.05). Sustained increases in FEV~ and PEFR as well as decreased use of rescue medication beginning at week 1 were also observed. At week 1 the increase from baseline in FEV~ in pranlukast-treated patients ranged from 220 m[ (75 mg dose) to 300 ml (450rag dose) (p < 0.05). An improvement in symptoms of allergic rhinitis was also observed. Pranlukast was well tolerated with an adverse experience profile similar to placebo. This study demonstrates that pranlukast is an effective oral treatment for asthma in patients with concurrent asthma and allergic rhinitis.

Abstracts

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1306

Additive Benefits of Concurrent Salmeterol and Fluticasone propionate Therapy in Asthma. W Stricker, S Weinstein, P Chela'ins~', A Woodring, B Prillaman, T Shah, Rolla, MO, Huntington Beach, CA, N Dartmouth, MA, Glaxo Wellcome Inc., Research Triangle Park, NC Recent treatment guidelines advocate the concurrent use of anti-inflammatory and bronchodilator therapy for optimal management of asthma. To determine if concurrent use of long-acting beta-agonist Salmeterol (S) and inhaled corticostcroid fluticasone propionate (FP) improves asthma control compared to individual agents, 136 patients receiving only short-acting beta agonists with an FEV~ of 50-80% predicted and >15% reversibility were randomly assigned to 1 month treatment with placebo (P), S 42 meg, FP 88 meg, FP 220 mcg, S and FP 88, or S and FP 220 twice daily. Mean baseline and change in AM lung function tests before and after treatment are displayed below.

Patients (n) Baseline FEV~ (L) % predicted A AM FEVI (L)

P

S

FP 88

FP 220

S/ FP88

S/ FP220

23 2.4 68 0.0

21 2.8 70 0.3

23 2.9 69 0.3*

23 2.5 65 0.3*

25 2.3 67 0.6*

21 2.6 69 0.7*+#

*p < 0.05 vs P; + p < 0.03 vs S: # p < 0.04 vs FP 220 Greater improvements in lung function were seen when S and FP werc used together compared to their use alone with the results for S and FP 220 being statistically significantly greater than the individual components. These preliminary rcsults indicate that concurrent use of S and FP may provide better asthma control than the individual agents and support treatment guidelines recommending the use of these two classes of medications together for optimal asthma management.

1307

Superior Improvement in Quality of Life for Patients with Nocturnal Asthma receiving Salmeterol Xinafoate 42 p~g BID (SLG) versus Placebo (PL). F Cox,* B Bowers,* B Friedman, ** V Petroeella, * A Emmett, * K Rickard, * *Glaxo Wellcome Inc., RTP, N.C.; **Fountain Valley, CA. To evaluate differences between treatment groups in asthma-specific quality of life (QOL), the Asthma Quality of Life Questionnaire (AQLQ) was administered to 474 adult and adolescent patients reporting significant nocturnal symptoms associated with moderate persistent asthma. Patients were enrolled in two parallel group, randomized, double-blind, placebo-controlled, multicenter 12-week studies comparing inhaled SLG 42 ixg BID and PL BID. Patients were allowed to continue fixed regimens of inhaled corticosteroids or theophylline and supplemental albuterol use was allowed "'as need". The AQLQ was administered at baseline, Weeks 4, 8, 12 and at the point of withdrawal if a subject was withdrawn from the study. At baseline, there were no AQLQ score differences between treatment groups. At Weeks 4, 8, and 12 the mean global and domain score changes from baseline for SLG were significantly greater (p -< 0.011 than for PL. The following summarizes the mean score changes from baseline to Week 12:

Mean Change From Baseline to Week 12 (SE)

Activity Limitation Asthma Symptoms Emotional Function Environmental Exposure Global

SLG (n = 240)

PL (n = 234)

1.1l 1.63 1.38 1.12 1.35

(t.55 0.86 0.52 0.59 0.66

(0.08)* (0.09)* (0.11)* (0.101" (0.08)*

((I.07) (0.09) (0.09) (0.09) (0.07)

*p < 0.01, based on mean score change from baseline using F-test

S319

In conclusion, SLG was superior to PL ("as need'" albuterol) for improving QOL in patients experiencing nocturnal symptoms associated with moderate persistent asthma and consistently met and exceeded the reported criteria for a moderate (1.01 or a large (1.5) QOL change. I ~Juniper et. al. J Clin Epid. 47(1):81-87, 1994.

1308

Chronotherapeutic Effect Of Inhaled Steroids on Asthma at 8AM or 5:30 PM versus QID Dosing. DJPincus MD, TR Humeston BS, RJ Martin, M D National Jewish Center for Immunology and Respiratory Medicine Denver Colorado We have recently demonstrated that QID dosing of inhaled steroids and QD dosing at 3PM had equivalent beneficial effects in mild to moderate asthma. Since administration at 3 PM is inconvenient, the present study was undertaken to assess whether a single administration at 8 A M or 5:30 PM would be equally beneficial when compared to conventional QID dosing. In an ongoing study, subjects were given 4 weeks of triamcinolone acetonide as 200 mcg QID (n = 21) or 800 meg QD at 5:30 PM (n = 18) or 800 mcg at 8 A M (n = 181. Preliminary data shows that the three treatment groups are comparable at baseline. For AM PEFR, the 8 AM group had statistically less improvement than either other group. For PM PEFR, there was a trend toward least improvement in the 8 AM group. There are no significant differences between groups for change in FEV], PC20, or 13 agonist use. The data suggests that QD dosing at 5:30 PM may be as effective as QID, whereas 8 AM is not as effective as either of the other time points. Thus the optimal window of time for QD dosing of inhaled steroids may be between 3:00 PM and 5:30 PM.

S320

1309

Abstracts

J ALLERGY CLIN IMMUNOL JANUARY 1997

Defining threshold steroid dose and correlation with absolute eosinophil counts in steroid-dependent asthmatics. GB Moss, DL Hamilos, and PE Korenblat.Washington University School of Medicine, St Louis, MO. Although the association between asthma and esoinophilia is well-established, the utility of the absolute eosinophil count (AEC) as a marker of disease activity is less certain. We studied a group of asthmatic patients who had failed multiple attempts at weaning oral steroids despite the use of inhaled steroids and adjuvant therapies. All patients were referred to the Barnes-Jewish West County Asthma Center. Most patients were successfully weaned off systemic steroids by maximizing high dose inhaled steroids. Systemic steroids were tapered by <12.5% per week to a dose of 5 mg/day and then by 1 mg/week until a symptom exacerbation occurred. Morning AECs were obtained periodically during the taper. Patients took the oral steroid at 3 PM. Adjuvant medications, such as long-acting [3-agonists or methotrexate, were also used in some patients, Five subjects failed weaning and demonstrated persistent hypereosinophilia, despite being on inhaled steroids (>2000 p,g/day). Each patient was stable at a certain dose of oral steroids in terms of symptom scores and use of rescue medications. When the oral steroid dose was decreased slightly below the stable dose, the AEC rose followed by an increase in asthma symptoms in 4 of the patients 12 to 28 days thereafter. We propose that the term "steroid-dependent asthma" should be limited to patients who cannot be weaned from oral steroids despite high-dose inhaled steroids. Systematic tapering of oral steroids in these patients allows for determination of a threshold dose at which eosinophilia increases and asthma symptoms exacerbate within a short period of time. In general, AECs below 600 are associated with stable asthma. A similar threshold AEC may apply to patients on inhaled steroids alone.

Stable Threshold Exacerbation Steroid Steroid Days after Subject Dose AEC Dose AEC taper GO JW MF KM MBF

7 mg 10 mg 4 mg 5 mg 32 mg

490 320 747 455 53

5 8 3 4 24

mg mg mg mg mg

820 900 1209 1570 688

12 14 14 * 28

*Patient received a steroid burst for sinusitis.

1310

Encasing of Mattresses in Children with Asthma and House Dust Mite Allergy. S Halken, 1), U Niklassen 2), LG Hansen 3), F Nielsen 2), A HOst 4), 0 Osterballe 3), MC Veggerby 5), L K Poulsen 5). Departments of Pediatrics S~nderborg Hospital 1), Kolding Hospital 2), Viborg Hospital 3), Odense University Hospital 4) & Laboratory of Medical Allergology, National University Hospital 5), Denmark. In a prospective, doubleblind, placebo controlled study the effect of a semipermeable mattress cover (Allergy Control) in children with asthma and house dust mite (HDM) allergy was investigated. 60 children (6-15 yrs) with asthma, a positive SPT against Der pI and a positive bronchial provocation test with Der pI were included. The children were randomized to placebo or active mattress and pillow covers. Clinical data and dustsampling from mattresses were obtained and adjustment of asthma medication was performed every 3 months during 1 yr. Dust samples were analysed for Der pI, Der fI and Der mI by means of species specific ELISA technique. 52 children completed 1 yr follow-up. In 3 children data concerning asthma medication were insufficient. At inclusion no difference in HDM conc., medication and lung function were found. In the active group (n = 28) a significant reduction in the total HDM conc. (mean: 77630-+7034 ng/g dust, p = 0.005) and in the dose of inhaled steroids (mean: 424---~227 pg/day, p = 0.003), and a significant increase in mean morning and evening PEF (317-+358 and 326--->363, p =

0.003) and log PD2o (3.20---~3.84, p = 0.02) was found. No significant changes in these parameters were found in the placebo group (n = 24). In conclusion, these mattress and pillow covers significantly reduced the conc. of HDM (Der pI + fI + mI) in mattress dust and the dose of inhaled steroids in children with asthma and HDM allergy.

1311

Fluticasone Propionate (FP) 1000 Itg bid Appears to Improve Glucocorticoid (GC) Receptor (GCR) Binding Affinity While Reducing Eosinophil Cationic Protein (ECP) Levels & Oral GC Requirements in Severe Steroid Dependent Asthma. SR Nimmagadda, JD Spahn, H Nelson, DYM Leung, SJ Szefler. Denver, CO. This 1 yr, double-blind, placebo-controlled, single site pilot study examined the effect of high-dose FP on GCR binding affinity, ECP levels, and GC dose reduction. FP 1000 Izg, FP 500 Izg, or placebo was administered to 13 steroid dependent asthmatics bid. PBMC GCR binding affinities (Kd) utilizing [3H]-dexamethasone radioligand binding assays, ECP levels, and GC doses were recorded at baseline, 4 & 6 wks, 6 mo, and 1 yr. Patients failing treatment due to poor asthma control were assigned openlabel FP 1000 ixg bid. At 6 mo, all 4 patients on placebo had required entry into the open-label arm of the study, while 2/5 patients on FP 500 p.g, and none of the patients on FP 1000 Izg bid entered the open-label arm. Improvements in all study parameters were seen in all groups with the greatest changes noted among those on FP 1000 p~g bid as seen in the Table (data presented as mean _+ standard error of mean).

Change in Kd, ECP, Oral GC from baseline to 6 wks Group (n) Placebo (4) 500 Izg bid (5) 1000 jzg bid (4)

A Kd nM

A ECP ix/L

A oral GC

-15.4 _+ 4 -5.8 + 4.6 - 2 3 _+ 9

-3.6 + 4.0 -7.6 + 6.4 -12.1 + 13

- 1 mg/d - 8 mg/d - 9 mg/d

Significant differences were not detected due to the small number of patients in each group, but trends for greater improvements were noted with FP treatment at 1000 p~g bid. In conclusion, trends in oral GC dose reduction, reduced ECP levels, and improved GCR binding affinity were noted with FP 1000 ~g bid. The results obtained from this pilot study warrant further research on FP in severe, steroid dependent asthma.

J ALLERGYCLIN IMMUNOL

Abstracts

$321

VOLUME 99, NUMBER 1, PART 2

1312

Effect of nedocromil sodium on bronchial response to histamine and allergen challenge in asthmatics. Z Siergiejko, S Chyrek-Borowska, Z Zietkowski Department of Allergology and Internal Diseases, University Medical School, Bialystok, Poland Nedocrnmil sodium has recently been considcred to be a drug with a wide spectrum of the activity. It can block chloride channels on a number of cell types. The purpose of our studies was to evaluate the protective effect of a single dose of nedocromil sodium on bronchial responses to histaminc and allergen challenge in asthma patients. The effect of hmg-term uedocromil treatment on specific (sBR) and non-specific bronchial reactivity (BR) was also investigated. Cross-over, placebo controlled studies were carried out on 16 allergic asthmatics, sensitive to Dcrmatophagoides ptcronyssinus (Dpt). A schedule of the studies is presented below.

1314

Nguyen, DA Guerreiro, TF Reiss, BS Friedman, BA Knott, AAMGRC, San Diego, CA; IMTC, Inc, Prairie Village, KS; ASTHMA, Inc, Seattle, WA: Merck & Co, Inc., Rahway, NJ Previous studies in adults with exercise-induced bronchoconstriction (EIB) have demonstrated that montelukast, a new, potent cysteinyl leukotriene (CysLT~) receptor antagonist, inhibits EIB at the end of a once daily dosing interval. This study was designed to determine if montelukast inhibits EIB in 6- to- 14- year olds. In a randomized, double-blind, crossover study, twenty-seven asthmatics (20 males, 7 females) with a pre-challenge FEV~ ->70% of the predicted wdue and a post-exercise FEV~ fall of at least 20% on two occasions, receivcd placebo or montelukast 5 mg (chewable tablet) once daily in the evening, for two days. In the evening, approximately 20-24 hours after the last once-daily dose, a standardized exercise challenge was performed. The montclukast group demonstrated a significant improvement compared with placebo in: area below the percent change in FEV~ versus time curve (AUCo_61mm0 (-589.72% 9 min and 264.6(1%. min for the placebo and montelukast groups, respectively [p=0.013]), and maximum percent fall in FEV~ after exercise ( 26.11% and -18.27% for the placebo and montclukast groups, respectively [p 0.009]) and a borderline significant improvemenl in the time to recovery (27.98 minutes and 17.76 minutes for the placebo and monte[ukast groups, respectively [p=11.079]). Montelukast was generally well tolerated in this study. We conclude that montelukast (5 mg/day), compared with placebo, inhibited exercise-induced bronchoconstriction (58.77% inhibition of A U C , ,,~,mm)in 6- to 14- year old asthmatics at the end of a once-daily dosing interval.

Successive days of the studies 1-2 3-17 18-19 20-23 24-25

26-81

n-8, * n = 16 ,i n 16 *~ Ned 2 • 2 ** PT **t PT *'2 n = 8 PI2 ~ 2

82-83

,3 **;

84-139

n 8 P1 2 • 2 n = 8

140-142

*) **3

Ned2 x 2

*-BPT with histamine. **-BPT with Dpt, PT-previous therapy, PI-placebo, Ned-Nedocromil sodium, .l**l_Bp T 31) min after 4 mg of nedocromil, *2.**2-BPT 30 rain after 2 puffs of placebo, *-~.**)-BPT - 24 h after last dose placebo or nedocromil, 2 • 2 - 2 times 2 puffs daily. The single dose of nedocromil effectively prevented histamine or allergen bronchoconstriction. The prolonged therapy diminished sBR, BR and reduced the intensity and frequency of LAR. In some patients previously demonstrating dual asthmatic response, EAR only was observed. After placcbo no changes in sBR and BR were noted. 1315

1313

Delayed Asthmatic Response [DYAR], its Clinical Feature and Pharmacologic Modulation. Z Pelikan, M PelikanFilipek, JH Oostenbrink, Breda, The Netherlands The 48 patients with allergic bronchial asthma, developing the DYAR to "inhalant" allergens (onset within 26-30, maximum within 30-48, resolving within 56 hours after the challenge) were randomly selected and divided into 4 groups. In each patient 2 protection tests (PT) were performed, in group l (n ~ 12) with Disodium cromoglycate (DSCG) and Nedocromil Sodium (NDS), in group I1 (n = 12) with DSCG and Beclomethasone dipropionate (BDA), in group lIl (n 12) with NDS and BDA and in group IV (n 12) with Salbutamol (SBT) and Budesonide (BUD). The drugs were given in the form of pressurized aerosol in the following daily doses: DSCG 4 x 2 puffs x 5 mg (40 rag), NDS 4 • 2 puffs • 2 mg (16 rag), BDA 4 • 2 puffs • 100 mcg (800 meg), BUD 4 • 2 puffs • 50 meg 14/)0 meg), SBT 4 • 2 puffs • 1011 mcg (800 mcg). The treatments were started 28 days before and continued up to 56 hours after the challcnge. The study design was double blind, crossover. Results: (1) DSCG (n - 24) and SBT (n 12) have not demonstrated any significant protective cffects on the DYAR (p > 0.05 respectivcly p > 0.2); (2) NDS (n - 24) has prevented the DYAR significantly (p < I)./)5); (3) BDA (n 24) and BUD (n - 12) have prevented DYAR highly significantly (p < 0.001 respectively p < 0.Ill). The different effects of the drugs studied on the DYAR suggest an involvement of specific mechanisms in the DYAR, such as the cell-mediated mechanism(s) upon participation of the T-lymphocytcs, which would differ from those mechanisms presumed to be inw)lved in the immediate lIAR) and late (LAR) asthmatic responses.

Montelukast, a leukotriene receptor antagonist, inhibits exercise-induced bronchoconstriction in 6- to- 14- year old children. JP Kemp, RJ Dockhorn, GG Shapiro, HH

Effect of intratracheal nebulization of steroids on antigen challenge induced cell migration into the lungs of sensitized Brown Norway Rats. BM Taylor. JM Justen, BKJ

Leong, CP Sabaitis, DA Rop, WE Fleming, IM Richards, Pharmacia & Upjohn, Kalamazoo, MI, USA. Brown Norway Rats were sensitized by intramuscular injection of ovalbumin (OA) mixed with aluminum hydroxide. Two weeks later, the sensitized rats were challenged with aerosol inhalation of a 1% OA aqueous solution. An immediate and a late phase response were observed. Bronchoalveolar lavage examinations showed that the delayed response was characterized by an influx of eosinophil rich inflammatory cells into the lungs. In evaluating the effectiveness of an inhaled anti-inflammatory drug, a miniature nebulization probe was inserted into the trachea of a sensitized rat, whereby, a pre-determined dose of a drug solution was nebulized at 2 ixl per puff of air (~2 mL) into the lungs. The Mass Median Aerodynamic Diameters were 8.3 to 9.4 ixm. When water soluble dexamethasone was administered, a dose dcpendent inhibition of eosinophil influx was observed 24 hr post challenge. In additional studies, when sensitized rats were dosed with 1 Ixg/kg per dose for 3 in 24 hr, an 84.2 + 4.2% reduction of eosinophil influx was observed. When a single 1 mg/kg dose was given 1 hr before challenge, a 59.7 • 8.4% reduction was obscrvcd. After methyl prednisokme suleptanate was administered at a dose of I mg/kg at 1 hr before and 7 hr after challenge, a 63.2 • 10.1% reduction from the control was observed. Thus, this intratracheal nebulization technique allows the aerosol delivery of metered doses of a compound directly into the lungs of small animals and should be of great utility for efficacy evaluation of inhaled aerosol drugs.

S322

1316

Abstracts

Inhaled Fluticasone Propionate Dry Powder Administered Either Once Daily or Twice Daily Via The Diskus Is Safe And Effective In Pediatric Patients With Chronic Asthma.

S. Weinstein, S. Galant, W Silvers, B. Lanier, J. Grossman, A. Brown, A. Hamedani, K. House, D. Clements, S. Harding, Huntington Beach, CA, Orange, CA, Englewood, CO, Fort Worth TX, Tucson, AZ, Glaxo Wellcome, Inc., Research Triangle Park, NC. A 12-week, multicenter, double-blind, parallel-group trial evaluated the safety and efficacy of inhaled Fluticasone Propionate (FP) powder 100 mcg BID (FP BID), FP 200 mcg QD (FP QD) and Placebo (PLA) administered via the 60-dose powder Diskus device in 242 pediatric patients (173M, 69F) aged 4-11 with chronic asthma. Patients enrolled included 78 PLA, 80 FP BID and 84 QD. Clinic visits were scheduled every 1-2 weeks. Overall mean baseline FEV 1 was 70-73% of Polgar predicted and PEF was 74-75% of Polgar predicted, with no significant difference between groups in baseline pulmonary function. Patients were enrolled with baseline therapy of anti-inflammatory agents (inhaled corticosteroids or cromolyn) [ICS] or beta-agonist therapy [BA] alone. Patients were dropped if they met predefined criteria for lack of efficacy (LOE). Significantly more PLA patients discontinued due to LOE than in either FP group (p = 0.001). Analysis of mean change from baseline at endpoint FEV~ and diary PEF indicated both FP dose regimens were significantly better than PLA (p -< 0.001), but not significantly different from each other (p -> 0.219). The types of adverse events were consistent with those seen with other ICS. Overall, FP administered either QD or BID was well tolerated and improved lung function in patients previously treated with ICS or BA therapy. 1317

Aseptic Meningitis Syndrome Following Intravenous Immunoglobulin Therapy in Steroid-Dependent Asthma.

James M. Stocks, MD, E. Richard Stiehm, MD* G. Wendell Richmond, MD ~, The University of Texas Health Center at Tyler, *UCLA Medical Center, #Rush Presbyterian-St. Luke's Medical Center. The syndrome of aseptic meningitis (ASM) due to intravenous immunoglobulin therapy (IVIG) given as treatment for various immunologic disorders has been previously reported. A double-blind, placebo-controlled, randomized, multi-center clinical trial designed to investigate the safety and therapeutic effect of Venoglobulin-I~ (10%) (Alpha Therapeutic Corporation, Los Angeles, CA) was conducted in patients with severe, steroid-dependent asthma. Forty-nine patients were enrolled and received at least one of seven monthly infusions of placebo, 1 g/kg, or 2 g/kg IVIG. Three of the twenty-one patients (14%) who received IVIG at the 2 g/kg dose developed ASM within 24 hours of their first IVIG dose. ASM was not identified in other patients though less severe headaches were more frequent in patients receiving the 1 g/kg and 2 g/kg doses of IVIG (33% and 27%, respectively) as compared to placebo (16%, p = 0.02). In the cases of ASM, headache, meningismus, photophobia, and fever were observed, and a neutrophilic leucocytosis (128 to 566 WBC/Ixl) was documented in the CSF. All three patients responded to supportive therapy though one patient was found to have pseudotumor cerebri and took 61 days for the headache to resolve. Pretreatment with NSAID medication and keeping the rate of infusion to less than 0.08 ml/min/kg did not prevent ASM. High-dose IVIG therapy (2 g/kg) appears to be associated with increased risk for the development of ASM. 1318

J ALLERGY CLIN IMMUNOL JANUARY 1997

Influence of salmeterol treatment upon Nitric Oxide level in exhaled air and bronchodilator response to terbutaline in children with mild asthma. G Fuglsang, J Vikre-

Jorgensen, L Agertoft, S Pedersen, Kolding Hospital, Kolding, Denmark. Aim: To evaluate how continuous treatment with salmeterol influences Nitric Oxide (NO) level in exhaled air and the bronchodilator response to terbutaline. Methods: 22 children, aged 7 to 15 years (mean = 11 years), with mild asthma were treated with inhaled salme-

terol 50 txg bid and placebo for three weeks in a randomized double blind cross-over study. These treatments were followed by treatment with inhaled budesonide 200 ixg bid for 3 weeks. On the last day of each period NO level was measured in exhaled air and a cumulative dose-response experiment with terbutaline (cumulative dose 1475 pxg)was performed. Results: Nitric Oxide levels were unaffected by salmeterol treatment but significantly reduced during budesonide therapy (p < 0.001), mean NO being 10.7 ppb (placebo), 12.7 ppb (salbutamol) and 5.2 ppb (budesonide), respectively (the corresponding maximal NO levels were 19.5, 22.9 and 9.4 ppb). Baseline lung functions after salmeterol treatment were significantly higher than baseline after placebo (P < 0.05). Therefore, the lung function data were fitted into a non-linear mixed-effects 'partial agonist' pharmacodynamic model, which ignores the faulty baseline spirometry. The calculations showed that the salmeterol treatment had a significant effect on the terbutaline doseresponse curve (EDs0 was increased by an estimated factor of 41 (95% confidence interval 6.7-254). Conclusion: Salmeterol treatment does not affect NO levels in exhaled air but it significantly changes the dose response curve to terbutaline.

1319

Low Dose Inhaled Fluticasone Propionate Administered Once Daily or Twice Daily via the Diskus is as Safe and Effective as Beclomethasone Dipropionate in Steroid Naive Patients with Asthma. J Selner, H Boltansky, P Chervinsky,

D Pearlman, A Pedino~, S Weinstein, J Wolfe, A Stevens, B Prillaman, A Hamedani, S Harding, E Field. Denver, CO, Washington, DC, North Dartmouth, MA, Aurora, CO, Princeton, NJ, Huntington Beach, CA, San Jose, CA, Research Triangle Park, NC. A 12-week, multicenter, randomized, double-blind, double-dummy parallel-group trial compared efficacy and safety of fluticasone propionate (FP), beclomethasone dipropionate (BDP) and placebo (PLA) in 299 steroid naive asthma patients aged 12 and up previously treated with beta-agonist therapy. FP was administered via Diskus, a new 60-dose dry powder device, at blister doses of 100mcg BID (FP-BID) and 200mcg QD (FP-QD). BDP was given at 168mcg BID (exactuator dose) via metereddose inhaler. Visits were scheduled every 1-2 weeks. Subjects were dropped for lack of efficacy (LOE) when meeting predefined criteria for changes in FEV~, PEF, or nighttime awakenings. Fewer patients on FP-BID (n = 5) and FP-QD (n = 7) were dropped for LOE vs PLA (n = 19; p<0.01). There were no significant differences between BDP vs PLA treated patients in LOE withdrawal rates. Mean FEV l at baseline was 2.56-2.62L (67.7-68.8% predicted). After 1 week of treatment, FEV~ improved by 13-14% on all active treatments (p<0.05 vs PLA). FP-BID, FP-QD and BDP treatment significantly improved FEV 1 at endpoint (last treatment visit) (p<0.05 vs PLA). All active treatments significantly improved change in PEF from baseline to endpoint (p<0.005 vs PLA) with concurrent decreased use of beta-agonist (p<0.05 vs PLA). Adverse event rates were low and comparable across treatment groups. Overall, low dose FP-BID and FP-QD improved lung function with efficacy similar to BDP given at approximately double the dose.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

Abstracts

1320

Clinical Dose Response Study of Azmacort| HFA-134a in Adult Asthmatics. M Welch. W Sih,ers. J Smith, P Bagehi. L Wehrle, L Galuchie. S Levy, San Diego CA, Englewood CO, Rh6ne-Poulenc Rorer, Collegeville, PA The phase-out of chlorofluorocarbons (CFCs) necessitates the development of aerosol asthma medications with non-CFC propellants including HFA-134a. The primary objective of this study was to examine the dose response of Azmacort| HFA-134a at total daily doses of 150, 300, and 600 Ixg (bid); additionally, Azmacort| CFC was examined at the same doses. A total of 514 moderate asthmatics who required chronic inhaled corticosteroid therapy (ICS)were enrolled in this 8 week, multi-center, double-blind, placebo (PL)-controlled randomized, and parallel group study. During a 5-21 day baseline (BL) period, patients were removed from their ICS therapy and required to be symptomatic for randomization. In this study, Azmacort(~ HFA-134a showed a dose response for FEV,, AM PFR, 24-hour symptom scores (SxScore 0-12, 0 -= no symptoms), and 24-hour 6-agonist use. The rates of discontinuation due to ineffective therapy were similar for all active treatment groups and were markedly lower than for placebo. No serious adverse events related to any test drug were reported. Mean Change from BL to Endpoint for Select Efficacy Parameters Azmacort| HFA-134a (t~g/day) PL (N =76)

150 (N =73)

300 (N=77)

600 (N=75)

3.93 12.14 -0.73 -0.35 34

18.09 ~ 9.23* -1.52" 1.83" I0

12.26" 36.42* -2.03* 3.26* 9

22.01" 44.89* -2.57* -3.35* 7

FEVl (%) AM PFR (L/min) 24H SxScore (units) 24H [3-agonist (puffs/day) Discontinuations

*one-waylinear trend test p-value <0.05 vs. placebo These data show that Azmacort| HFA-134a is both well tolerated and effective at total daily doses of 150, 300 and 60(1 ixg when used to control symptoms of moderate asthma.

1321

A Twelve Week Placebo-Controlled Efficacy and Safety Study of Intal ~' 1 mg Formulated with CFC or HFA-227 as Propellant for the Treatment of Asthma. W Howland, D

BirdsalF, T UrvniaU.,

FE Cas~ ~and the hTtal Study Group.

IAustin, TX; -'Rochester, NY; 3RPR Pharmaceuticals, Collegeville, PA. Cromolyn sodium, a well-recognized treatment for asthma, is currently being developed as a CFC-free formulation using HFA-227 as the propellant. After a 2 week baseline period, a 12 week randomized, double-blind, placebo-controlled, parallel study compared the etficacy and safety of 2 mg CFC Intal and 2 mg CFC-free Intal QID in 377 patients at least 12 years of age. Eligible patients (approximately 125/group) had an FEV~ 50-90% and required inhaled beta-agonists at least three times per week for the preceding month. Patients recorded daily symptom scores, beta-agonist use and PEF on daily diary cards. The primary efficacy variable was total symptom score (SS): the sum of the daytime and nighttime asthma scores. Mean baseline and treatment week 1-12 variables are prcsented below: Mean values Baseline/Treatment Weeks 1-12 Placebo CFC Intal HFA Intal SS (0-10) Daytime score (0-5) Nighttime score (0-5) Albuterol Use (puffs/day)

3,54/3.24 3.57/2.82* 2.09/1.87 2.01/1.56"

3.57/2.83* 1.99/I.53"

1.49/1.39

1.57/1.30

1.59/l.26

4.08/3.40 4.03/2.50*

3.66/2.42*

$323

Mean values Baseline/Treatment Weeks 1-12 Placebo CFC Intal HFA I n t a l FEVI (liters)

2 . 6 0 / 2 . 6 4 2.72/2.79

2.82/2.95

*p < (/.05 Both formulations of Intal significantly improved total symptom score, daytime asthma score and albuterol use compared with placebo. There were no significant differences between the Intal formulations. No significant differences were demonstrated among all treatment groups for the incidence of adverse events or laboratory results. Conclusion: CFC-free lntaV~' 1 mg is an effective treatment for asthma; the safety and efficacy profile is comparable to the CFC formulation of IntaP~ 1 rag.

1322

Long Term, Regular Treatment with Salmetero| is Effective in Protecting Against Bronchial Hyperresponsiveness as Measured by Methacholine Challenge in Asthmatics.

R Rosenthal, P Chen,insky, A DeGraffl S Gahmt, P Goldberg, J Grossman, *J Kemp, P Korenblat, Z Munk, H Nelson, D Pearlman, *J Ramsdell. P Scanlon, G Shapiro, T Sim, D Tinkelman. M Vandewalker, J Wolff', MB Welch, C McChmg, TArledge, Y Wang, TRossing, E Stahl Fairfax, VA, Dartmouth, MA, Hartford, CT, Orange, CA, Indianapolis, IN, Tuscon, AZ, *San Diego, CA, St Louis, MO, Houston, TX, Denver, CO, Aurora, CO, Rochester, MN, Seattle, WA, Galveston, TX, Atlanta, GA, Rolla, MO, San Jose, CA, Glaxo Wellcome Inc, RTP, NC This 12-month study evaluated the effects of regularly administered inhaled salmeterol powder in the Diskhaler| on airway hyperreactivity. Asthmatics (n = 352) meeting entry criteria (stable asthma, ~12 years, FEV~ 70-90% predicted, PD2o FEVt methacholine -<7.5 mg/ml reproducible to 1/21oglt0 were randomized into this double-blind, parallel-group study of BID salmeterol (S) 50meg versus placebo (P). Methacholine Challenge (MC) technique and calibrated equipment were standardized between centers. The use of concomitant anti-asthma drugs was similarly regulated for both groups. MCs were conducted during the 2-week placebo lead-in period, at Treat-ment Weeks 4, 12, 24, and 52, and Post Treatment Days 1, 2, and 7. During treatment, S was more effective than P in decreasing bronchial hyperresponsiveness at all time points and statistically significant at Weeks 4 and 24 (p < 0.034). In general, S produced a one doubling dose improvement in PD2o from baseline. During posttreatment, the level of protection for S recipients declined (compared to during treatment). At 7 days post S therapy, the decline was to less than one doubling dose below baseline. The level of protection for P recipients remained the same compared to during treatment. This 12 month study demonstrated that regular administration of salmcterol powder 50meg BID by Diskhaler significantly and persistently decreases airway hyperresponsiveness as measured by MC and does not result in hyperresponsiveness posttreatmcnt.

$324

Abstracts

J ALLERGYCLINIMMUNOL JANUARY 1997

1323

Salmeterol Improves Asthma-Specific Quality of Life In Patients With Moderate Persistent Asthma. A Navak** Bowers B*, Cox F* V Petrocella, *, Emmett A, K Rickard. *Glaxo Wellcome Inc, RTP, N.C.; ** Normal, IL. To evaluate differences between treatment groups in asthma-specific quality of life (QOL), the Asthma Quality of Life Questionnaire (AQLQ) was administered to 536 patients with a history of persistent asthma symptoms. Patients were enrolled in two identical, randomized, double-blind, placebo-controlled, 12 week, multicenter studies comparing inhaled salmeterol 421xg BID (SLG) and placebo (PL) BID. Supplemental albuterol was allowed "as needed". Eligible patients were -> 12 years old, had an unmedicated FEV~ of 40-80% of predicted normal, and currently symptomatic. The AQLQ was administered at Baseline (prior to study treatment), and Weeks 4, 8, and 12. At Baseline, there were no differences between treatment groups for either the individual domain scores or the global score. At Weeks 4, 8, and 12, the global and domain scores mean change from baseline for SLG were significantly greater (p < 0.001) than PL. The following summarizes the mean change from Baseline to Week 12:

1325

(ng/ml) =.500 _ ~ 0 1 400

300 ~200 ~100 O

E

Mean Change From Baseline to Week 12 (SE)

Activity Limitation Asthma Symptoms Emotional Function Environmental Exposure Global

SLG (n = 262)

PL (n = 274

1.00 (0.07)* 1.44 (0.09)* 1.23 (0.10)* 1.09 (0.08)* 1.21 (0.08)*

0.62 (0.06) 0.73 (0.07) 0.66 (0.08) 0.58 (0.07) 0.66 (0.06)

before

In conclusion, SLG provided significantly greater improvement in asthma-specific QOL as compared to PL ("as needed" albuteroi) and consistently exceeded the reported criteria for a moderate change (1.0) in QOL.

To continue or not to continue BDP in stable asthmatic children: Can measurement of bronchial responsiveness help? K Matsuda, MCT Capulong, N Sakaguchi Y likura, H Saito, A Akasawa, National Children's Hospital Tokyo, Japan We investigated the bronchial responsiveness to methacholine of 27 asthmatic children in remission and maintained on beclomethasone dipropionate (BDP) . We evaluated whether bronchial responsiveness would significantly alter in patients whose BDP had been discontinued as compared to those who continuously used the drug. Twenty-seven asthmatic children with a mean age of 12.4 • 3.l years and have had no attack for months (duration of remission :4-48 mos; mean: 13.8 • 13 mos) were enrolled in the study. These patients were grouped into 2 : group A (N= 10),maintained on BDP; and group B (N=17),BDP was discontinued. Bronchial responsiveness to methacholine was measured by using the Astograph with the minimum dose of methacholine (Dmin) as the indicator of bronchial sensitivity. Methacholine challenge test was performed on all patients at the beginning of the study and 1 month after. Both groups were observed and evaluated clinically for 3months after 2nd challenge. They were excluded from the study if they developed an attack before the second test. There was no significant difference in the Dmin of the initial challenge between the groups, nor was it associated with the severity of asthma ,duration of remission and the serum IgE level. Dmin was virtually unchanged in groups A and B. Five patients in group I3 had an attack and 4 showed a decrease in Dmin in the 2nd test. Our study suggests that measurement of bronchial responsiveness may be a good parameter in evaluating not only the patients' clinical status but also their need for maintaining or decreasing anti-asthma drugs.

after

and sputum levels of eosinophil cationic protein (ECP) and IL-5 were compared before and 6 weeks after administration of oral theophylline concomitant with the change in PEF. The dose of theophylline was adjusted to keep the serum concentration in the therapeutic levels (10-20 txg/ ml). After treatment with theophylline, there was a significant fall in ECP level in sputum (geometric mean; 227 ng/ml vs. 74 ng/ml, p < 0.01) and a marginal increase in sputum IL-5 level in sputum (122 pg/ml vs. 304 ng/ml, p < 0.07). The number of eosinophils in sputum (median value; 350/1~1 vs. 75/ixl) and in blood (445/ixl vs. 226/~1), and the serum level of ECP (15.0 ng/ml vs. 11.2 ng/ml) decreased after the treatment, but the differences were not significant. We conclude that theophylline inhibits eosinophil activation counteracting the effect of IL-5 in mild asthma.

*p < 0.001 vs PL; Based on mean score change from baseline using F-test. The intent-to-treat-population was used for all analyses.

1324

Anti-inflammatory effects of theophylline (Uniphyl | in mild asthmatics. K. Sugiyama S. Motojima K. Tateishi S. Makino T. Fukuda Dokkyo University School of Medicine, Tochigi, Japan Theophylline has recently been reappraised as a antiinflammatory drug. We studied whether oral theophylline is really anti-inflammatory for mild asthmatics. In 6 asthmatic subjects (M/F = 4/2, average 34 years of age) who had been treated with a beta-agonist inhaler alone, the number of blood and sputum eosinophils, and the serum

1326

Linear Growth of Prepubertal Asthmatic Children Treated With Long-Term Inhaled Budesonide. IBBarlan, MBakir, F T~kenmez, MA Nursoy, M Bafaran, Marmara University Hospital, Istanbul, Turkey Increased use and earlier introduction of inhaled corticosteroids in the long-term management of asthma raised concern about suppression of linear growth in children. We therefore evaluated the height measurements of 94 asthmatic children (Group I) treated with inhaled budesonide (IBUD) in comparison with 26 mild asthmatics (Group II). The mean age of group I was 7.36 • 3.23 (range 2-15 years). The mean duration of IBUD therapy was 13 • 7.5 (6-45) months. The patients were divided into 3 groups with respect to daily dose of IBUD as less than 400 I~g (n = 18), 400-800 ~g (n = 70) and more than 800 ixg (n = 6). The mean age of group II was 6.88 • 3.20 (range 2-14 years). They were treated with inhaled albuterol on as required basis for a mean duration of 12.5 • 2.5 (6-30) months. Height measurements were carried out by the same standard stadiometer in the beginning and with 6-months intervals during the follow-up. Measurements were converted to z-score by use of the normal values for age and sex. The results showed that the mean height measurements (z-score) did not change significantly either in group I (-0.0258 • 0.9661 to -0.1117 • 0.9812) or in group II (-0.1221 • 1.1200 to -0.3093 • 1.0990) during the follow-up period. There was no significant change with respect to the dosage of IBUD in group II either (p > 0.05). We concluded that inhaled budesonide, with daily doses of 200-1200 Ixg, does not likely to cause an impairment in height gain during the treatment of asthmatic children in comparison with steroid-free mild asthmatics.

J ALLERGYCLIN IMMUNOL VOLUME 99, NUMBER 1, PART2

1327

1328

Abstracts

Salmeterol (42 ixg BID) Improves Clinical Outcomes in Adolescents and Adults With Nocturnal Asthma. L DuBuske, F Cox, B Bowers, A Ernmett, C Kalberg, V Petrocella, K Rickard, Allergy and Arthritis Family Treatment Center, Gardner, MA and GIaxo Wellcomc lnc, Research Triangle Park, NC Two identically designed, randomized, double-blind, 12week studies compared salmeterol (SLG) aerosol 42 Ixg BID to placebo (PLB) aerosol BID in 474 patients with nocturnal asthma [mcan % predicted FEV t 61.1 (SLG) and 58.9 (PLB)] aged 12 and over. During run-in, all patients demonstrated nocturnal symptoms on 6 of 14 nights and -> 15% decrease in peak flow rate (PEFR) upon nocturnal awakening. All patients used alhuterol (ALB) aerosol as needed to relieve acute symptoms. Patients kept a daily record of AM and PM PEFR, asthma symptoms, and supplemental ALB use. At all 12 treatment weeks, SLG significantly increased mean AM PEFR, the % of symptom-free days, the % of nights with no awakenings, and the % of days and nights with no supplemental ALB use (all parameters, p -< 0.004). Over weeks 1-12, AM PEFR significantly (p < 0.001 ) increased by 38 L/rnin with SLG as compared to no change with PLB (prn ALB). SLG significantly decreased (p < 0.001) diurnal PEFR variation by 19 L/rain as compared to no change with PLB. The % of nights with no awakenings was significantly (p < 0,001) increased from 28% at baseline to 66% with SLG over weeks 1-12 as compared to 2891 at baseline to 40% with PLB. A total of 73 and 86 asthma exacerbations were reported with SLG and PLB respectively. Two (2) and 10 withdrawals due to lack of efficacy occurred with SLG and PLB respectively. The safety profiles were similar in both treatment groups. Theophylline (THP) users comprised 23% and 24% of the SLG and PLB populations, respectively. SLG and PLB effects on AM PEFR were similar in both THP and non-THP users. This study shows that regular SLG therapy (42 txg BID) results in improved lung function, reduced diurnal variation in PEFR, and better daytime and nighttime symptom control as compared to PLB (prn ALB).

$325

does not effect HPA function as determined by the short cosyntropin stimulation test. 1329

Salmeterol (42p~g BID) Improves Clinical Outcomes in Adolescents and Adults With Moderate to Severe Asthma. J Murray, B Bowers, F Cox, A Emmett, C Kalberg, V Petrocella, K Rickard, Vanderbilt Medical Center, Nashville, TN and Glaxo Wellcome Inc, Research Triangle Park, NC Two identically designed, randomized, double-blind, 12week studies compared salmeterol (SLG) aerosol 42/xg BID to placebo (PLB) aerosol BID in 538 patients with moderate to severe asthma (% predicted FEV~ 40-80%; mean of 62%) aged 12 and over. During run-in, all patients demonstrated symptoms and a ->15% diurnal peak flow rate (PEFR) variation. All patients used albuterol (ALB) aerosol as needed to relieve acute symptoms. Patients kept a daily record of AM and PM PEFR, asthma symptoms and supplemental ALB. At all treatment weeks, SLG significantly increased (p < 0.001) mean morning PEFR, the % of symptom-free days, and the % of nights with no awakenings when compared to PLB (prn ALB). SLG significantly (p < 0.001) decreased daytime and nighttime supplemental ALB use over weeks 1-12 when compared to PLB. Over weeks 1-12, AM PEFR significantly increased by 54 L/rain in SLG as compared to 20 L/rain in PLB. Diurnal PEFR variability significantly (p < 0.001) decreased from 67 L/rain at baseline to 25 L/rain in SLG as compared to a decrease from 68 L/min at baseline to 45 L/min in PLB. Over weeks 1-12, the % of nights with no awakenings significantly (p < 0.001) increased from 42% at baseline to 70% in SLG as compared to an increase from 43% to 49% in PLB. A total of 47 and 64 asthma exacerbations were reported in SLG and PLB respectively. Three (3) withdrawals due to lack of efficacy occurred in SLG and 11 in PLB. The safety profiles were similar in both treatments. This study shows that regular SLG therapy (421xg BID) results in improved lung function, reduced diurnal variation in PEFR, and better daytime and nighttime symptom control as compared to placebo (prn ALB).

1330

Comparison of Triamcinolone Acetonide HFA Vs. CFC Formulations on the Short Cosyntropin Test in Children. J Caldwell, E Brunel. M Rouse. S Vaccaro and M Gillen, Gainesville Clinical Research Center and Rhone-Poulenc Rorer, Gainesville, FL and Collegeville, PA. The proposed phase-out of CFC propellants has necessitated reformulation of inhalational aerosols using "'ozone friendly" propellants. Azmacort [triamcinolone acetonide (TAA)] has been reformulated using HFA-134a. This study evaluated the influence of this change in propellants on the effects of TAA on the hypothalamic-pituitary-adrenal (HPA) axis. 110 male and female children, ages 6-12, entered 1 of 5 treatment groups: TAA HFA 450, 900 or 1350 meg/d, marketed TAA CFC 900 mcg/d or placebo. HPA function was assessed by the short cosyntropin test at baseline and after six weeks of treatment. A (I.25 mg dose of cosyntropin was administered intravenously and serum cortisol levels were measured 30 and 60 rain. later.

Group Mean Change in 1 hr. Post Cosyntropin Serum Cortisol (Wk 6 minus Baseline) TAA TAA TAA TAA Placebo CFC 900 HFA 450 HFA 900 HFA 1350

Mean (mcg/dl) n p-value

-0.13 23 --

-2.42 22 0.0461

1.67 22 0.1283

2.40 20 0.0521

-2.01 18 0.0951

All doses of TAA caused a drop in post cosyntropin cortisol levels, although only marketed TAA was statistically significant (p < 0.05) vs. placebo. No patient had a negative response to cosyntropin indicating that the mean changes may be clinically irrelevant. No differences appeared among the active treatment groups. In conclusion, the reformulation of TAA with HFA-134a

Inhaled Corticosteroid prescribing in very young children. AJ White l and DH Richards,: IUniversity of Southampton, UK; 2Glaxo Wellcome plc, Uxbridge, UK Inhaled corticosteroids (ICS) are the drug of choice to treat allergic inflammato~ conditions such as asthma although their use in paediatric patients has always been approached more cautiously, perhaps due to concern over side effects. We present data collected using a structured questionnaire from 687 doctors (64% primary care physicians, 19% respiratory physicians and 17% paediatricians) in France, Holland, Italy, Spain and the UK. The completed questionnaires provided comprehensive data on the next ten patients who presented to the physician with symptoms of asthma/obstructive airways disease. Of the 6782 patients who were recruited, 4194 had a diagnosis of asthma, 812 (19%) of whom were aged 8 or under. Table I. ICS use in patients (Pts) with asthma (dose is the total r e c o m m e n d e d daily dose of ICS)

Age range (years)

1 to 4

Tot. asthma Pts (n) No. of asthma Pts taking ICS (%) Mean dose (Ixg) range (ug)

368 184 (50%) 353 25-4000

5 to 8

1 to 8

444 812 222 406 (50%) (50%) 331 342 50-1500 25-4000

All ages

4194 2393 (57%) 428 25-4000

The data show that 5(1% of children with a diagnosis of asthma aged up to 8 years old were taking ICS. This was seen in both the children aged 1-4 years and those aged 5-8 years. ICS are not thought to be prescribed frequently to very young children, those aged 1-4 years. However our data seem to suggest the contrary. They show that ICS are prescribed to children aged 1-4 years with the same frequency as to children who are twice as old, at similar doses.

$326

Abstracts

J

ALLERGYCLIN IMMUNOL JANUARY 1997

1331

Comparison of the effects of beclomethasone 17-propionate (BP) and fluticasone 17oL-propionate (FP) on allergen-induced activation of PBMC from atopic asthmatics. N.Powell, S J Till, A B Kay & C J Cotrigan. London, UK Background: BP (administered as the dipropionate prodrug) and FP are effective topical corticosteroids in routine use for the treatment of atopic asthma. We hypothesised that this was partly attributable to the inhibition of secretion of pro-eosinophilic cytokines from allergen-specific T-cells Objectives: To evaluate the relative potency of BP and FP at inhibiting proliferation and cytokine expression (IL-3, IL-5, GM-CSF) by PBMC from Der p-sensitive atopic asthmatics in response to specific allergen in vitro. Methods: For cytokine production, PBMC isolated from 6 sensitized atopic asthmatics were stimulated at 5 • 106 cells/ml with 2.5 ~,g/ml of Der p and IL-3, IL-5, GM-CSF were measured in culture s/n on day 6 by ELISA. For proliferation, PBMC were stimulated at 0.5 • 106 cells/ml with 25 txg/ml Derp and cellular proliferation measured on day 7, by 3H-thymidine incorporation. All cultures were performed in the presence of serial log dilutions of BP and FP (10-6M to 10-12M, or drug vehicle control). Results: BP and FP inhibited allergen-induced T-cell proliferation and cytokine secretion in a dose dependent fashion. FP was considerably more potent than BP (IDso 0.1 and 10nM respectively for IL-5 secretion) Conclusions: BP and FP suppress the elaboration of eosinophil-active cytokines from allergen-specific T-cells from sensitized atopic asthmatics in vitro. This may be relevant to the clinical efficacies of these drugs. FP is considerably more potent in this regard than BP.

1332

Oropharyngeal Complications of Budesonide (BUD) Inhalation Via The Turbuhaler (TBH) or Pressurized Metered Dose Aerosol With Nebuhaler (NEB) Spacer. JH Toogood, F White, J Anderson, J Baskerville. London Health Sciences Centre and University of Western Ontario, London, Canada. Problem: The potential of the TBH dry powder inhalation device for increasing the incidence of oropharyngeal complications with BUD therapy requires assessment because the TBH does not possess the particle trapping action of the NEB. The latter has been shown to reduce BUD's oropharyngeal deposition and associated complications in comparison with the presstirized aerosol without a spacer. (AM REV RESP DIS 1984;129:723-9). Method: Asthmatic adults inhaled BUD twice daily, 0.4, 0.8, 1.6, 2.4 mg. per day, two weeks at each dose level, in an 8 week randomized, open, parallel groups trial comparing the TBH(n=30) vs NEB(n=28). Every two weeks the oropharynx was inspected, swabbed and cultured for Candida. Symptoms and nystatin usage were recorded in a daily diary. Results: The Candida colony count increased with BUD daily dosage (p=.0007, ANCOVA). There was no difference between the TBH vs NEB devices (p=.29). Clinically identifiable thrush did not occur. Voice huskiness and sore throat were less frequent with the TBH: averaging 0.9 and .39 days per week respectively in the TBH group, and 0.99 and 1.8 days per week in the NEB group. Conclusion: The TBH does not increase the incidence or severity of the oropharyngeal complications of BUD relative to the pressurized aerosol plus NEB spacer. It is not known to what extent this would also hold if the BUD were administered more frequently eg. QID.

1333

Salmeterol Safe Compared to Current Treatments as Measured by Electrocardiography. BE Swearingen, GL Burke, KA Rickard, Glaxo Wellcome, Inc., Research Triangle Park, NC, The Bowman Gray School of Medicine, Winston-Salem, NC This 44-week, multicenter, randomized, double-blind, placebo-controlled study compared the cardiovascular safety of salmeterol (S) 42meg, b.i.d, to placebo (P) plus current asthma therapy (CT) and to P plus reduced CT in 405 patients (206 male). Patients (pts) with persistent

asthma requiring at least two prescription asthma treatments in addition to any inhaled corticosteroid therapy and demonstrating reversibility of ->15% were enrolled into the study. Following an initial randomization to S or P, pts were randomized to wean (W) or not-to-wean (NW) non-steroid CT. The resulting four groups were: P + W (n = 128), P + NW (n = 128), S + W (n = 123), and S + NW (n = 26). All pts were supplied albuterol for treatment of acute symptoms. ECG tracings were recorded at baseline, after 17 weeks, and at study conclusion. These tracings, blinded to treatment allocation, were evaluated after study completion by two cardiologists not associated with the study. There were no differences among treatment groups with respect to ECG changes from baseline. Fifteen (15) patients experienced unfavorable changes from baseline [P + W:n=4(3%),P+NW:n=7(5%),S+W:n=4(3%), S + NW: n = 0] while eight (8) pts had favorable changes [P+W:n=2(2%),P+NW:n=3(2%),S+W:n= 3 (2%), S + NW: n = 0]. Of the fifteen (15) pts experiencing an unfavorable change from baseline, six (6) pts had ventricular premature contractions (P + NW: n = 2, S + W: n = 4). Five (5) pts had supraventricular ectopics (P + W: n = 1, P + NW: n = 4). One (1) pt had Twave inversion (P + W: n = 1). Three (3) pts had prolonged QTI (P + W: n = 2, P + NW: n = 1). We conclude that no differences in ECG abnormalities exist between salmeterol and current asthma therapies.

1334

Comparison of Four Portable Compressor/Nebulizer Symptoms. D T Loffert, PARI Respiratory Equipment, Richmond, VA. Five replicates of 4 commercially available portable compressor/nebulizer systems from 3 sources were studied: (DeVilbiss Traveler, Omron Micro-Air, PARI Dura-Neb 2000 and PARI Walkhaler). Each compressor was operated with the nebulizer included by the manufacturer. We compared Delivery rate (M1/Min), Percent Output in the Respirable Range (PORR), and Respirable Particle Delivery Rate (RPDR). All nebulizers were filled with 2.5 ml of saline. PORR was measured by continuous sampling by Malvern MasterSizer X Laser analyzer. The nebulizers were sampled at a simulated inspiratory flow rate of 20 liters per minute. M1/Min varied from 0.10 to 0.35 ml/min. The Traveler (0.10) had the lowest ml/min while the Dura-Neb 2000 (0.35) had the highest. PORR varied 37.25% to 56.89%. The Walkhaler (56.89%) had the highest PORR while the Micro-Air (37.25%) had the lowest. To combine these characteristics we calculated (RPDR) = M1/Min multiplied by PORR. Mean RPDR was compared between nebulizers by analysis of variance. The Dura-Neb 2000 (0.20ml/min) had the highest RPDR while the Traveler (0.04ml/min) had the lowest (means significantly different at p < 0.0001). We conclude that the M1/Min, PORR, and RPDR of the commercially available portable compressor/nebulizer systems vary greatly. These factors affect the treatment time as well as the total dose of drug delivered.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1335

Abstracts

double blind study was approximately 1 year and the open label extension is finishing its third year. The subjects were monitored by FEV 1, symptom scores, maintenance dose of oral steroids and the number of hospitalizations for asthma. Subjectively, the 11 asthmatics who remained in the study felt a marked reduction in their asthma symptoms, as well as an improved quality of life. Their rate of hospitalization for asthma decreased from 1 admission/patient/year to 0.1 admissions/patient/year, representing a 10 fold reduction. The mean FEV1 for 13 of the 14 patients was 1.64 pre-BT and 1.93 while on BT. representing a 29% improvement. The mean dose of prednisone for the 13 subjects was 20 mg/D pre-BT and 0.77 rag mg/D afterwards. One subject remains on daily oral prednisone, but the dose has been reduced from 45 mg/D to 10 mg/D. Ten subjects were on a mean dose of 570 mg/D theophylline before the study. Four subjects remain on theophylline after three years in the open label extension, for a mean theophylline dose of 170 mg/D/subject. Three subjects were dismissed from the study, with 2 of them having a long enough trial to observe benefit from BT. The first subject was noncompliant and there is no data for this subject. The second subject was started on another steroid inhaler by an outside physician. The third subject had frequent URTIs, Despite being dropped from the study, two of these subjects did show improvement in their FEV t 131% & 120%) while on BT and both were weaned from oral prcdnisone. Budesonide Turbuhaler| a topically potent inhaled glucocorticosteroid appears to be effective in this small number of steroid-dependant subjects. The improvement consists of decreased or elimination of systemic steroid use as well as decreased theophylline use, improved FEVt and marked reduction in hospitalization for asthma.

Evaluation of the effect of inhaled [~ 2-agonist in children with asthma and age-matched controls. H Mochizuki, M

Shigeta, H Arakawa, M h~to, K Tokuyama, A Morikawa, Department of Pediatrics, Gunma University School of Medicine, Maebashi, Gunma, Japan To evaluate the effect of inhaled 13 2-agonist against bronchoconstriction, we studied [3 2-agonist-induced decrease in respiratory resistance (Rrs) during methacholine inhalation challenge using oscillation technique. Three hundred and eighty five atopic children with asthma (mean, 10.5 years) and 24 age-matched disease controls (mean, 9.1 years) were subjected to methacholine inhalation challenge. After inhalation challenge, we calculated parameters in dose-response curve, that is, the linear slope of Rrs increased (St), and the linear slope of Rrs decreased after inhalation of salbutamol (r-St). In asthma and control subjects, r-St was correlated with St (p < 0.001, p < 0.001, respectively), r-St in asthma was lower than that in controls, though there was no significant difference between St value in asthma and that in controls. These data suggest that the effect of inhaled 13 2-agonist is related to bronchial reactivity (St), and inhaled [3 2-agonist is less effective in asthmatic children than in control subjects. 1336

Once-Daily Evening Theophylline in Nocturnal Asthma Patients on Inhaled Steroid and {3-Agonist. WW Busse, R Dockhorn, K HaSher, L Sandstrom, PG Lacouture, U of Wisconsin, Madison, Wl; IMTCI, Lenexa, KS; Purdue Frederick, Norwalk, CT. To determine whether an additive effect of theophylline was achieved in nocturnal asthma with inhaled steroid and 13-agonist a double-blind, placebo-controlled, crossover trial was conducted at two centers. Subjects with >3 nocturnal awakenings/wk, an overnight decrease in peak flow ->20% and current treatment with inhaled steroid and i3-agonist were enrolled. A 14-day titration with once-daily evening theophylline (Uniphyl| Tablets) achieved a peak STC of 10-20 mcg/ml. After a 7-day washout, subjects received Uniphyl or Placcbo for 28 days, washed out, then crossed over to thc second treatment. Forty-eight (48) subjects were randomized.

Baseline (N = 45)

FEV~ (L) FVC (L) P E F R (L/min)

Day 28 Uniphyl Placebo (N = 38) (N = 42)

ANOVA

2.85 _+ 0.13 3./)7 _+ 0.14 2.86 + 0.14 p = 0.001 3.86 + 0.18 4.04 + 0.18 3.87 _+ 0.18 p = 0.004 438 ~+ 20 487 _+ 28 438 + 27 p - 0.05

In addition, home Daily Peak Flow significantly improved (p < 0.004) with Uniphyl in both AM and PM. Symptoms generally improved with both groups; slightly more with Uniphyl. Two-thirds of adverse events were not related to study medication; haft were mild; headachc (34% vs 14%) and nausea (23% vs 5%) were greater with Uniphyl. Therefore, in patients managed on inhaled steroids and [3-agonists, the addition of once-daily evening Uniphyl provided increased benefits, i.e., significantly improved PFTs and stabilized symptoms. 1337

Long-term Use of Budesonide Turbuhaler| on Steroid Dependent Asthmatics. Led~brd DK, Rosenbach KP, Lockev RF University of South Florida & JA Haley VA, Tampa FL Budesonide is a highly potent, topical corticosteroid with low systemic bioavailability. The Turbuhaler| is an effective dry powder inhalational device which delivers 29% of an inhaled dose to the lower airways compared to MDIs which deliver 11%. Budesonide Turbuhaler| (BT) has been demonstrated to be an effective treatment lk~r steroid dependent asthma. The purpose of report is to discuss the long term experience of glucocorticosteroid dependent asthmatics with Budesonidc Turbnhaler| Fourteen subjects from the double-blind placebo controlled study were recruited to participate in an open label extension of the double blind BT study. The duration of the

$327

1338

Open Label, Home Infusion Trial of High Dose Gammaglobulins in a Group of Inner City, Steroid Dependent Asthamtics. D. Grafino, K. Shah, H. Aguila, M. Santiago, T. Zecca, A. Buggs, G. Jordan, J. Oleske. Children's Hospital of New Jersey, Newark, New Jersey Asthma affects approximately 7% of the general pediatric population. Its prevalence and morbidity are significantly higher in minority inner city children. Compliance with multiple medications, necessary for optimal disease control, is very difficult in this setting and steroid (CS) use is frequently necessary for the management of symptoms. Based on the promising results of previously published studies, we enrolled a group of 6 severe, CS-dependent poorly compliant, minority asthmatic children, ages between 10 and 18 years, in an open label trial of high dose (2 gm/Kg) intravenous gammaglobulines (IVIG) administered at home monthly for 1 year. Consent for the treatment was obtained. Immune function studies and pulmonary function tests were obtained initially and every 3 months until the end of the study. Use of CS, acute exacerbations, E R admissions and a quality of life assessment score were recorded throughout the study. Of the 6 original patients, one dropped out after 4 months without explanations. Steroid use was completely discontinued in 3 of the remaining patients, with no change in CS use in the others. The FEV1 improved by more than 20% in 4 of 5 patients, and normalized in 2 of 5. Subjective improvement was noted in of 5 of 6 patients within 6 months of treatment. Home infusions allowed excellent compliance with the IVIG without loss of school days. No severe acute reactions were reported. We conclude that high dose W I G can be useful in the management of some steroid dependent, poorly compliant inner city asthmatic children and that home infusion can be a cost saving and safe alternative to in hospital infusions.

$328

1339

1340

Abstracts

A Comparative Study of the Efficacy and Administering Technique of Salbutamoi Delivered From Conventional Metered-Dose Inhaler and Breath-actuated Device in Asthmatic Children. Nganthavee W,, Vichyanond P, Visitsunthom N, Tuchinda M, and Udomsubpayakul U, Mahidol University, Bangkok, Thailand. Background: Breath-actuated device such as the Autohaler has been proposed to be simpler for use and at least as effective as metered-dose inhaler (MDI) in asthmatic children aged 6 years and over. Objective: To assess bronchodilatory effect of salbutamol delivered from the Autohaler compared with MDI and placebo MDI in patients with mild to moderate asthma between the age of 6-14 years and to assess the techniques of using Autohaler and MDI. Methods: In a randomized, double-blind, placebo-controlled, crossover study, the effects on lung functions from salbutamol (200 Ixg) delivered via Autohaler or MD1 were assessed in 21 patients at 0, 15, 30, 45, 60, 90 minutes 2, 4, 6 hours on 3 separate days. The baseline FEV~ from the three study days were within 20% variation from one another. Prior to the study, the children were taught correct methods for use of MDI and Autohaler until they could demonstrate the correct techniques for both devices. These techniques were subsequently evaluated by the investigator during the following three successive days. Results: Mean percent improvement over the baseline of FEV~, FVC, FEFzs_75%, PEF via Autohaler were generally greater than those via MDI and placebo, but were only statistically significant for FEVI, FVC (Autohaler greater than MDI) at 30 minute time point (p < 0.05). No difference was observed between the three treatments (Autohaler, MDI, placebo) after 4 hours administration of salbutamol indicating the effect of salbutamol (200 ug) began to decline 4 hours thereafter. Area under bronchodilation-time curve of FEV1, FEF, s_~s%, from Autohaler were greater than that of placebo (p < 0.05), and Autohaler was greater than MDI, but did not reach statistical significance. Area under bronchodilation-time curve for FVC, PEF did not differ between treatments. Coefficient of variations for use of MDI was greater than from that of Autohaler (21.79% versus 13.10%) indicating MDI methods are more difficult to accomplish than Autohaler. The total mean technique scores for using Autohaler was greater than MDI, however this did not reach statistical significance (p > 0.05). Conclusion: Salbutamol via Autohaler or MDI provided a significant bronchodilatory effect in children with mild to moderate asthma. There is a propensity for a greater bronchodilatory effect from salbutamol given via Autohaler than via MDI. The administering techniques of using of Autohaler were more simple and children exhibited less error with Autohaler than with MDI. Comparing The Safety Of Generic (GEN) Versus Standard (STD) Antiasthmatic Inhaled Steroids (I-S). JH Toogood, J Baskerville, AB Hodsman. London Health Sciences Centre and Lawson Research Institute, London, Canada. Problem: There is an urgent need for a standardized assay procedure to compare the systemic toxicities of GEN versus STD formulations of I-S such as beclomethasone or budesonide for regulatory purposes. To estimate the assay's statistical power and sample size requirements, agreement is required as to what difference between the systemic activities of the test drugs is deemed to be "clinically important". Method: We measured bone mineral density (BMD) by densitometry in a cross sectional survey of 69 asthmatic patients (mean age = 59.9 years _+ 13.3 SD) treated with I-S 10.1 years _+ 5.5 and prednisone (10.7 years _+ 9.7). Controlling for age, sex, physical activity, prednisone and estrogen exposure, we identified a linear decline of BMD on the daily dose of I-S (p = .013) (JACI 1995;96:157-66). We compared the regression of BMD on I-S dosage with published estimates of the risk of fracture as a function of declining BMD (Drugs & Aging 1993;3:391-9). Result: This indicated that a 20% excess in the assayed toxicity of the GEN over the STD drug would imply a 10 20% increase in the lifetime risk of fracture at I-S doses of

J ALLERGY CLIN IMMUNOL JANUARY 1997

1,0 and 2.0 mg. per day, respectively. In our clinical practice 34% of adult asthmatics require >1.0 mg/day (JACI;1992: 89(2):186). Conclusion: Given the importance of osteoporosis as a complication of long term steroid usage, such an increment could reasonably be considered to signify a "clinically important" increase in risk. We propose a _+ 20% confidence limit to demarcate "clinically important" differences between the systemic potencies of GEN vs STD I-S formulations, as determined from relative potency studies.

1341

Intravenous Pranlukast (Ultair TM) Inhibits LTD4-Induced Bronchocnnstriction in Patients with Asthma. LJ Smith, DK Jorkasky, A Carr, SC Boike, Northwestern University, Chicago IL, and SmithKline Beecham Pharmaceuticals, Phila., PA The effect of pranlukast (Ultaiff M, SB 205312, ONO1078) on LTD4-induced bronchoconstriction was examined in seven patients with mild to moderate asthma in a double-blind, randomized, four period crossover study. Daring each treatment period (periods separated by at least a one week wash out) patients received an intravenous infusion of placebo or one of three active total doses of pranlukast: 10 mg, 30 rag, or 60 mg given as a 30-minute loading infusion followed by a two-hour constant rate infusion. Bronchoprovocation testing with increasing doses (0.5 to 1000 ug/mL) of inhaled LTD4 was conducted at approximately one hour following the start of the infusion of pranlukast or placebo. The inhibitory effect of pranlukast or placebo on LTD4-induced bronchoconstriction was measured by PC2oFEV 1 (the interpolated concentration of LTD 4 causing a 20% decrease in FEV1). The shift in PC20FEV 1 from placebo associated with administration of each dose level of pranlukast was estimated as the ratio of the adjusted geometric means for pranlukast/placebo. During intravenous dosing, with pranlukast, PCz0FEVI was increased in a dose-related fashion relative to placebo:

Dose (rag) 10 30 60

Shift in PC2oFEV1 (Multiple of placebo effect) 95% CI 12.83 27.72 100.02

5.71,28.81 12.34, 62.23 44.54, 224.58

These results demonstrate that intravenous administration of pranlukast blocks the effects of inhaled LTD4 in a dose-dependent fashion, and confirm previous results using oral pranlukast.

J ALLERGYCLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2 1342

Abstracts

Pranlukast (Ultair'") Inhibits Cold Air-Induced Bronchoconstriction in Patients with Asthma. ME Strek, J Sedy, J Solway, DK Jorkasky, B Jones, SC Boike, University of Chicago, Chicago, IL and SmithKline Beecham Pharmaceuticals, Philadelphia, PA The effect of pretreatmcnt with pranlukast (Ultair'", SB 205312, ONO-1078), a leukotriene D 4 (LTD4) receptor antagonist, on the bronchospastic response to hyperventilation of cold air was assessed in a double-blind, randomized, two period crossover study. Nonsmoking patients with mild to moderate asthma received pranlukast 450 mg or placebo orally twice daily ior six days with a single dose on the seventh day of each study period. There was a washout pcriod of at least one week between study periods, Isocapnic hyperpnea of cold air was performed in patients (n= 12) at four hours after the morning dose on Day 1 and Day 7 of each treatment. On average, praniukast increased PDe~V~ (the minute ventilation causing a 20% decrease in FEVt) on Day 1 and Day7 relative to placebo.

1344

Point Estimate

95% CI

Day 1 Day 7

23f/r 34%

4%, 44% 2%, 75%

hi vitro IgE production was inhibited in a dose dependent fashion by TGF-~ in the presence of IL-4. The effect of TGF-[3 was partially overcome when cells were cultured with anti-CD4(/and IL-4. By contrast, lymphocyte proliferation after 5 days was only partially suppressed after the addition of TGF-[3 to IL-4 and/or IL-4 plus anti-CD40 stimulated cultures. TGF-[3 concentrations of 100 ng/ml only produced 50% suppression. In conclusion, TGF-[3 may be an important regulator of in vitro lgE synthesis and lymphocyte proliferation.

A similar effect on respiratory heat loss was noted. These results indicate that pranlukast protects against cold air induced bronchoconstriction and further define the activity nf this oral leukotriene receptor antagonist.

1343

A Randomized, Double-Blind, Placebo-Controlled, DoseResponse Study with Formoterol Turbuhaler| J.P. Kemp, P. Chen,insl,~, H. Nelson, Allergy and Asthma Research, San Diego, CA.. Allergy Associates, North Dartmouth, MA., National Jewish Center for Immunology and Respiratory Medicine, Denver, CO. Methods: Following a one week baseline screen period, formotcrol fumarate dihydrate was administered via Turbuhalcr to three groups of patients at 6, 12 or 24 mcg BID respectively, and compared to a placebo Turbuhaler group for one week. Serial spirometry, vital signs, 12 lead ECG, and serum potassium and glucose before and after dosing for up to 12 hours were completed at the start of treatment (Day 1) and end of treatment (Day 8). Peak expiratory flows, symptom scores and use of rescue medication were measured during the baseline and treatment week. Holter monitoring was also completed during the baseline and treatment phase. A 15% reversibility or greater was demonstrated by patients before randomization. Results: A total nf 165 patients were randomized and 156 completed trcatmenl. Mean age was 31 years and baseline FEV~ was 2.3L (66% predicted). The 12-hour average area was statistically significantly improved for all doses of formoterol compared with placebo on both test days. Median time to onset of response, defined as a 15% increase in FEV~ over baseline, was within 3 rain. for all formoterol groups. Median duration of response was 10.2 hr. (6meg), 9.5hr. (12meg) and 11.8 hr. (24mcg). Mean increases in morning PEF were statistically significantly greater in all three formoterol groups compared to placebo, showing remaining effects 12 hr. after drug intake. Symptom scores and use of breakthrough therapy were statistically significantly improved and reduced, respectively, for all doses of formoterol. There were no clinically relevant differences between the three formoterol groups and placebo for 12-lead EGCs, Holter recordings, vital signs, potassium, glucose, or laboratory values. The most frequent adverse events were headache and tremor. No serious adversc events occurred, Conclusions: A rapid onset and long duration of response was observed with all three doses of formoterol Turbuhaler. A significant difference between doses could not be detected. All three doses were well tolerated.

TGF-[3 as a Regulator of in vitro IgE Synthesis. KJ Nastasi, WR Valenski, and HG Herrod. Crippled Children's Foundation Research Center, UT, Memphis, TN A number of factors such as IL-4, IFN-y, and TGF-[3 have been reported to influence lgE production. Previous studies by Armitage et. al. using tonsillar B-cells stimulated with IL-4 and CD40 ligand showed suppression of IgE synthesis after the addition of TGF-~. We report the effect of TGF-[3 on itl vitro IgE synthesis by peripheral blood mononuclear cells (PBMC) in 10 studies from 6 donors. We also report the effect of TGF-[3 on PBMC proliferation in 2 donors. PBMC were cultured with 1L-4, anti-CD40, IL-4 plus anti-CD40, IL-4 with TGF-13, and IL-4 plus anti-CD40 with TGF-~ for 10 days. The results of in vitro IgE synthesis are illustrated in the table below as percent suppression after the addition of varying concentrations of TGF-[~ in ng/ml:

TGF-[3 .01 .05 .1 .5 1 5 l0 50 100 [L4 2.3 5.6 27 62 74 89 84 97 100 IL4+ o~CD40 - 6 20 25 42 56 60 62 74 72

Average increase in PD2oVE relative to placebo Timepoint

S329

1345

Interleukin-10 Regulates Cytokine Production and Inflammation in a Murine Model of Allergic Bronchopulmonary Aspergillosis. G Gmnig I, B Seymour I, V Kurup 2, MW LeacM, DB Cor~y4, D RennicU; ~DNAX Research Institute, Palo AItn, CA; 2VA Medical Center, Milwaukee, W1; ~Schering-Plough Research Institute, Lafayette, N J; 4Department of Medicine, University of California, San Francisco, CA. The role of IL-10 in T helper 2 responses is still controversial. However, because IL-10 suppresses inflammatory reactions, it may be of therapeutic interest in allergic airway diseases. We sensitized ILIOKO and WT mice with Aspergillus filmigatus antigens intranasally (i/n) for 3 to 5 times or intraperitonealty (i/p) for 4 times and once i/n. Mice were from the outbred (C57BL/6 x 129 Sv) or C57BL/6 genetic backgrounds. The absence of IL-10 had the most dramatic effect in outbred mice. Fifty 60% of the IL10KO outbred mice died after the third i/n sensitization while mortality was rare in WT mice. Compared to WT outbred mice, bronchoalveolar lavage (BALl fluids of i/n sensitized IL10KO mice had much higher levels of IL-5 and IFN-y. However, lung lesions were induced to a similar degree nf severity in both groups of mice suggesting that mortality in IL10KO outbred mice was related to the overproduction of cytokines. C57BL/6 IL10KO mice sensitized i/p and once i/n had remarkably exaggerated responses as well. Their lung lesions, BAL eosinophilia, and BAL-IL-5 levels were greatly increased compared to WT mice. In contrast, WT and IL10KO C57BL/6 mice sensitized i/n had similar lung lesions, BAL-eosinophilia, BAL cytokinc levels (1L-4, IL-5; IFN-y levels were negligible), total serum IgE titers, and airway hyperrcactivity. In conclusion, our studies suggest that depending on the genetic background and the route of sensitization, IL-10 plays a major role in downregulating cytokine production and inflammation elicited byA~spergilh~sfitmigatus antigens.

$330

Abstracts

J ALLERGYCLIN IMMUNOL JANUARY 1997

1346

Expression of Th2-type cytokine transcripts by human buccal epithelial cells. Smith JK Dykes R, Krishnaswamy G, Joyner W, Berk SL. East Tennessee State University, Johnson City, Tennessee. Although it is known that epidermal epithelial cells secrete a variety of cytokines, little attention has been given to cytokine production by buccal epithelial cells (BEC), an important component of the mucosal barrier. Using reverse transcriptase polymerase chain reaction (RT-PCR) we have examined cytokine mRNA transcripts of 25 BEC samples taken from healthy donors. After oral rinsing with phosphate buffered saline (PBS), BEC were collected by gently rubbing the buccal mucosal with sterile dacron swabs and dispersing the samples in sterile siliconated tubes containing 10 ml of PBS. Samples were washed three times with PBS containing penicillin (50 U/ml) and gentamicin (50 ~g/ml). BEC preparations containing ->2 percent mucosa[ associated mononuclear ceils were further processed by density gradient separation on Percoll, which yielded ->98% purity at the 10-30% interface. BEC samples containing 5-10 • 105 cells were then analyzed for IL-2, IL-4, IL-5, IL-6, IL-8, IFN-% GM-CSF and TNFc~ mRNA transcripts by RT-PCR using beta actin as a housekeeping gene. Eighty-eight percent of BEC samples expressed IL-8, 74% IL-5, 20% GM-CSF, 12.5% IL-4, and 4.2% IL-6. Absent from samples were IL-2, IFN~/ and TNF~ transcripts. We conclude that BEC constitutively express IL-5 and IL-4, Th2-type cytokines potentially capable of enhancing type 1 hypersensitivity reactions. BEC also regularly express the pro-inflammatory cytokine, IL-8, and, less commonly, GM-CSF and IL-6. Thl-type cytokine transcripts (IL-2 & IFN-~,) and TNFc~ were not expressed.

1347

Reduced IL-10 Secretion by T Cells Expressing Mutant CFTR: A Role for Ca 2+ and Cl- Channels. RB Moss, Y Hsu, L Olds, Stanford Univ Medical Ctr, Stanford CA We have reported (RB Moss et al, Clin Exp Immunol in press) that CD4+ T cell clones [TCC] from controls and CF patients with ~F508 mutations transcribe CFTR mRNA, express cytoplasmic CFTR and display equivalent Ca2+-mediated C1- current; however, TCC from patients with CF but not controls display defective cAMP-mediated C1 current. CF-derived TCC preserved mitogen and antigen proliferative responses but selectively secreted - 5 0 % less IL-10 compared to control TCC after activation with concanavalin A (p = 0.04) or anti-CD3/phorbol ester (p = 0.05). This difference was independent of atopy. Secretion of interferon-% IL-2, and IL-4 was comparable after both forms of activation, while IL-5 was reduced in CF TCC following anti-CD3/PMA. We hypothesized that enhanced Ca 2+ entry on T cell activation due to defective CIchannel-mediated regulation of Ca 2+ entry could account for this selective IL-10 secretory defect. By FACS analysis of cellular IL-10 production, CF TCC showed reduced sensitivity to Ca 2+ ionophore/phorbal ester. Secretion of IL-10 from PBMC after LPS activation was inhibited by the C1- channel inhibitor DIDS, while transcription of IL-10 mRNA and cytoplasmic IL-10 production was unaffected. Mutant CFTR may down-regulate secretion of IL-10 in CF cells via CI- channel-dependent regulation of Ca 2+ influx after activation, which uniquely affects IL-10 secretion.

1348

Development of an in vitro cell culture system to examine cytokine mechanisms in inflammatory lung disease using ex vivo lung inflammatory cells derived from premature infants with hyaline membrane disease (HMD). KY Kwong, CA Jones, C Lecart, N Khuu, P Minoo, and R.A. deLemos, Los Angeles, California. Mechanisms of cytokine mediated lung inflammation have often been proposed based upon in vitro manipulations of distinct cell lines. We report the successful development of an in vitro cell culture system, using hronchoalveolar lavage derived lung inflammatory cells, from premature infants with HMD. Lung inflammatory cells (predominantly macrophages and neutrophils) were obtained from premature infants with HMD, placed in complete media and incubated for 24 hours in 21% 02 and 5% CO z. Lung inflammatory cells ex vivo, continued to pro-

duce proinflammatory cytokine protein (IL-lb, IL-8 and TNFa). In addition they bad the capacity to respond to exogenous lipopolysaccharide (LPS) with increased expression of TNFa, IL-lb, IL-8 and IL-10 protein as compared to culture in complete media alone. Exogenous IL-10 co-cultured with LPS stimulated ex vivo lung inflammatory cells down regulated IL-lb expression in a dose dependent fashion and IL-8 at higher concentrations. Anti TNFa and anti IL-lb antibodies co-cultured in a similar fashion inhibited the expression of IL-8 in the lung inflammatory cells in the presence of LPS stimulation. Cell survival post 24 hours culture cultured in complete media only and co-cultured with other factors as described were between 50-60%. The various cytokine responses observed in our system were consistent with those reported by other investigators using distinct in vitro cell lines. This cell culture system provides a unique opportunity to manipulate lung inflammatory cells ex vivo, derived from an in vivo model of lung inflammation (HMD); and hence may provide a more accurate insight into in vivo mechanisms involved in inflammatory lung diseases.

1349

Schistosoma mansoni egg antigen (SEA) triggers the release of IL-4 from basophils of naive human donors in an IgE-mediated way. H Haas, FH Falcone, B Gibbs* U

Amon* M Schlaak Forschungszentrum Borstel, Borstel, *University Lfibeck, Ltibeck, Germany We have recently shown that basophils from nonimmune human donors release IL-4 after stimulation with SEA. Since this effect was abrogated by low-pH stripping of surface-bound IgE from the basophils and was reconstituted by resensitizing the cells with stripping supernatants or serum, we supposed SEA-induced IL-4 production was mediated via IgE. To prove this supposition, IgE was purified from human sera using anti-IgE sepharose and employed for resensitization of stripped basophils. As expected, IL-4 was induced, when the eluate fraction was used for resensitization. In contrast, the effluent fraction had no effect. Considering that IL-4 production is induced by crosslinking lgE bound to the high-affinity IgE receptor on basophils, we conclude that SEA induces IL-4 production by crosslinking receptor-bound IgE. It is unlikely that the interaction between SEA and IgE is antigen-specific, since IL-4 was released from the basophils of all donors (n >50), whilst none of the donors had ever suffered from schistosomiasis. We would rather favour a nonantigenspecific--e.g, lectin-like--interaction between SEA and IgE.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

Abstracts

1350

Effect Of Theophylline On lnterleukin 13 Production By Allergen-Stimulated Mononuclear Cells of Asthmatics. G Sansonc, S Lai. S Wang, S Rockitter, M Frieri, Nassau County Medical Center, East Meadow, NY Nitric oxide (NO) plays a key role in many inflammatory conditions, including asthma. We have recently reported that in vitro peripheral bMod mononuclear cells (PBMCs) release No and that theophylline (15 Ixg/ml) significantly reduces this spontaneous production of NO (J Allergy Clin Immunol 97:255, 1995). lntcrleukin (IL)-13, a novel T cell-derived cytokine, also appears to inhibit NO secretion both in animal and human models. Thus, the aim of the study was to investigate whether thcopylline (5-15 ~tg/ml) could affect the production of IL-13 in PBMCs of asthmatics and whether a functional relationship between NO and IL-13 existed. A total of 12 subjects were evaluated: 5 mild-to-moderate asthmatic patients, as assessed by clinical symptoms and skin test (ST) reactivity, and 7 normal mm-atopic controls. Following 6% dextran sedimentation, PBMCs were isolated by Ficoll-Hypaque centrifugation and werc incubated at a dcnsity of I • 1(1"cclls/ml. After a 72-hour incubation, culture supernatants were collected and assayed for NO and IL-13 levels using Gricss reaction and ELISA. Both levels of NO and IL-13 wcrc significantly elevated in asthmatics compared to normal controls (0.76 • (l. 17 IxM vs. 2.72 % 0.83 btM and 39.89 + 4.39 pg/ml vs. 56.61 • 12 pg/ml, p < 0.05). In asthmatics, all doses of theophylline failed to affect IL-13 production whereas, on the contrary, thenphylline (15 p~Jml) significantly reduced the production of NO from 2.72 + 0.83 p~M to 0.74 + 0.18 I~M (p < 0.05). No correlation was found between NO and IL-13 levels. These results suggest that theophylline blocks the release of NO by PBMCs of asthmatics independently from I L- 13 production.

1351

Giucocorticoids inhibit cytokine secretion and proliferation by T-cells. IC Crocker, S Newton, RG Townls Creighton University, Omaha, NE T-helper type 2 (TH2) cells, which secrete a range of cytokines, are known to orchestrate the inflammatory response which characterizes atopic disease. As glucocorticoids have a variety of effects on T-cell lhnction, this study was undertaken to determine the effects of mometasone furoate (MF), beclomethasone dipropionatc (BDP), and hydrocortisone (HC) on proliferation of PBMC and cytokine secretion by cultured TH2 cell lines. Five TH2 cell lines were established by continuous culturc of PBMC from atopic donors with specific aero-allergen. Ttt2 cells were pre-treated with 10 ~ to 10 I~'MMF, BDP, or HC for 24 h. Afterwards, 1%: PHA was added and the cultures continued for 48 h. The media was harvested and analyzed for cytokincs by ELISA. To assess the effects of the glucocorticoids on lymphocyte proliferation (n = 5), fresh PBMC were incubated with either He, BDP, or MF for 24 h. PHA (0.1%) was added as a stimulus for 48 h and proliferation detected by [SH] thymidine incorporation. Glucocorticoids inhibited secretion of cytokines and proliferation in a dose-dependent manner without diminishing cell viability. MF was signilicantly more potent than BDP, which was more potent than HC. IC-50

Test IL-4 IL-5 Proliferation

Hydrocortisone Beclomethasone Mometasone 1.(7 I~M 1.8 btM 220 nM

32 nM 38 nM 6 pM

<100 pM <100 pM <100 pM

In conclusion, MF, BDP, and HC inhibit cytokine secretion and proliferation, but MF is much more potent, suggesting that MF may be more effective in the treatment of allergy than BDP or HC.

S331

1352

The Flavonoid Quercetin Induces Anti-inflammatory Cytokine (IL-13) and Inhibits Pro-inflammatory Cytokine (TNFo0 Gene Expression by Normal Peripheral Blood Mononuclear Cells (PBMC). Nair MPN, Hou J, Sweet A, Kandaswami C, Middleteon E Jr., Schwartz SA. Dept. of Medicine, State University of NY at Buffalo, Buffaln, NY 14203 USA. The flavonoids comprise a large class of low molecular weight secondary plant metabolites, which are known to exert significant anti-tumor, anti-allergic and anti-inflammatory effects. We have recently shown that quercetin, a polyhydroxylated flavonoid, significantly inhibited lipopolysaccharide induced tumor necrosis factor (TNFc0 production by normal PBMC. IL-13 is a recently cloned cytokine secreted by activated T cells and is known to suppress several inflammatory cytokines such as IL-I, IL-6 and TNFcc In the present studies we investigated the effect of quercetin on constitutively expressed TNFc~ and IL-13 gene expression by normal PBMC. PBMC cultures received either media alone, or different concentratkms of quercetin (0.1 to 100 b~M). After 24 hr of incubation, total RNA was extracted and reverse transcribed. The newly synthesized cDNA products were mixed with the house keeping gene, G3PDH, primer pair plus a set of TNFcx/IL-13 specific cytokinc primers in the same tube using a semiquantitative polymerase chain reaction (PCR) assay. The PCR products were separated in 1.2% agarose gel containing ethidium bromide. Our results showed that quercetin significantly inhibited TNFc~ gene expression and significantly induced IL-13 gene expression by normal PBMC in a dose-dependent manner. Our results provide direct evidence that anti-inflammatory effects of quercetin may be mediated through the induction of the anti-inflammatory cytokine, IL-13 and inhibition of the pro-inflammatory cytokine, TNFo<

1353

IL-4 and IL-5 Receptor mRNA Expression in Acute and Chronic Atopic Dermatitis. R. ,4. Taha I, D.Y.M. Leung 2, M. Boguniewicz 2, E. Minshall and Q. Hamid. MeakinsChristie Laboratories, McGill University, Montreal, Canada and 2Dept of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, USA Atopic dermatitis lAD) is associated with tissue eosinophilia and the infiltration of CD4-positive T lymphocytes within acute and chronic skin lesions. While an increased expression of IL-4 and IL-5 mRNA has been reported in AD, it is unclear whether these cytokines act locally to influence the pathophysiology of this disorder. The aim of this study was to investigate the expression of IL-4 and IL-5 receptor mRNA in acute and chronic AD compared to uninwflved skill and tissue from normal controls. We have used the technique of in situ hybridization to determine IL-4 and IL-5 receptor mRNA expression in skin biopsy specimens of acute and chronic skin lesions and uninvolved skin from patients with AD (n 5) Acute and chronic skin lesions exhibited a significant increase in the number of IL-4 and IL-5 receptor mRNA-positive cells compared to uninvolved skin, and skin from normal controls (p < 0.05). Chronic skin lesions had a significantly greater number of IL-5 receptor mRNA-positive cells when compared to acute skin biopsies (p < 0.05). The uninvolved skin of individuals with AD showed a slight increase in the numbers of cells expressing IL-4 receptor mRNA when compared to normal skin, hut this failed to reach significance (p > 0.05). In conclusion, these results provide evidence to support a local action of IL-4 and IL-5 in AD.

$332

1354

1355

Abstracts

J ALLERGY CLIN IMMUNOL JANUARY 1997

Atopie Dermatitis in Children: Expression of CD30 in Cutaneous Biopsies and Evaluation of Serum Levels of Soluble CD30. G. Cavagni, *D, Brugnoni, ~F. Facchetti, R. Altobelli, A. Borghi, M. Gardenghi, M. Duse, L.D. Notarangelo, *C Tosoni, *R. Cattaneo, A.G. Ugazio, 1st. Clinica Pediatrica, Servizio di Immunologia Clinica,* Anatomia Patologica lI--Universit/~ di Brescia--Italy. Owing to the involvement of the Th2 cells in the pathogenesis of atopic dermatitis (AD), we have evaluated the expression of CD30 both on cutaneus biopsies and in sera of affected children. In fact CD30 is believed to be the marker of Th2 cells. Soluble low affinity receptor for IgE (sCD23) and soluble Interleukin 2 receptor (sIL2R) were evaluated too. Our study included 10 children with AD in an acute phase. In all children skin prick tests, total and specific IgE and eosinophil count were performed; moreover the ultrastructure investigation was performed. 13 healthy children were evaluated for the same parameters. Results: all the patients had atopic characteristics. As compared to controls, AD patients showed significantly higher levels of sCD30 (52 • 22 U/ml vs 10 • 8; p < 0.003), sILR (4595 _+ 2415 pg/ml vs 2176 _+ 625; p < 0.03) and sCD23 (172 _+ 132 pg/ml vs 47 _+ 32; p < 0.02). These data suggest a strong involvement of both Th2 cells and IgE in AD. In cutaneus biopsies CD30 was expressed in only two patients who showed severe AD and marked eosinophilia. In conch~sion, this may suggest that CD30 + Th2 cells infiltrate the skin of the affected subjects only during active phase of AD. Plasma levels of gammalinolenic acid in children with atopic dermatitis and non-atopics. S Wolf-Abdolvahab, M Focke, W Hemmer, F Wantke, R Bracun, M G6tz, R Jarisch. Dermatologic & Pediatric Allergy Clinic, Vienna, Austria. The aim of the study was to assess whether there are differences in the plasma levels of unsaturated fatty acids (UFA) between children with atopic dermatitis (AD) and non-atopics. Levels of UFA in plasma were determined in 28 patients with AD and in 13 non-atopics serving as controls. The fatty acids (FA) were extracted from plasma samples with methanol and chloroform (1:2), derivatized to cumarin fatty acids, and then determined by high pressure liquid chromatography. Mean levels of gammalinolenic acid (GLA), the proportion of GLA, arachidonic acid (ARA), and linoleic acid (LA) in total FA, and the ratio of LA to ARA were calculated: Atopics GLA GLA/FA ARA/FA LA/FA LA/ARA

4,05 nM/l 0,14% 0,89% 0,79% 0,94

(• (• (• (+0,38) (•

Controls 3,07 nM/l (• 0,13% (• 0,57% (+0,09) 0,91% (• 1,54 (+0,70)

The results did not show significant differences in UFA composition between patients and controls. In some individuals from both groups, however, plasma levels of GLA were very low. The data do not clearly support the hypothesis that a deficiency of GLA is involved in the pathogenesis of AD. Substitution might be effective only in patients with low levels of UFA.

1356

Hypereosinophilia, elevated lgE and Interleukin-5 levels in neonatal atopic dermatitis. JM Spergel, l j Boyce,2 and LC Schneider, 1 Children's HospitaF and Brigham and Women's Hospital, 2 Boston, MA Atopic dermatitis is characterized by severe pruritus, elevated IgE and pathologic skin changes. Skin lesions are characterized by infiltration of activated macrophages and T cells expressing IL-4 and IL-5, consistent with Th2 response, seen in many allergic processes. Here, we report elevated serum IL-5 levels and bioactivity in a six month old male with atopic dermatitis, indicating that IL-5 may

play role locally and systemically in atopic dermatitis with hypereosinophilia. N.D. is a 6 month old male, who presents with severe eczema and failure to thrive. Laboratory analysis and skin biopsies eliminated Histiocytosis X, hyper IgE syndrome or other immune deficiency as a diagnosis. N.D. on presentation had an (AEC) absolute eosinophil count of 12,000 cells/ram3 and IgE of 1381 U/ml. He had strongly positive CAP (Pharmacia) to milk, soy, egg, wheat and peanut. IL-5, detected in his serum by antibody neutralization assay (for eosinophil viability sustaining activity), and ELISA was 22 pg/ml at time of initial evaluation. Treatment of his atopic dermatitis with aggressive skin care and on diet of elemental formula led to a decrease in his AEC and IgE to 1300 cells/ram3 and 773 U/ml, respectively. The clinical improvement with a decrease in eosinophil count was accompanied by diminished IL-5 level by ELISA, to 12 pg/ml. Therefore, serum levels of IL-5 may correlate with the severity of atopic dermatitis with hypereosinophilia and may play a role in pathogenesis of atopic dermatitis with hypereosinophilia by promoting eosinophilopoiesis, eosinophilic survival and activation.

1357

High Dose Intravenous Immunoglobulin ( M G ) in Atopic Dermatitis and Hyper IgE Syndrome. Wakim ME, Alazard M, Yajima A, Speights D, Saxon S, Stiehm ER. Departments of Pediatrics and Medicine, UCLA School of Medicine, LA, CA. We gave patients with HIE (n = 1) or severe AD (n = 9) IVIG as a 10% solution (Venoglobulin l| Therapeutic Corp.) at a dose of 2 grams per kilogram every 30 days for seven months and followed their clinical, laboratory, and immunological parameters. Five patients were on systemic steroids prior to the course of IVIG. Therapy was completed in 9 of the 10 patients; one patient withdrew because of hypertension and transient renal insufficiency. Skin disease improved slightly in 6 patients; remained unchanged in 2 patients, and worsened slightly in 1 patient. The average daily prednisone dosage was 6.8 rag/day prior to treatment and 5.1 nag/day during IVIG therapy (p = .1250). The 3 patients with abnormal pulmonary function showed mild improvement of pulmonary function during treatment, but returned to baseline during follow-up. Flow cytometric studies showed no consistant pattern of change in monocytes, T cells or B cells (including CD23 + B cells). IgA and IgM levels were unchanged. The mean serum IgE levels went from 3221 + 2454 IU/ml (SD) before IVIG to 2944 + 2491 IU/ml (p = .4609) during IVIG and then to 2231 • 2229 IU/ml (p = .1484) during the 6 month follow up period. In vitro IgE production of peripheral blood mononuclear cells (PBMC) following IL-4 and anti-CD40 stimulation before IVIG was 6.6 -+ 3.1 ng/ml (SD) and 4.3 • 3.1 ng/ml (p = .1641) after 6 IVIG treatments. There were no significant trends in lymphocyte proliferative responses to PHA, Candida, or tetanus. Radioallergosorbent testing showed no remarkable changes from positivity to negativity. We conclude that IVIG was of marginal clinical benefit in these patients and did not significantly alter IgE levels, IgE synthesis, or other measures of immunologic function.

Abstracts

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1358

Probiotics Downregulate Intestinal Inflammation in Allergic Children. H Majamaa, E Isolauri, Medical School and Tampere University Hospital, Tampere, Finland. The gastrointestinal microflora is an important constituent of the gut mucosal defence barrier. The aim of the study was to evaluate the clinical and immunologic effects of cows milk elimination with (n 15) and without (n 16) the addition of Lactobacillus GG (5 .',< 10~ cfu/g formula) in an extensively hydrolysed whey formula in children with atopic eczema and cow's milk allergy. The severity of atopic eczema was assessed by clinical scoring (SCORAD). The concentrations of faecal c~-I antitrypsin, tumour necrosis factor-c~ (TNF-c<) and eosinophi[ cationic protein (ECP) were determined as markers of intestinal inflammation before and after dietary intervention. The clinical score of atopic dermatitis improved significantly in children treated with extensively hydrolysed whey formula fortified with Lactobacillus GG. The concentration of c~-I antitrypsin decreased significantly in this group (p 0.03) but not in the group receiving extensively hydrolysed whey formula without Lactobacillus GG (p = 0.92). In parallel, the concentration of faecal T N F ~ decreased significantly in the group receiving Lactobacillus GG fortified whey formula, from 661 (113-1120) to 32 (9-101) pg/g (p = 0.002), but not in those receiving the extensively hydrolysed whey formula only, from 550 (124-1814) to 446 (125-937) pg/g (p = 0.33). The concentration of faecal ECP remained unaltered during therapy. The results of the present study suggest that probiotic bacteria may downrcgulate the hypersensitivity reactions and intestinal inflammation in patients with atopic eczema and food allergy. By promoting endogenous barrier mechanisms probiotic bacteria might have a role in the treatment of lood allergy.

1359

Childhood Atopic Eczema--A Study of its Impact on Family. AS Kemp, JC Su, GA Varigos, TM Nolan. Departments of Dermatology, Immunology and Paediatrics. Royal Children's Hospital, Melbourne, Australia We aimed to document the impact on the family of childhood atopic eczema. The impact on family questionnaire of Stein and Riessman was used. This questionnaire uses four subscales which assess: 1). Financial Burden, 2). Familial/Social Impact, 3). Personal Strain, 4). Mastery. 48 Children aged 4 months to 15 years (mean age 4.6 years) attending the Dermatology Clinic at the Royal Children's Hospital with atopic dermatitis were selected sequentially. Patients were graded according to severity scale described by Rajka and Langeland as mild, moderate and severe and compared with a control population of 46 consecutive children with insulin depending diabetes from the Diabetes Clinic. The impact on family scores were compared by two sample t test analysis. Results-The impact on family score for all children with eczema 2.25 (95% CI 2.1-2.4 n = 48) was significantly higher (p < .0(112) than that seen for diabetes 1.85 (95% CI 1.7-2.0 n = 46). Subdivision of eczema into groups of different severity showed that scores for moderate 2.3t (n = 20) and severe eczema 2.61 (n = 10) were significantly higher than children with diabetes, moderate (p < .003) severe (p < .0002) while mild eczema had a score equivalent to that seen in diabetes 1.97 (n = 18 p < 0.41). Conclusion-The social and emotional effects of atopic dermatitis have not been well quantitated. This study demonstrates that the impact on family scores are significantly higher for moderate and severe cases of eczema than that of another chronic childhood disease, insulin dependant diabetes. Even in milder cases the impact on family score was equivalent to that of insulin dependant diabetes. Atopic dermatitis is not necessarily a minor skin disorder but a major handicap with significant personal social and financial burdens on the family.

S333

1360

Circulating CLA + Lymphocytes from Atopic Dermatitis Patients Contain Increased Percentage of Cells Bearing Staphylococcal-related T Cell Receptor Variable Segments. M] Tortes MD, PhD., J Gonzalez PhD., M Blanca MD, PhD., MJ Carvajal PhD., M D Giron MD, JL Corzo MD, A Martinez-Valverde MD, PhD., LF Santamaria PhD. Spain. Atopic dermatitis lAD) is a T-cell mediated skin inflammation. Staphylococcus aureus (Sa) colonization is present in over 90% of AD cutaneous lesions. It has been suggested a role for Sa superAntigens (SaSag) in AD pathogenesis. The cutaneous lymphocyte-associated Antigen (CLA) is a skin homing receptor of T lymphocytes associated to the cutaneous immune response. Using a panel of TCR V[3 specific monoclonal antibodies we have studied by flow cytometry, whether CLA + cells from AD patients present a selective expression for Sa-related TCR VI3 segments. Results indicate that AD patients have higher percentage of circulating CLA+CD3 + lymphocytes compared to controls. When we analyzed different TCR VI3 segments in both CLA- and CLA- lymphocytes we found that only patients with active AD and cutaneous Sa colonization expressed higher percentage cells positive for the TCR V~2 and Vl35.1 segments in the CLA + but not in the CLAsubset. These V[3s are recognized by SaSag. Moreover, we found increased percentage of HLA-DR ~ cells in CLA+V[35.1 § lymphocytes of patients with active AD and with Sa infection. However, patients with no active AD were very similar to healthy controls regarding TCR V[3 and HLA-DR phenotype in circulating CLA+ lymphocytes. This suggests that SaSag, present in the skin of AD patients, may may be modulating skin-homing T lymphocyte activation and response in AD patients. To determine the exact role of this SaSag in the CLA ~ T cell response and activation in vitro studies with isolated skin-homing T cells will be necessary.

1361

Increased Frequency of CD30-Expressing CD4 + T Cells in Blood of Atopic Dermatitis. Y Adachi, Y Onoue, J Yamarnoto, YS Adachi, D Seki, M Morohashi, G Murakami, T Miyawaki, Departments of Pediatrics and Dermatology, Toyama Medical and Pharmaceutical University, Toyama, Japan Enhanced and sustained responses of Th2 cells have been proposed to be involved in allergic diseases. It has been shown that Th2 cell lines favors surface expression of CD30, which is a member of the TNF/nerve growth factor superfamily. To elucidate a pathological role of Th2 cells in allergic diseases, we here examined whether T cells expressing CD30 might circulate in the blood of allergic individuals. Peripheral blood mononuclear cells were obtained from 27 patients with atopic dermatitis (1-35 years of age) and 14 non-atopic subjects (4-38 years of age). Expression of CD30 on T cell populations was evaluated by three-color immunofluorescence analysis. CD30 was appreciably expressed on a few but substantial number of CD4 + T cells, especially of the CD45RO-positive memory or activated phenotype, but CD30 expression on CD8 + T cells was negligible. We found that the percentages of CD30-positive cells among CD45RO+CD4 * T cells were significantly higher in atopic patients than non-atopic donors (4.2 _+ 0.6% vs. 0.7 -+ 0.2%, p < 0.001), seemingly indicating the possible participation of Th2 cells in the pathogenesis of atopic disorders. Actual production of Th2 cytokines by these CD30-positive CD4 + T cells is under investigation.

$334

1362

Abstracts

Chronic urticaria as a presenting sign of hairy cell leukemia. LS Clore Jr and CT Stafford Allergy-Immunology Section Medical College of Georgia, Augusta, GA. Chronic urticaria is a common clinical disorder that is idiopathic in over 75% of cases. Less commonly, urticaria may be the presenting manifestation of an allergic or infectious disease, endocrinopathy, inherited syndrome, or autoimmune disorder. Rarely, urticaria may be a sign of underlying malignancy, including leukemia. C.C. is a 48 year old white female who was referred for evaluation of recurrent urticaria for 3 years. The pruritic, erythematous wheals were pinpoint, and appeared to be precipitated by heat, stress and effort. Prick tests were negative except to D. pteronyssinus. CBCs over the past 5 years revealed WBCs of 2,300-5,000 cells/ram3. Skin biopsy revealed interstitial edema with infiltration of eosinophils and mast cells consistent with urticaria. The impression was probable cholinergic urticaria, for which hydroxyzine was prescribed with fair symptomatic control. One year later, she presented with bright red blood per rectum. Repeat physical exam revealed lymphadenopathy and splenomegaly. Subsequent lab studies showed pancytopenia. Endoscopy was normal except for small, nonbleeding hemorrhoids. Bone marrow biopsy revealed histologic evidence of hairy cell leukemia that was treated with 2-chlorodeoxyadenosine (2-CDA). Upon initiation of chemotherapy her pruritus and urticaria subsided. Recent CBC revealed Hgb 9.2 g/dL, platelets 290,000 cells/ram 3, and WBC 4,100 cells/ram3. Peripheral blood smear showed no hairy cells. A literature search failed to reveal any previous reports of urticaria associated with hairy cell leukemia. However, the association of urticaria with neutropenia may be a clue of a possible malignancy. This case illustrates the need for a thorough evaluation of patients with chronic urticaria to include a comprehensive medical history, complete physical examination, and close follow-up with pertinent laboratory studies based on clinical clues.

1363

Mesalamine in the Treatment of Severe Refractory and Corticosteroid Dependent Chronic Idiopathic Urticaria. AE Gorenberg, D Gorenberg San Bernardino, CA Severe refractory chronic idiopathic urticaria (SRCIU) is a frustrating problem for both patients and their physicians. Typically, patients have no response to H1 antagonists and often require systemic glucocorticosteroids (CS) chronically to control their symptoms. Sulfasalazine has previously been reported to control CS dependent SRCIU. Mesalamine is the active compound that is produced in the gut from sulfasalazine, and it has not shown the potential serious side effects that sulfasalazine has. We report 4 patients with SRCIU who had not responded to combinations of H~ antagonists, but who did respond to mesalamine. All patients had previously failed therapy with loratadine 10 mg/day and subsequently failed therapy with hydroxyzine 200 rag/day in combination with cyproheptadine 16 rag/day. Two of the patients were using prednisone 20 mg daily for control of their symptoms. The patients were started on mesalamine 500 mg QID and their dose was increased to a maximum of 1500 mg QID over the course of 10 days. All patients had a complete remission of their urticaria by the third week of treatment, and subsequently remained free of symptoms without the use of antihistamines or prednisone. Mesalamine may be useful in the treatment of SRC1U. A formal study of a larger group of patients is ongoing.

1364

Autoimmune Thyroid Antibodies and Thyroid Function Tests in Chronic Urticaria and Angioedema. AFFirm, AL Levy, CHBano~; Allergy & Asthma Centers of Charleston, PA, Charleston, SC. Anti-thyroid antibodies (Abs) have been documented in individuals with chronic urticaria and angioedema. Previous studies have found positive serologies in 3-6% of normal patients (pts) and 12-14% of urticaria pts seen in a primary care setting. A prospective study assessed the prevalence of anti-thyroid Abs in pts referred to a specialty practice in Allergy and Clinical Immunology with refractory urticaria and angioedema. The presence of abnormal

J ALLERGY CLIN IMMUNOL JANUARY 1997

thyroid function testing (total T4, TSH) and anti-thyroid Abs (antimicrosomal, anti-thyro-globulin) was assessed in 131 pts, including 38 (29%) men and 93 (71%) women. Thyroid function tests were abnormal in 20 (15.3%) pts, including an elevated T4 in 8 (6.1%), decreased T4 in 1 (0.8%), elevated TSH in 11 (8.4%), and decreased TSH in 6 (4.6%) pts. Anti-thyroid serologies were abnormal in 27 (20.6%) pts. Antimicrosomal Abs were elevated in 18 (13%) pts; anti-thyroglobulin Abs were elevated in 21 (16%). This study documents a higher prevalence of abnormal thyroid function tests and autoimmune thyroid Abs in pts with chronic severe urticaria referred to Allergy and Clinical Immunology specialists. A relationship may exist between autoimmune thyroid disease and recently described anti-IgE rc Abs. Hence, pts referred for refractory urticaria and/or angioedema should have thyroid function tests and anti-thyroid antibody titers. 1365

lmmunohistochemically evaluation of Eosinophil Cationic Protein (ECP) in chronic idiopathic urticaria. A Motolese, C Sevignani, C Vaschieri. Department of DermatologyUniversity of Modena We investigated Eosinophil Cationic Protein (ECP) immunohistochemically expression in skin biopsies of 12 patients with chronic idiopathic urticaria. The presence of eosinophils and ECP were studied using the markers EG-1 and EG-2 antibodies, which recognize, respectively, the storage form of ECP and storage and secreted form of ECP. The number of cells EG-2 + was counted and the mean from three sections was calculated in comparison with the mean of ceils obtained from Haematossilin-Eosin (H.E.) stained sections. We studied also the number of circulating eosinophils and serum levels of ECP in all the patients. The control group consisted of 5 non-atopic healthy subjects, 2 patients with atopic dermatitis and 2 subjects affected by bollous pemphigoid. With EG-2 more eosinophils were detected than was expected from eosinophil counts with H.E. EG2 specifically stains ECP with 3 different patterns: cytoplasmatic or extracytoplasmatic granular single cell staining; fibrillar staining in collagen fibers and ECP staining at endothelium vessel level. All the 12 patients had normal eosinophil blood counts, whereas the serum ECP value had increased in 6 subjects. The demonstration of EG-2 expression in lesional skin of urticaria patients suggests the involvement of activated eosinophils in chronic idiopathic urticaria and a possible role for the cytotoxic ECP molecular in the evolution of the skin lesions in chronic urticaria.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1366

1367

Anisakis simplex sensitisation in patients with chronic urticaria and acute recurrent urticaria. M Caloto, P L6pez, MM AlcEizar, R Cuetos, T Sainza. ML Baeza, H.G.U. Gregorio Marafi6n. Madrid. Spain. The goal of this study was to evaluate the possible relation between chronic and acute recurrent urticaria and sensitisation to anisakis simplex (AS). We selected 92 patients (60 females, 32 males), mean age 43 + 16 years old, with chronic urticaria (CU) or acute recurrent urticaria (ARU): total IgE, specific IgE (CAPFEIA) to A.S. Ascaris lumbricoides (AL), Echinococcus granulosus (Ech) and Toxocara (Tx), skin prick test with commercial AS extract, stools exam for ova and parasites and rest rutinary tests were performed. 1. High total scrum lgE (>120 KU/ml) was found in 56.52%. 2. Percentages of positive CAP to the different parasites were: AS 48.9%, AL 20.7cS~, Ech 5.4%, Tx 3.3%. 3. Skin prick test was performed to 68 of 92 patients and it was positive in 33.3% of them. 4. A significant statistical correlation between serum levels of specific IgE to AS and AL was found (r 0.52, p < 0.0001 ). 5. AL ova or AS grubs were not found in any stools samples, 6. There were not difference in AS sensitization between CU and ARU. All patients were treated with antihistaminics therapy. A clinical evaluation of 25 patients 3 months later reported: 9 patients had been eating fish: 5 were asymptomatic and 4 subjectively improved; 16 patients had stopped eating fish: 8 became asymptomatic and 1 remained similar. Only 1 patient had related his symptoms to fish and becamc asymptomatic upon fish avoidance. Conclusions: 1. CU and ARU patients have a high rate of AS sensitisation. 2. Most of the patients sensitised to AS are also sensitised to AL probably because of cross-sensitivity. 3. The clinical evaluation of the patients who had stopped eating fish during 3 months was not different to the rest of the group. Latex allergy and decreased work ability index among health care workers. Kujala VM, Karvonen J, L~iirii E, Kanen,a L, Estlander T, Reijula KE, University of Oulu, Oulu, Finland In this study the association between natural rubber latex (NRL) sensitization and work ability index (WAI) among health care workers was investigated. Furthermore, the diagnostic sensitivity and specificity of a postal questionnaire as a screening device of NRL allergy was evaluated. The study population consisted of 32 female health care workers with an occupational latex allergy, and 51 control subjects who were individually matched for age and occupation. A self administered two-part questionnaire including seven items of the WAI, as well as questions on glove-related symptoms was mailed to the subjects. The median age for NRL allergic subjects was 40 years (range 23 to 62), and the diagnosis of occupational latex allergy had been made 6 years (range 2 to 16) before the present study. The WAi scores were on average lower among the sensitized subjects as compared with their nonsensitized controls. Even after removing the contribution of the presence of allergic eczema, diagnosed by physician, from the original WAI score the proportion of NRL allergic subjects and the control subjects in the good work ability category were 34% and 53%, respcctively. Ten health care workers (31%) had changed occupation and one early retirement had occurred after sensitization to NRL. The sensitivity and specificity of the present self administered questionnaire as an indicator for latex allergy was 84% and 89%,, respectively. In conclusion, there is a clear association between NRL allergy and decrease in the WAI among health care workers, which can not bc explained by age, gender or profession.

Abstracts

S335

1368

Therapeutic with intravenous gammaglobulin in patients with chronic idiopathic urticaria and positive intradermal skin test to autologous serum. E Toma= A Pregal, MH Clode, A Palma-Carlos. Hospital Santa Maria, Lisbon, Portugal. Chronic idiopathic urticaria patients have been reported to have positive intradermal skin test to autologous serum due to the existence of circulating histamine releasing autoantibodies (anti-IgE or anti-lgE receptor), which are responsible for the disease in 25% of the cases. In order to evaluate the therapeutic efficacy of intravenous gammaglobulin in this situation, the authors performed intradermal skin tests with autologous serum in 33 patients with chronic idiopathic urticaria (10 male, 18-68 years old, mean age 48.8; 23 female, aged 13-67, mean 43.9), having 9 (27%) a positive test. Six out of these, with severe urticaria, requiring permanent or frequent use of systemic corticotherapy, were selected and given intravenous gammaglobulin, 400 mg/Kg monthly. Five of the patients improved significantly immediately after the first administration and thereafter they only needed intermittent therapy with antihistamines. The authors concluded that intravenous gammaglobulin may be an useful therapeutic approach in severe, unremitting cases of chronic urticaria with positive skin tests to autologous serum.

1369

A Case of Acute Synoviai Fluid Eosinophilia Associated with Delayed Pressure Urticaria (DPU). ATDobracki, JL Baldwin University of Michigan, Ann Arbor, MI A 30 year old man presented with a 16 mo history of nearly daily painful swellings that involved multiple joints as well as areas away from joints especially feet, hands, arms, and forearms. These swellings were more pronounced at the end of the day and more severe with repetitive activity. There was also a 6 month history of several urticarial flares. Three weeks prior, one episode of urticaria with fever of 103 after exposure to streptococcal pharyngitis resolved with antibiotic treatment. Review of previous laboratory data revealed: Synovial fluid from left knee 2/4/96 with 36% eosinophils and skin biopsy of an urticarial lesion 3/25/96 showing perivascular inflammatory interstitial dermatitis with mixed inflammatory infiltrate with eosinophils. Two CBCs done on 12/ 18/95 and 4/2/96 showed no eosinophils. Lyme titres, stool for O&P, ANA, RF, ASO, RPR, C3, C4, CIINH, HepB sAb, HIV were all negative. SPEP, CRP and blood chemistries were all normal. Physical exam was normal except for one 15 x 2 cm urticarial lesion on the posterior thorax as well as an erythematous, slightly edematous right fourth toe with some exfoliation. Physical urticaria challenge for delayed pressure urticaria (DPU) was positive. The patient was initially treated with prednisone with complete resolution of his pattern of daily swellings. A search of the literature found one additional case of DPU with eosinophilic synovitis which responded to antihistamine therapy. The patient was placed on cetirizine 10 mg/d. His response to therapy is being followed. We conclude that the coexistence of two rare disorders in the same patient suggests a common etiology.

S336

1370

Abstracts

Oceupacional Contact Allergy to Herbicide Glyphosphate. A.RodHguez, S.Echechipia, ]M OLaguibel, BE Garcia and A. Sanc hez-Pala cios. Farmers and other agricultural workers are exposed to a wide variety of chemical, biologic, and physical hazards to work. Herbicides are no frequently used in place of hand labor or machine cultivation to control unwanted plants. Glyphosphate (Roundup) is a nonselective postemergence herbicide. We reported the case of a 42-year-old male, with no past history of allergic reactions, who developed eczema on face, neck and both hands due to his proffesional activity in farm, handling different pesticides. Skin prick test with a battery of common inhalant and food allergens were all negative. Total serum IgE was 105 KU/L In patch testing with standard contactants (ICDRG), group of plants, pesticides and repellents (acaricides, animal repellents including rodenticides, fungicides, herbicides and insect repellents), only Glyphosphate 2% pet. showed a positive response (2+). Allergic contact dermatitis had been reported in workers mixing or loanding this herbicide, although it seems to be a rare sensitizer.

1371

Allergic sensitization to dexpanthenol and exacerbation of eczema by orally ingested pantothenic acid. C Botzi, W Hemmer, M Focke, R Bracun, M GOtz, R Jarisch, Dermatologic & Pediatric Allergy Centre, Vienna, Austria. Dexpanthenol, an alcoholic derivative of pantothenic acid (vitamin B5) employed in topical pharmaceutics and cosmetics for its stimulating effects on skin cell proliferation, is a rare cause of allergic contact dermatitis. Case report." A 33-year old woman presented for chronic dermatitis of the face, with dispersed lesions also on her trunc and arms. Patch testing with standard allergens and special series (emolients, perfumes, preservatives, acrylates, metals, rubber chemicals) was negative. History revealed that she used a panthenol containing baby creme for skin care. A 2+ + reaction (72h) was obtained by patch testing the product as is. Subsequent testing with the ingredients revealed a 2+ + reaction to dexpanthenol 5% pet. After omission of the commercial product the dermatitis improved but did not clear. Dispersed lesions remained periorally, and recurrent eczema resembling nummular dermatitis was found at the patient's wrists. Oral challenge with 3 • 60 mg Ca-D-pantothenate for 4 days resulted in severe exacerbation of hand lesions and flare-up of older sites. Observance of a diet low in vitamin B5 resulted in long term improvement of eczema, with worsening after intake of B5-rich nutrition. The data suggest that sensitization to a contact allergen may be the etiologic background of nummular dermatitis. Nummular dermatitis has been rarely linked up with contact allergy. In the present case, normal nutritional intake of pantothenic acid appeared to be sufficient to sustain chronic eczema.

1372

CD4 + T cells Regulate and CD8 + T cells are the Effectors of Contact Dermatitis to Urushiols in Mice. A E De loannes, A M Kalergis, MI Becker, M Torres, J Garbarino ~, C L6pez. Departamento de Biologfa Celular y Molecular. Facultad de Ciencias Biol6gicas, Pontiffcia Universidad Cat61ica de Chile. ~Universidad Federico Santa Marfa, Chile. Urushiols are pro-electrophilic contact sensitizers that are activated intracelularly by the APC. The role of T cells subpopulations in the development of the allergy was investigated by immunodepleting with Mabs directed to T cell markers. The immune response to the urushiols was determined by measuring the ear swelling with an engineer's micrometer from Mitutoyo, Tokio, Japan. Since naive mice exhibit a significative primary response to the epicutaneous application of the allergen, we tested the inflammatory response in Balb/c scid/scid. Immunodeficient mice show also an inflammatory response to the urusbiol but 50% less intense. A deep immunodepletion of either CD8 + or CD4 + T

J ALLERGY CLIN IMMUNOL JANUARY 1997

cells rendered the response of the animals to background levels. Conversely, mice treated with a low dose of the anti-CD4 Mab, exhibited a higher response than the control animals. Our results strongly suggest that CD8 + T cells are the effectors, whereas CD4 + T cells may up- or down-regulate the response to urushiols in mice. (Granted by Fondecyt 1-93-0654 and 1-96-0510.)

1373

Comparison of the Quintest| and Multi-test| for Allergy Skin Testing. TA Mahr, R M West, LA Rahn. Gundersen Lutheran Medical Center, LaCrosse, WI The Lincoln Multi-test| is a commonly used disposable, plastic skin test device. We compared this device to the newly developed Bayer Quintest| which is disposable and has surgical steel tips in a linear plastic handle. The Quintest has not been previously compared to other available methods for performing cutaneous prick testing. Testing was done using saline, histamine, DP mite, DF mite, ragweed mix, grass mix, tree mix, maple, birch, oak, mold mix, feathers, weed mix, dog, and cat. These were placed on the forearms with each device. Only 1 physician read the tests. Wheal and flare was evaluated at 15 minutes, and was rated on a scale of 0 - 4+. The end points of the study were (1) wheal and flare response of each device, (2) speed of application of the above set of skin tests, (3) the perceived ease of application of the device, and (4) the patient's subjective assessment of discomfort from the device. A total of 35 patients were tested. The median age was 8 years, with a range from 5 to 34 years. There were 19 males (54%) and 16 females (46%). There was a perfect correlation between devices with the negative control, however, there was slightly greater number of histamine tests by MT 4+, which by QT were 3+ (p = .002). All other allergens tested had good agreement between the 2 devices. Using a scale of 1 = fast, 2 = some difficulty, 5 = slow, the speed of application was rated far better for the QT(avg. 1.7) than the MT(avg. 2.9) (p < .001). Utilizing a similar scale for ease of application 1 = easy, 3 = some difficulty, 5 = difficult, there was a trend toward greater ease of application with the QT(avg. 1.8) than the MT(avg. 2.7) (p = .002). In regards to pain, the scale used was 1 = no pain, 3 = discomfort, 5 = hurt! There was also a tendency of patients to rate the QT(avg. 2.4) less painful when compared to the MT(avg. 3.1) (p < .001). We conclude the two devices to be similar in reproducibility of skin test results, with the Quintest having a tendency to be both faster and easier to apply, and also causing less discomfort to this selected population.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

Abstracts

1374

Skin reactivity to histamine in Intensive Care Unit (ICU) patients. V de Benito, MP Lopez-Saez, A Prieto, T Sainza, S Olalde, H G U Gregorio Marafl6n, Madrid, Spain. Diminished skin reactivity to histamine is commonly found in very old patients and in those taking certain drugs. Little is known about skin reactivity in critically sick patients. We performed prick and intradermal tests (PT and ID) with histamine phosphate (10 and 1 mg/ml respectively) and physiologic saline in the volar surface of the arm to 15 randomly selected patients from ICU. None of the currently administered drugs were among those described to interfere with histamine. We used as a control group 15 volunteers. All control subjects had a positive reaction to histamine (wheal >3 ram) both in PT and [D. Four out of the 15 ICU patients did not elicit any reaction to histamine, they had a mean age (MA) of 57.5 (group 1). Three ICU patients showed a flare without a wheal, their MA was 67.3 (group 2) while the 8 which positively reacted (group 3) had a mean wheal diameter for PT and ID of 6 and 8 mm respectively, their MA was 56. All 4 patients in group 1 were in coma (Glasgow - <8), two of them caused by brain damage, two in group 2 were in coma without a neurologic cause and 5 in group 3 were in coma, which was caused by brain damage in 3 of them. We conclude that 26.6% of ICU patients had a negative test to histamine (PT and [D) while all patients with positive PT to histamine also reacted to ID. This has to be taken into account when drug skin tests are necessary. All negative patients were in coma. If this unresponsiveness has to do with special treatment doses, specific illness or high stress has to be determined.

1375

The Effect of Systemic Glucocorticoids on Cutaneous IL~ Production, Endothelial E-selectin Expression and PMN Infiltration after Bacterial Endotoxin Injection in Human Subjects. J Max, BS Bochner, RP Schleimer, LA Beck, Johns Hopkins University, Baltimore, MD Glucocorticoids (GC) have numerous anti-inflammatory properties, but their mechanism of action has not been well elucidated in vivo. It has been suggested that GC inhibit the generation of endothelial-activating cytokines. In the present study, wc assessed the effects of a systemic glucocorticoid on the expression of endothelial E-selectin, IL-I[3 and PMN infiltration following endotoxin injection (10 E.U., Dr. Hochstein, FDA) in man. Fourteen healthy subjects were randomized to receive either placebo or prednisone (30rag BID < 5 doses) followed by intradermal injection with bacterial endotoxin the morning of the last dose. Skin biopsies taken two hours post-injection were examined for expression of endothelial E-selectin and PMN infiltration. In addition, tissue specimens were homogenized and assayed by ELISA for IL-I[3 (limit of detection 0.125 pg/m[). Results were as shown:

Placebo Prednisone

IL-1[~

E-selectin

PMN

(ng/gm total protein)

(% total vessels +)

(cells/ mm 2)

160_+41 298+65

45 • 52_+8

149_+53 166_+35

Endotoxin injection induced the expression of endothelial E-selectin and the recruitment of PMN as well as IL-11~ production in both placebo and prednisone-treated subjects. There were no significant differences between the two groups in any of the measured parameters. In this endotoxin model of inflammation systemic glucocorticoid pretreatment did not inhibit the expression of endothelial E-selectin, PMN infiltration or the tissue production of

IL-16.

S337

1376

Acquired lgA and IgG2 deficiency in chronic mucocutaneous candidiasis. Gallagher KT, Roberts RL, Hyman C, Stiehm ER. UCLA Department of Pediatrics, Los Angeles, CA 90095 and Yucaipa, CA 92399. Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent candidal infections of the skin, nails and mucous membranes secondary to heterogeneous defects in T cell mediated immunity but with intact antibody function. We report a 13 year old boy diagnosed with CMC who developed IgA and lgG2 deficiency at twelve years of age. He presented at age 2 with a history of oral thrush unresponsive to nystatin, diaper rash, and paronychia of a thumb and finger. Endoscopy revealed Candida and Rhodotorula esophagitis. Laboratory evaluation revealed IgG of 612 mg/dl, IgM 18 mg/dl, lgA 38 mg/dl, negative delayed cutaneous hypersensitivity to candida and low in vitro lymphocyte proliferation to Candida antigen. T and B cells were within normal limits and PHA proliferation was low normal. His Candida infections responded well to ketoconazole but he developed two episodes of zoster, recurrent apthous ulcers, and chronic verruccal lesions (warts) of the hands. At age twelve he developed recurrent pneumonia with effusion and splenomegaly. Laboratory studies revealed IgA <12 mg/dl, lgG 632 mg/dl, lgG1 532 mg/dl, IgG2 4 mg/dl, IgG3 96 mg/dl, IgG4 <1 ~g/dl, IgM 76 mg/dl, CD3 1367 cells/p,1, CD4 701 cells/p,l, CD8 508 cells/pJ, CD4:CD8 1.38, CD19 105 cells/pJ, and CD16/56 263 cells/~l. Pneumococcal antibody titers were nonprotective post immunization. He has been free of bacterial infections since beginning monthly lVIG but has had progression of his verruccal lesions and progressive lymphopenia (945 cells/l~l). This and eleven other reported CMC patients with antibody deficiency provide evidence for a greater spectrum of immune defects associated with CMC.

1377

Development of IgE-Mediated Sensitization to Contaminating Fetal Calf Serum Proteins During Immunization with Human Renal Carcinoma Vaccines. LA Beck, H

Sampson, M Columbo, JW, Simons, EM Ja~'ee, C Weber, HI Levitsky, DM Pardoll, FF Marshall *KM Leiferman, BS Bochner, Johns Hopkins Univ., Baltimore, MD and *Mayo Clinic, Rochester, MN We evaluated two patients in a Phase I trial of a granulocyte-macrophage colony-stimulating factor (GMCSF) gene transduced, autologous renal cell carcinoma vaccine for the Rx of metastatic disease. Patients were seen because with repeated dosing they developed immediate and late phase (LP)-Iike reactions at skin sites injected with autologous normal kidney cells (NK) or renal carcinoma cells (RC). Reactions worsened with each subsequent challenge, raising the possibility that allergic sensitization to some antigen(s) in these preparations had occurred, Skin testing was performed with dilutions of autologous NK and RC prepared by mechanical disruption or enzymatic digestion (collagenase). Since fetal bovine serum (FBS)-containing media was used in cell cultures, patients were also skin tested to FBS. Similar wheal/flare diameters were seen with both mechanically-dispersed and enzymatically-digestcd NK and RC preparations, suggesting that collagenase was not a significant allergen. Both patients had positive skin tests to FBS with one reacting even at a dilution of 1:500 million. Skin testing of three controls with FBS was negative. Skin biopsies 24 hr after RC injections revealed extensive infiltration and degranulation of eosinophils as in the LP. In vitro histamine release with patients but not control basophils was seen with FBS and vaccine preparations that contained FBS. GEL separation of FBS followed by immunoblotting with patient serum and antiIgE identified several bands. End-terminal sequencing of the most prominent band (45kD) revealed it to be bovine ul anti-trypsin. This would suggest that FBS proteins are responsible, in part, fur the cutaneous reactions seen with this vaccine. Further tumor vaccine trials have substituted autologous serum for FBS to avoid this problem.

S338

1378

1379

1380

Abstracts

Autoimmune Progesterone Dermatitis: Effective Prophylactic Treatment with Danazoi. E. Shahar, R. Bergman, S. Pollack. Rambam Medical Center and B. Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel. Background." Autoimmune progesterone dermatitis is a rare condition with characteristic urticarial skin eruptions occurring during the perimenstrual period or following progesterone treatment. Various treatment modalities have been suggested for this condition but none has been successful. Objective: To determine the efficacy of the anabolic androgen danazol in the prophylactic treatment of ster o i d - a n d antihistamine--resistant autoimmune progesterone dermatitis. Diagnosis was confirmed by skin biopsy and intradermal skin test with progesterone analogue. Case reports: Two young women with recurrent episodes of perimenstrual urticarial pruritus were treated by twice daily administration of danazo1200 mg during the perimenstrual period. During follow-up for the last 14 months no urticarial rash was observed. Conclusion: Danazol may be administered in the perimenstrual period for prophylaxis of severe attacks of autoimmune progesterone dermatitis. Collaborative Study of the Genetics of Asthma, Atopy and HLA: Linkage of HLA to Mite-Sensitive Asthma. 12th International Histocompatibility Workshop & Conference: HIM and Allergy workshop-MN Blumenthal, L Caraballo, W Cookson, ST Holgate. M Howell, F lnacio, C Lahoz, DM Marsh, G Peltre, A RttflTlli, L Friedhoff, SS Rich University of Minnesota, USA, University of Cartagena, Colombia, Oxford University, England, University of Southampton, England, Institute of Immunology, Portugal, University of Madrid, Spain, Johns Hopkins University, USA, Institut Pasteur, France, National Research Council, Italy, Bowman Gray School of Medicine, USA It is apparent that genetic factors play a central role in bronchial asthma and allergies. Many studies demonstrate that asthma, atopy and their intermediate phenotypes show familial aggregation and are often associated with specific HLA antigens. The objective of this study is to collect families with two or more siblings with mite-sensitive asthma, characterize clinical features of the family members, and determine genotypes in the HLA region using PCR-based methods. Families were collected from multiple centers, but data from six with complete HLA typing were analyzed. A total of 256 families (1384 individuals) provided genotypic (allowing HLA haplotype formation) and phenotypic data. Using affected sib-pair analyses with HLA haplotype, significant evidence for linkage was found for mite sensitivity based on DerPI (p = 0.06, mean IBD sharing = 0.56) and DerPII (p = 0.01, mean IBD sharing = 0.62). Suggestive evidence for linkage of asthma and HLA was found ("ever diagnosed with asthma", p = 0.07, mean IBD sharing = 0.55). No evidence for linkage with HLA haplotype was demonstrated with other allergens or with total IgE. These data suggest that genetic susceptibility to asthma and mite-sensitive asthma may be mediated partly by genes in the HLA complex on 6p21.3. It is unclear how the HLA-linked susceptibility regions interact with other such regions (5q, l l q 12q, 14q) or whether the results are a function of the ascertainment criteria (two affected sibs with mite-sensitive asthma). Candidate genes in this region may provide insight into the autoimmune aspects of asthma and atopy. Distribution of the 132-Adrenoceptor Polymorphisms in Atopic and Non-atopic Patients. ML Kowalski MD, PhD, G Woszczek MD, M Borowiec MSc, Dept Clinical Immunol and Allergy, s POLAND. Several point mutations within the coding region of the human 132-adrenergic receptor ([32-AR) have been described, and some of them are associated with a significant change in the function of [32-AR. Since an impairment of the I3a-AR function was implicated in atopy, we studied the distribution of two most important 13~-AR polymorphisms in atopic patients.

J ALLERGY CLIN IMMUNOL JANUARY 1997

The study included 60 atopic patients with seasonal asthma and/or rhinitis (sensitisation to pollens confirmed by positive SPT), and 48 healthy controls with negative personal and family history of allergy, and negative SPT to a battery of inhalant allergens. Genomic DNA was isolated from peripheral blood, and 132-AR genotype was assessed by allele-specific PCR amplification with pairs of primers specific for two polymorphic sites: one at nucleic acid 46 (Arg/Glyl6), and the second at nucleic acid 79 (Glu/ Gin27). The generated PCR products sizes were 913 bp and 442 bp for Arg/Glyl6, and Glu/Gln27 respectively. 132-AR polymorphic forms at amino acid position 16 and 27, both in homozygous and heterozygous form, were present with similar frequency in atopic and non-atopic subjects:

Non-atopics n = 47 Atopics n = 60

Arg 16

Gly 16

Gin 27

Glu 27

28

36

36

35

27

49

45

37

No relationship of the total serum IgE levels to the 132-AR polymorphisms were observed. We concluded, that 132-adrenoceptor polymorphisms are not associated with the presence of atopic phenotype.

1381

Expression and B-cell epitope mapping studies of the Lep d 2 allergen. Paul Whitley, Margit Schmidt, Lotta Elfman and Marianne van Hage Hamsten, Department of Laboratory Medicine, Division of Clinical Immunology, Karolinska hospital, S-171 76 Stockholm, Sweden. Lepidoglyphus destructor is an important non-pyroglyphid mite species in Europe that is a predominant allergen in farming environments. The major allergen of L. destructor, Lep d 2, is a protein of 15 kD and is recognized by about 90% of sera RAST positive to this mite species. The cDNA of two isoallergens of the 15 kD protein have been sequenced and the protein has been expressed in a number of different heterologous protein expression systems. In order to map the B-cell epitopes, the full length protein and truncated forms of the protein have been expressed and purified from E. coil as Glutathione-S-transferase (GST) fusion proteins. The full length (125 amino acids) GST fusion protein reacts strongly with patient IgE in dot blots and Western blots but the truncated proteins, of which the longest is 108 amino acids, are not recognized by patient IgE. A possible explanation for this result is that the major B-cell epitopes are situated in the C-terminal portion of the protein. Presently we cannot rule out the possibility that some folding feature of the whole protein is important for recognition by patient IgEs. The results of the expression of Lep d 2 and B-cell epitope mapping studies will be presented.

Abstracts $339

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1382

Group A) B)

humans, VLA-4 is expressed on all leukocytes except neutrophils (PMN). This unique pattern of expression has led to the hypothesis that VLA-4 interaction with its endothelial ligand, vascular cell adhesion molecule-1 (VCAM-I), is a primary mechanism for selective recruitment of leukocyte subpopulations in inflammatory disease. Although this pattern of VLA-4 expression has been assumed to be consistent for all species, in the present study we demonstrate constitutive VLA-4 expression on rat PMN. Flow cytometric analysis of rat PMN utilizing the mouse anti-rat VLA-4 mAbs TA-2 and MRc~4, as well as the mouse anti-human VLA-4 mAb L25, demonstrated a consistent, low level of VLA-4 expression (e.g. 2.3_+0.3 mean fold fluorescence above background IgG with TA-2). Despite low levels of VLA-4 expression on rat PMN, PMN VLA-4 was demonstrated to mediate specific adhesion to VCAM-l-transfected Chinese hamster ovary (CHO)-cells, as rat PMN adhesion to VCAM-1 transfected cells was significantly greater than adhesion to non-transfected CHO-cells (5.5_+ 1.2 vs 2.6+-0.5, n=6, p<0.05), and adhesion to human VCAM-l-transfected cells was completely inhibited by the VLA-4 mAb TA-2 (2.8~0.6, p<0.05). Thus, rat PMN constitutively express a4 integrius and can bind to VCAM-1. Unlike most species, use of c~4 integrin antagonists in the rat may directly affect PMN recruitment responses in vivo.

HLA Genetic Studies of the Selective IgE Response Response Penicillins. M Torrecilla MD, A Jurado MD, C Muhoz MD, B Cardaba MD, M Palomeque MD, J de la Fuente MD, C Lahoz MD, PhD., J Fernandez MD, PhD., M Blanca MD, PhD. Spain. Experimental evidence indicates that the immunological response to haptens is linked to MHC Immune response genes. In addition it has been shown in atopic subjects an association between the IgE response to allergens and HLA class I1 antigens. Recent data indicate that there is a subgroup of subjects with allergy to penicillins that show a selective response to amoxicillin. These are skin test positive only to AX determinants and have good tolerance after the administration of different penicillins not related to AX. The IgE antibodies of these subjects recognise as the relevant part of the epitope a structure mainly formed by the side chain of the AX conjugate. This represent a well defined population where the genetic response to the hapten can be studied by HLA studies. A group of 38 subjects (A) who developed a selective response to AX and had good tolerance to BP was studied. Analysis of class II-DR antigens was made by FRLP. A control group (B) of sex and age matched subjects was used. Results were: DR1

DR2

DR3

DR4

DR5

DR6

DR7

DR8

16.21 21.62 16.21 35.13 24.32 13.51 40.54 2.70% 21.2 23.2 25.1 30.2 20 25.7 31.3 6.1% A statistical analysis showed no difference in the proportion of DR genes distribution (Chi square analysis with Yates correction). These results indicate that the selective response to AX is not HLA-DR restricted and that the genetic control of this response can be associated to other genes.

1383

Evaluation of the Immunologic Features of a Timothy Grass Pollen Allergen Chemically Modified with a Synthetic Polymer. A Babakhin, S Andreev, V Shustova, I Gushchin, H Nolte, ? L M DuBuske, * Institute of Immunology, Moscow, Russia, ?University Hospital of Copenhagen, Denmark, *Immunology Research of New England, Fitchburg, MA, USA The aim of this study was to evaluate immunologic features of a pollen allergen chemically modified with a synthetic polymer. Water soluble conjugate of Timothy grass pollen allergen (G6) and the non-toxic copolymer N-vinylpyrrolidone and maleic anhydride (VMA) modified with epsilon-aminocapronic acid (EACA-VMA) were investigated in (CBAxC57BL/6) F1 mice. Mice which were immunized 3 times at 3-week intervals with 10 ~g of G6 failed to demonstrate anti-G6 IgE-response but induced anti-G6 lgG antibodies comparable to that of unmodified G6. Conjugate G6-EACA-VMA could not trigger active (in mice) or passive (in rats) anaphylaxis. Analysis of the dose-response curves using microfiberglass-based whole blood basophil histamine release assay showed that at least 100 fold increased concentration of modified allergen G6-EACA-VMA was needed to achieve the same level of histamine release from basophils of patients sensitive to Timothy grass pollen compared with basophil response to non-modified allergen (G6). In an ELISA inhibition assay the capacity of IgE was significantly reduced; a 50% inhibition achieved if G6-EACA-VMA was utilized at 100-fold greater concentration than unmodified G6. These results demonstrate the possibility of reducing allergic response without affecting immune activity of an allergen; a potential approach in the creation of safe and effective extract for allergen-specific immunotherapy.

1384

Rat neutrophils constitutively express ~4 integrins and bind to vascular cell adhesion molecule-1. KL Davenpeck, SA Sterbinsky, BS Bochner, Johns Hopkins Asthma and Allergy Center, Baltimore, MD The integrin very late activation antigen-4 (VLA-4, e~4131) has been demonstrated to play an important role in recruitment of leukocytes to sites of inflammation. In

1385

Variable Loss of Oxidative Burst Function in Stored Neutrophils Obtained by Leukopheresis. TO Harville, JM Cook, R Johnson, BA Waters-Pick, MJ Laughlin, J Kurtzberg, Duke University Medical Center, Durham, NC Clinical experience has demonstrated that infections in patients with disorders of neutrophil function (eg CGD) or numbers (eg due to chemotherapy) can be treated with infusions of freshly leukopheresed donor neutrophils. Leukopheresis and preparation for infusion can take several hours, which has felt to impinge on the effective life of neutrophils (circulation half life of - 8 hours). Therefore, in some cases, leukophereses with subsequent infusions have been performed several times each day, but usually no less frequently than every 24 hours. To determine whether donor leukophereses could be spaced further apart, we questioned: How long could neutrophils be stored for subsequent daily infusions? To study this, we chose to measure oxidative burst activity by fluorescence intensity shift of dichlorofluorescin to dichlorofluorescein after PMA stimulation. Assays were performed at 24 hour intervals on fresh and stored aliquots from 3 subjects. Neutrophils were harvested into Hespan/Tricitrasol by standard leukopheresis. Paired specimens of leukopheresed neutrophils were irradiated with 2500 rads of gamma irradiation (which did not alter their function) and stored at either room temperature or 4C. Viability was determined by trypan blue exclusion with >98% viability for >4 days in all samples. Donor 1 had good oxidative function for 48 hours and Donor 2 for 72 hours. In contrast, Donor 3 lost oxidative function after 24 hours, despite >98% trypan blue exclusion for 96 hours. Conclusions: Viability of stored neutrophils by trypan blue exclusion does not indicate retention of oxidative burst function. Leukopheresed neutrophils from some donors can be stored for 48-72 hours, allowing several days of infusions from one leukopheresis. Oxidative burst assays should be performed to determine whether leukopheresed neutrophils remain functional after the first 24 hours of storage.

S340

1386

Abstracts

Use of Monoclonal Antibodies for the Stability Study of Der p and Der f in Mite Extracts. T Liu and Y Lin, Center for Biologics Evaluation and Research, FDA, Bethesda, MD Allergenic extracts are complex mixtures of proteins and other organic and inorganic molecules of which only a few have allergenic activities. Most frequently used methods for potency measurements of allergenic extracts, RAST or ELISA competition, depend on the use of allergic patient serum pools and reference extracts. It is highly desirable to have an independent assay method to determine the stability of the reference extracts for these assays. Using purified mite allergens as standards, the the stabilities of group I and II allergens in CBER's mite reference extracts, Dermatophagoides pteronyssinus and D. farinae, were determined by Sandwich ELISA. Samples were stored at 4 ~ C, room temperature, 37 ~ C and 50~ C and the amounts of Der p l/If, DerfI/II were determined at different time intervals. Derp I/II were not stable at any of the storage temperatures, but the degradations were slower at lower temperatures. Derf I was stable for at least 3 years and Derf II for 9 months when stored at 4 ~ C; both Derfl/lI were relatively stable for 9 months at room temperature but degradations started at one year. Neither proteins remained intact at 50~ C. Immunoblot data, though not quantitative, showed the same trend of degradation. Competition ELISA relative potency determinations for these stability samples also showed a similar trend when 4~ samples were used as references. These results indicated that group I and II allergens in mite extracts, containing 50% glycerol, are not stable when stored at 4 ~ C for over one year.

1387

Reduced L-Selectin Expression in Patients with Hyperinmunoglobulinemia E Syndrome. CC Forero, L Vargas, D Garcia de O, F Montoya, PJ Patifto, A Jaramillo, Laboratory of Immunology, School of Medicine, University of Antioquia, Medellin, Colombia. The initial interaction of leukocytes with vascular endothelium in inflammation sites depends on the expression of adhesion molecules of the selectin family. This process, called rolling, is essential to permit adherence, chcmotaxis, trans-endothelial migration and accumulation of ]eukocytes as response to a specific stimulus in a inflammatory microenvironment. With the purpose of establishing a relation between chemotactic deficiency observed in patients with Hyperimmunoglobulinemia E syndrome (HIES) and the role of L-selectin cellular adhesion molecule, we evaluated the basal expression of this glycoprotein in the membrane of both circulating granulocytes and lymphocytes of patients with HIES. The results of this study show a significant reduction of the L-selectin expression on quiescent and activated granulocytes and lymphocytes from patients with HIES, which could help us to understand the pathogenesis of the immune defects that have been observed in these patients.

1388

Progression of Episodic Edema Associated with Eosinophilia into Hypereosinophilic Syndrome with Cardiac Fibrosis. Gurjit K. Khurana Hershey, * Gerald J. Gleich/" and Catherine S. Tripp, * *Division of Immunology and Rheumatology, Dept. of Pediatrics, Washington University School of Medicine, St. Louis, MO and ?Division of Allergic Diseases, Mayo Clinic and Foundation, Rochester, MN We report a 14 year old African American female patient who presented with episodic edema and eosinophilia and went on to develop cardiac pathology consistent with hypereosinophilic syndrome. Episodic edema and associated with eosinophilia has been described previously by Gleich et. al. as a benign syndrome without evidence of other organ involvement. In addition, they responded to glucocorticoids. Our patient first presented at the age of 11 with intermittent swelling of her arms, legs and face associated with fatigue and decreased activity. The episodes occurred 1-2 times a month and resolved spontaneously after approximately 7 days. The only laboratory abnormality associated with these episodes was profound

J ALLERGY CLIN IMMUNOL JANUARY 1997

hypereosinophilia. Her only physical finding was diffuse symmetric non-pitting edema. Prednisone was effective at reducing her symptoms and normalizing her eosinophilia. Four years after her initial presentation, she developed episodic non-radiating substernal chest pain. An EKG revealed ST segment elevation and inverted T waves suggestive of a myopathic process. An echocardiogram and serial CPK MB's were normal. An endomyocardial biopsy revealed endocardial fibrosis and organized thrombosis consistent with hypereosinophilic syndrome. This patient represents the first description of an insidious progression from benign episodic edema associated with eosinophilia to cardiac manifestations consistent with hypereosinophilic syndrome. Therefore, some patients who initially present with only diffuse edema and eosinophilia may develop more ominous major organ involvement many years after the initial presentation.

1389

Week-to-week Variation in the Measurement of Specific IgE Concentration over Two Years. K Richter, D Feigl, J Kuehr University Children's Hospital, Freiburg, Germany To quantify longitudinal variation in the measurement of specific IgE, the same serum sample was examined in weekly intervals regarding specific IgE concentration to four inhalant allergens. Initially, a large serum sample of a male atopic adult was divided into aliquots of 300 Ixl and stored at - 2 0 ~ C. Over a period of 23 months specific IgE antibodies against cat dander (el), Dermatophagoides pteronyssinus (dl), birch pollens (t3) and grass pollens (g6) were measured in about weekly intervals (CAP FEIA, Kabi-Pharmacia, Sweden). In total, the serum was analyzed on n = 76 occasions. No time trend (increase or decrease of values) was found. The median (5-95% interval) was 1.6 U/ml (1.2-2.2 U/ml) for el, 0.2 U/ml (0.1-0.5 U/ml) for dl, 21.1 U/ml (17.8-25.4 U/ml) for t3 and 66.0 U/ml (47.3-94.4 U/ml) for g6. In order to calculate variation coefficients (VCs), few outliers had to be eliminated and log transformation had to be performed. VC (n of values) was 3.9% for log_g6 (n = 74), 36.9% for Iog el (n = 74), and 3.2% (n = 75) for log_t3. For dl normalized distribution could not be achieved. Values equal or above 0.7 U/ml were found in 100% (n = 76) for g6, in 100% (n = 76) for el, in 100% (n = 76) for t3, and in 2.6% (n = 2) for dl. In conclusion, a storage effect over a period of 23 months appears to be neglectible, at least if sIgE concentrations exceed 1.6 U/ml and the serum sample was stored permanently at - 2 0 ~ C. Longitudinal variation might be higher for lower sIgE concentrations, however, a highly reproducible discrimination in terms of negative vs. positive (cut-off point of 0.7 U/ml) can be expected within a relatively small concentration range (on average 0.2-1.6 U/ml).

J ALLERGYCLIN IMMUNOL

Abstracts

$341

VOLUME 99, NUMBER 1, PART 2

1390

1391

Isolated Depressed C4 level in a patient with Physical Urticaria. GA Wheeler, C W Bassett, BA Silverman, L T Chiaramonte, Long Island College Hospital, Brooklyn, NY. This is a case report on a 33 year old white male with a history of aquagenic pruritus and persistently depressed C4 levels. The patient initially presented with nasal allergy and was started on immunotherapy for multiple aeroallergens. He also gave a history of having recurrent episodes pruritus with exposure to water since adolescence. He also had a wigue history of hip and joint pains. His aquagenic pruritus improved significantly after symptomatic treatment with cctirizine 10 mg per day. He denied any angioedema. C4 levels wcrc consistently decreased (8-14 mg/dl). CINH level and function were normal. CH50 was 41% of normal. Clq and ESR were within normal limits. C4 production is governed by 4 genes and individuals with absence of any number of these genes may have decreased C4 levels. Individuals with complete C4 deficiency are rare but homozygous deficiency for C4a or C4b is relatively common. Homozygous deficiency for C4 has been reported in 3-12% of the normal population with racial differences between Whites and Blacks. C4 deficiency has also been reported in association with SLE and it has been postulated that patients deficient in C4a, which binds preferentially to proteins, may fail to process proteincontaining immune complexes properly and that this might predispose them to SLE. Studies have shown an increased frequency of C4a deficiency in patients with SLE compared to controls. C4b deficiency has been shown to cause increase predisposition to bacterial infections. There have been no reports of C4 deficiency being associated with urticaria and we suggest that there may be a group of patients with physical urticaria who havc C4 deficiency.

ranged from 2 to 7 years and thc follow-up in our clinic ranged from 1 to 4 years. Five patients are male and 4 are female. None has a positivc family history for malignancies, immunodeficiency, autoimmunity t~r metabolic diseases. In addition to lymphadenopathy and hepatosplenomegaly, 6 patients have persistent thrombocytopenia, 4 have persistent anemia and 5 have severe neutropenia. Altogether, 7 of 9 patients have one or more hematological abnormality. IgM levels range from 125 to 6,400 mg/dl. IgG levels range from 1,200 to 5,3511 mg/dl. One patient is IgA deficient: in the others, levels range from 36 to 412 mg/dl. All serologies, including H1V, have been consistently negative, Despite their prohmged clinical course, all patients remain stable. Patients with severe anemia and neutropenia receive chronic steroid and IVIG treatment. Efforts continue to determine if all patients have different manifestation of one common etiology or if various causes can be identified in patients with different hematological manifestations. 1393

HLA-Phenotype in the Estimation of the Risk's Factors of Bronchial Asthma. A L Bondarenko, Medical Institute, Kirov, Russia The purpose of the research is to determine the immunogenetic markers of atopic and infectio-allergic asthma. A total of 193 unrelated Kazakh patients with bronchial asthma were subjected to this study. 180 healthy unrelated Kazakh composed control group. Serological typing was performed with the NIH method using reagents of StPeterburg Scientific Research Institute of Haematology and Blood Transfusion and ~Behring Mannheim", Germany. Our investigations defined that the predisposition to atopic bronchial asthma is connected with HLA-BS, B12, Cw2, infectio-allergic- HLA-B13, Cw4. Bronchial asthma is associated with HLA-B7 in women and HLA-B12 in men. Hormonodependencc developed rarely in presence of HLA-B5. A correlation was found between the presence of certain HLA-antigens and clinico-biochcmical and immunological features of infectio-allergic and atopic asthma. Family studies were also carried out in 25 patients. The revealed associations between the immunogenetic markers and bronchial asthma allowed to determine the risk's group and work out the rec~mmendations on profession-social orientation persons with definite HLA-phenotype. On the basis of the obtained data it has become possible to carry out the prognostic the different forms of bronchial asthma and the course of disease.

1394

Study of Specific IgM, IgG, IgA and lgE to Toxoplasma GONDII Antigens and sCD23 in Sera of Toxoplasmosis Patients. M. Romero, i M Ferrero, i CC Castro, z E G Wolff, j H Pizzi, 2 JC Mui~o, z tSecci6n Alergia e Inmunologia, Misericordia Hospital: 2Parasitology Departament 9 UNC, C6rdoba, ARGENTINA. The purpose of this work is to study the relationship of specific IgM, lgG, IgA, and IgE antibodies to toxoplasma antigens and sCD23 in sera from toxoplasmosis disease patients. Wc studied 23 patients which fulfilled the definition criteria to acute (a) and chronic (b) form of toxoplasmic infection, and compared 25 aged, matched, health controls (c). We measured specific IgM, IgG, lgA, and IgE to toxoplasma gondii antigens (Tga); and sCD23 from scra of (a), (b), (c) groups by ELISA tests. We found 18 out of 23 patients in (a) and 5 out of 23 in (b) P < .0005. The group (a) presented specific IgG to Tga in 14 out of 18 cases: one case presented only specific IgE to Tga. In 11 out of 18 cases presented in overlap with specific IgG, IgE, IgM and IgA to Tga in, and 3 out of 18 cases presented only specific IgA to Tga. The (b) presented specific IgG to Tga in 4 out of 5 cases, in I out of 5 patients were IgG, IgM, IgA and IgE to Tga negative. The control group did not present specific IgG, IgM, IgA and IgE to Tga. The sCD23 level was 7.50 _+ 2.39 ng/ml in (a) and 2.02 _+ 1.4 n~ml in (b). The control group (c) presented 3.51 + 1.06 ng/mI, P a vs b/c < .(1005. The highest level of sCD23 in acute group (a) and the specific IgG, lgM, IgA and IgE to Tga features, suggested a polyclonal B activation by the infectious agent.

Study of sCD23 Levels and Specific IgE to Cytosol Acidic Fraction of Trypanosoma Cruzi. S. Gea, ~ RM G6mez,2 M Ferrero, ~ CC Castro.: E G Wo!~ ~-JR Gagliardi, z M Romero,: D losa, JC Muifio. 2 qmmunology Depart.F.C.Q UNC,

ZAIlergy & Immunology Section, Misericordia Hospital, C6rdoba, ARGENTINA. The aim of this work is to study the relationship of sCD23 levels and specific IgE antibodies to cytosol acidic antigenic fraction of Trypanosoma cruzi(CAFT) in sera from Chagas disease patients. We measured sCD23 and specific IgE to CAFT by ELISA tests. We studied 65 Chagas patients defined by clinical and ser~)logical profile, and 3(1 aged, matched health controls (c). Thirty six out of 65 patients were in chronic chagas disease (a), with specific IgG to CAFT (>1:256). Twenty nine out of 65 were in acute form (b), with specific IgM to CAFT (1:64). The (c) were negative to specific lgM and lgG to ('AFT in all subjects. The sCD23 levels were: in (a) 3.97 + 0.98 ng/ml; in (b) 6.36 _+ 0.76 ng/ml, and in (c} 3.40 _+ 1 ng/ml. Pb vs a/c < .{1005; P (a) vs (c) NS. The specific lgE to CAFT was found (+) in 4 out of 36 in (a) and 3 out 29 in (b) P NS. The highest sCD23 levels in acute form, suggested a polyclonal activation by a specific antigen of an infectious agent, trypanosoma cruzi; but the number of patients with specific IgE to CAFT were low in both acute and chronic Chagas' disease. 1392

A Syndrome of Persistent Lymphadenopathy, Splenomegaly and Hypergammaglobulinemia of Unknown Origin in Russian Children. GI Kovalev, I V Kondratenko, A A Bologov, MA Kouznetsova, AJ Usatchova, RU Sorensen *, IB Resnick, Research Institute Pediatric Hematology, Moscow, Russia, Louisiana State University, New Orleans, LA*. Lymphadenopathy, hepatosplenomegaly and hypergammaglobulinemia can be seen in patients with HIV infection, infectious mononucleosis, the X-linked lymphoproliferative syndrome, lymphoreticular malignancies and various other infectious, malignant and metabolic diseases. We have observed this disease manifestations in 9 Russian children in whom no etiology has been identified. The age of onset ranged from 1 to 10 years, the disease duration

$342

1395

Abstracts

Determination of major IgE-binding epitopes of a latex acidic allergen, Hey b 5. L-S Hsieh, B M Martin, A E Doherty, Y Lin, CBER and NIMH, Bethesda, MD Proteins of natural rubber latex (NRL) and latex gloves can sensitize exposed individuals and elicit severe hypersensitivity reaction. Among these proteins, an acidic protein designated as Hev b 5, present in both NRL and latex gloves, has been cloned and purified (Sep/Oct. J. Bio. Chem. 1996). This protein is rich in glutamic acid and proline in the repeated motif of XEEX or XEEEX (X can be any amino acid, but most frequently Lys and Ala residues). Half of the latex allergic patients react to this protein, including health care adults and spina bifida children. To analyze which region of the molecule carries the main IgE binding epitope(s), we purified Hev b 5 by ion-exchange and reversed-phase chromatography. Subsequently we digested Hev b 5 with protease enzymes, such as trypsin, endoprotease Asp-N, and endoprotease glu-C. Peptides from protease digested protein were eluted by High Performance Liquid Chromatography and subsequently sequenced. Eluted peptides were spot-blotted on nitrocellulose membranes and later incubated with the individual patients' sera, which previously demonstrated reactivity to this acidic allergen. The IgE reactive spots were detected by a chemiluminescence-horse radish peroxidase system. Common epitopes were mapped by overlapping the IgE reactive peptides. This demonstrated that the major IgE epitopes are close to the C-terminal segment. Fine mapping to define the essential amino acid residues are underway using overlapping synthetic peptides.

1396

Antigenic determinants in ficin dominate latex-specific IgE responses. JF Halsey and PB Williams, IBT Reference Laboratory, Lenexa, KS. Ficin (EC 3.4.22.3),a cysteine protease with an estimated molecular weight of 23.8-25.5 kD, is isolated from the sap of the ficus tree (Ficusglabrata). This protease is a member of a group of highly cross-reactive sulfhydryl proteases which includes papain, chymopapain and bromelain. Ficin has many commercial uses in the pharmaceutical, food/ beverage, textile, and cosmetic industries. Since it had been previously demonstrated that ficus tree sensitive patients are often latex sensitive, we investigated the immunochemical basis of this sensitivity with purified ficin. Ficinspecific IgE was measured with paper disks to which ficin was coupled by a diazo linkage. Ficin-specific IgE was detected in sera from a significant percentage of the H. braziliensis latex reactive sera. The incidence of ficin positive sera was 20/44 (45%) in latex positive sera and 0/10 (0%) in latex negative sera. Although ficin-specific IgE was observed in many sera with high titers of latex-specific IgE, not all high titer sera had ficin-specific IgE. In all of the ficin positive sera, the binding of IgE to the ficin disk was inhibitable with soluble non-ammoniated latex protein. These inhibitions ranged from 9% to 99.5% with a mean inhibition of 62% and 10/12 showing inhibitions greater than 42%. These findings suggest that ficin or a closely related protein is present in H. braziliensis latex and could represent one of the more important allergenic proteins in latex. In addition, since latex sensitive individuals often suffer food allergies, we speculate that ficin or a related protein may be involved. Since ficin is utilized in a variety of different industrial applications, its involvement in both sensitization and symptom production in latex sensitive individuals may be an important consideration.

1397

Concentrations of Hevein and Rubber Elongation Factor (REF) in Relation to Total Allergen Activity in Extracts of Medical Latex Gloves. T Palosuo, H Alenius, S. MgikinenKiljunen, T Reunala, K Turjanmaa, National Public Health Institute, Institute of Occupational Health, University Hospital for Skin and Allergic Diseases and Departments of Dermatology, Universities of Helsinki and Tampere, Finland Herein (4.7 kD) and REF (14.6 kD) are important natural rubber latex (NRL) allergens but little is known on to what extent these proteins are present in NRL products. We immunized rabbits with highly purified hevein and

J ALLERGY CLIN IMMUNOL JANUARY 1997

REF and, using the immune sera, set up competitive ELISA-inhibition tests for their quantification. Levels of hevein and REF were then compared to total allergen activity, measured by IgE-ELISA-inhibition method, in extracts (1:5; w/vol) of 19 NRL gloves commonly used in Finland in 1995-1996. Concentrations of hevein (range l ng to 32 Ixg per ml of extract) correlated highly significantly to total allergen content of the gloves (r = 0.93, p < 0.0001). Concentrations of REF showed narrower variation (range 1-440 ng/ml) and weaker correlation (r = 0.68, p = 0.0013) to total allergen content. In gloves with low total allergen activity ( < 10 arbitrary (AU)/ml) only low amounts of herein (range 1-8 ng/ml) and REF (range 1-18 ng/ml) were found. Extracts of gloves with high allergen content ( > 100 AU/ml) usually contained txg quantities of hevein but lower amounts (ng range) of REF. These results suggest that a major proportion of total allergen content, particularly in high allergenic NRL gloves, is attributable to herein. Hyperimmune antigenspecific rabbit antisera offer a convenient tool for the quantification of NRL allergens in rubber products.

1398

Molecular Identification of Cross-Reacting Allergens in NaturaIRubberLatexand Banana. J Mikkola, H Alenius, K Turjanmaa, T Palosuo and T Reunala National Public Health Institute, Institute of Occupational Health, Departments of Dermatology, University Hospitals of Helsinki and Tampere, Finland Patients allergic to natural rubber latex (NRL) frequently exhibit immediate hypersensitivity reactions to banana. Previously we showed that the main IgE-binding epitope of prohevein, a major NRL allergen, is present in its N-terminal fragment known as hevein. Hevein is a chitin-binding domain of prohevein and shows high structure homology to several plant proteins. In this study we examined if IgE antibodies against hevein-like structures could explain cross-reactivity between NRL and banana. Sera studied were from a rabbit immunized with purified hevein and from 16 NRL-allergic patients showing IgEantibodies to hevein in ELISA. In immunoblotting, heveinimmunized rabbit serum identified a 33 kD banana protein which also bound IgE-antibodies from 7/16 patient sera. In immunoblot inhibition, purified hevein (10 ng/ml) completely inhibited IgE-binding to the 33 kD banana protein in all 5 patient sera tested. However, no inhibition in IgE-binding to other banana proteins was observed. Five of the 7 patients with lgE antibodies to the 33 kD banana protein showed a positive skin prick test to banana and 5 of the 7 patients had experienced allergic reactions from banana. These results suggest that cross-reacting IgE-binding epitopes are shared by hevein and a 33 kD banana protein and offer a possible explanation at molecular level for the common coexistence of latex- and banana allergy.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1399

1400

Evaluation of Latex-Ragweed Cross-Reactivity by lmmunoblot Inhibition. JA McCullough, SG Rau, AH White, DR Ownby. Detroit, Michigan. Many studies have shown clinical and in vitro crossreactivity between latex (LTX) and other allergens, especially certain foods. The purpose of this study was to determine whether analyses of immunblot inhibitions could explain discrepancies between in vitro and skin test results for anti-LTX IgE. Nine ragweed (RW) skin test positive adults, with positive in vitro tests for LTX-specific IgE (Diagnostic Products Corp.) and no history of LTX scnsitivity, were divided into two groups (Gps). Gpl (n = 4) were LTX prick skin test positive and Gp2 (n = 5) were LTX skin test negative. Two of the Gp2 patients also had oral allergy syndrome and positive skin tcsts to foods. Gp3 (n = 6) were true latex sensitive pcrsons with positive histories, skin tests and in vitro tests. Sera were evaluated with immunoblots prepared from SDS-Page separations of non-ammoniated latex and ragweed extracts. Cross-inhibitions were performed with latex and ragweed extracts. Cat extract (ALK America) was used as the control extract in the inhibition experiments. The cat extract did not inhibit either the LTX or RW lgE binding to immunoblots. The immunoblot lgE banding patterns for all Gps were consistent with previously published data. With all sera, cross-inhibition between LTX and RW was evident, especially with bands at approximately 14.4 and 21,5 kD. Neither the banding patterns nor the cross inhibition results demonstrated consistent differences between sera from the LTX skin test positive and the LTX skin test negative individuals. We concluded that all of the sera studied contain IgE antibodies which are cross-reactive between some LTX and RW allergens; however, neither the banding patterns of immunoblots nor the cross-inhibition results distinguish between LTX skin test positive and LTX skin test negative individuals.

Cellular Immune Response to Purified Latex Proteins. A M Chiu, PS Murali, JN Fink. KJ Kelly, and VP Kump, Medical College of Wisconsin, VAMC, Milwaukee, WI Latex aflergy has increased with the advent of universal precautions and changes in glove manufacturing. Certain patient populations have been found to be at risk for developing sensitivity to latex, such as spina bifida patients (SB) and health care workers (HCW). Furthermore, the development of a reliable standard diagnostic reagent has been slow. The use of purified latex proteins and evaluation of cellular immune response will enhance accurate diagnoses and the standardization of latex reagents. We have purified and characterized latex proteins by SDS-PAGE, electroelution, and specific IgE binding. These proteins were further evaluated for T-ccll responses, including cell proliferation and cytokine and immunoglobulin production. The paticnts were SB and HCW with latex allergy, and normal controls. In proliferation studies, a significant response is a value greater than the mean + 2 SD of values from normal healthy controls in rcsponse to specific antigens. Our results reveal that 14kD, 18kD, 23kD, 25kD, and 66kD antigens were significant proteins when reacted with sera from latex sensitive patients. 86(6/7) of SB and 100%(8/8) of HCW had significant response to 14kD; 71%(5/7) of SB and 67%(6/9) of HCW responded to 18kD; 71%(5/7) of SB and 0%(0/10) of HCW responded to 23kD; 33%(2/6) of SB and 0%(0/8) of HCW responded to 25kD; and 60% (3/5) of SB and 43%(3/7) of HCW had significant response to 66kD antigen. We also showed that total IgE, IL-4, and IL-5 levels were increased in the sera of these latex sensitive patients, 17%(1/6) of SB and 43%(3/7) of HCW had elevated IgE levels; 17%(1/6) SB and 57% (4/7) HCW had elevated IL-4 tcvels; and 29%(2/7) of SB and 33%(3/9) of HCW had elevated IL-5 levels. These results indicate that specific latex allergens cause a cellular immune response in latex sensitive patients, and that this response may aid in the more efficient diagnosis of latex sensitive natients.

Abstracts

S343

1401

Temporal Changes in Latex Protein Concentrations in Latex Gloves. GM Liss MD, GL Sussman MD, D Beezhold, Ministry of Labour, University of Toronto, Toronto, ON, and Hamilton Study Group. Although the manifestations of latex allergy are wellrecognized, it is not known whether lower concentrations of latex protein concentrations will reduce the incidence of sensitization or whether protein concentrations have, in fact, been changing in recent years. During a study of latex sensitization among hospital workers in Hamilton, Canada, we examined the antigenic protein content in samples from the lots of latex gloves being used at 7 different times during a 1.5 year period, 1994-1995, by an indirect ELISA technique (LEAP method), expressed as Ixg/gm of sample. Linear regression was used to analyze changes in protein concentration over time. The latex protein concentration in powdered surgical gloves, initially mean 557 gg/gm, declined at a rate of 295 I~g/gm per year (p < 0.0001); the change in latex protein accounted for 38% of variance. The protein concentration in low protein powderfree gloves, initially less than 1 ~g/gm, also declined but by much smaller amounts. During the time period under study, the protein content of the powdered latex gloves examined decreased significantly by more than an order of magnitude. It is not yet known whether this will result in reduced rates of sensitization.

1402

Ocular challenge with natural rubber latex. O. Karl, A.I. Lauerrna, H. Alenius, 7". Palosuo and 7". Reunala University Hospital for Skin and Allergic Diseases, Institute of Occupational Health and National Public Health Institute, Helsinki, Finland Background. Natural rubber latex (NRL) cause allergic reactions in skin and mucous membranes. Recently, airborne spread of NRL allergens from gloves has been demonstrated. We performed ocular challenges tests (OCT) to examine the sensitivity of eye to NRL allergens. Methods. Three NRL-allergic patients and one nonNRL-allergic control subject were studied with OCT and skin prick test (SPT). The allergens were hevein, a major 4.7 kD NRL allergen (10 p~g/ml) and Triflex • latex glove extract (5 mg/ml protein). Serial OCT challenges were performed with tenfold increases from 10t' dilution. 8 Ix drop was used and contralateral eye was challenged with saline. SPT was performed with the same allergens. Results. OCT was positive with hevein at 105-102 and with Triflex R at 104-103 dilutions. SPT's were positive at approximately same concentrations. All controls were negative. Conch~sions. OCT seems to be a safe and reliable method for assessing NRL allergy. The results show that very minute amounts of hevein (pg) cause symptoms in NRL-allergic patients.

S344

1403

1404

1405

Abstracts

Atypical Case of Latex Allergy (AAA1 1996). C Randolph and B Fraser, Waterbury, CT A 51A-year-old white male presents an atypical instructive case of immediate hypersensitivity to latex without risk factors of atopy or genitourinary abnormalities or food sensitivity. He was previously healthy except for recurrent otitis media, for which he had bilateral myringotomy and tympanostomy at age 1 year (1992), again in 1993 with adenoidectomy, and again in 12/95, as well as hernia repair (1993) and hypospadias repair (1992), presents with a 11/2 year history of generalized hives with nasal and ocular pruritis and discharge with sneezing on exposure to latex gloves only. His mother, a housekeeping person at a local hospital, brought latex gloves home to be used as balloons by her son. Within minutes of exposure he developed hyperemia of the eyes and nose with sneezing and pruritis with generalized hives, which resolved with Benadryl beginning 18 months prior to evaluation. Five months prior to evaluation, while visiting his mother at the hospital, he developed similar symptoms within 20 minutes. He has had no history of atopy to inhalants or foods otherwise, although his mother has inhalant allergy symptoms, his maternal grandfather has inhalant allergy and asthma, and his maternal uncle has asthma. He was evaluated with perennial and seasonal (Allergy Lab of Ohio) inhalant allergy testing, including latex (600 au/ml glycerinated 50:50 with allergist's diluent derived from exam gloves from Bodyguard Triflex, Huntington, CA as RAST inhibited courtesy of Dr. John Yunginger, Mayo Clinic) by Multitest (Lincoln Diagnostics, Decatur, IL) with +1 (5 mm wheal/10 mm flare) but negative to inhalants. Challenge with Triflex Bodyguard examination glove with cot applied to the finger and then wet with water • 30 minutes with no cutaneous reaction on the finger. However, on application of the wet cot to the face and eye area, he developed hyperemia of conjunctiva, sneezing, rhinorrhea, as well as localized urticaria in the perioral area. He was treated with Benadryl 1 tsp with improvement in 15 to 20 minutes. Antibody to latex was determined to be class II (140% Smith Kline Beecham compared to standard) in positive range. Latex Allergy: Epidemiologic Study of 1351 Hospital Workers. G.L. Sussman MD, G.M. Liss MD and Hamilton Study Group. Our objective was to study the prevalence of latex allergy, associated risk factors, and characterize airborne and other latex exposures in health care workers at two Hamilton Hospitals. Of the 1351 participants (66% participation rate) the prevalence of a positive latex skin test was 12.1% (95% confidence interval (CI) 10.3%-13.9%). The prevalence did not vary by gender, age, hospital or smoking status but subjects who were latex positive were significantly more likely to be atopic (P<0.01). Participants who were latex positive were also more likely to have positive skin tests to foods (P<10 9). The prevalence of latex sensitivity was highest among lab workers (16.9%) and nurses and physicians (13.3%). The prevalence of latex skin test positivity was greater in higher tertiles of glove use for sterile (surgical) gloves (P<0.005) but not exam gloves. This large cross-sectional study of health care workers more clearly defines the epidemiology of latex sensitization. Latex Exposure Causing Scarring Ulcerative Dermatitis. Smith C, Garcia M, Kim I~ Long Beach Memorial Medical Center, Long Beach, CA. and Harbor-UCLA Medical Center, Los Angeles, CA. Two types of allergic reactions to latex containing products have been described, i.e. contact dermatitis (T cellmediated, Type IV hypersensitivity) and Ig E mediated. However, scarring ulcerative dermatitis as an immunologic reaction to latex has not been well described. We report a case of a 57 year old female who worked as a nurse for 14 years with scarring ulcerative skin lesions for several years. These chronic disabling skin lesions were later noted to be related to latex exposure. Her Alastat and Pharmacia CAP latex assays were both negative, however her prick test to

J ALLERGY CLIN IMMUNOL JANUARY 1997

latex was positive (1 in 6 extracts). Because of her persistent skin condition, patch testing to latex glove was done. Within 24 hours of patch test, the 1 cm square test area was noted to be a 16" • 5" erythematous patch and she presented in mild anaphylaxis i.e. respiratory distress. Over the next two weeks, the area of patch test as well as her existing skin lesions in arms, face and neck became progressively erythematous and ulcerative. Ten months later, these lesions persisted as non healing ulcers. This pattern of progression of her skin lesions from erythema to chornic scarring ulcerations is similar to what she has been experiencing through the years. This case raises two important points. First, that latex exposure either precipitated this chronic scarring ulcerative skin lesions or aggravated an underlying skin lesion condition, the mechanism which is not well-defined. The second point is, if there is a positive prick test to latex even in an asymptomatic person with negative in vitro latex tests, the authors feel that patch testing is contraindicated because of the risk of anaphylaxis.

1406

% % % %

Atopy in Spina Bifida: Correlation with Parental Atopy. H. Azzam, S. Shah, C. Keenan, D. Kalman, R. Gleeson. Philadelphia, PA, Wilmington, DE. Background: Prior studies identify a prevalence of natural rubber latex sensitivity (NRLS) 10%-72% in spina bifida (SB) populations. NRLS had been found to be increased in atopic individuals. Prevalence of atopy is found to be high in SB, although the reason for this is unclear. Method: In an unselected population of SB patients, history of atopy and NRLS were elicited in 115 patients, parental history of atopy were elicited in 87 patients. Results: Number of patients (115); Mean Age (10,49 years); Age Range (1-21 years), Male/Female Ratio (59/ 56). Results were compared to published prevalence data. Atopy in SB

Previous Studies

Observed

of SB with NRLS of SB with Atopy NRLS SB with Atopy SB Parents/Atopy

10-72 12-44 ? ?

25/115 (23.3%) 34/108 (31.5%) 14/25 (56%) 28/87 (32.2%)

Family History

Gen. Population

Observed SB Patient

No Atopic Parents 1 Atopic Parent 2 Atopic Parents

9%-14% 38%-58% 59%-79%

11/59 (18.6%) 13/25 (52%) 3/3 (100%)

Conclusion: These data confirmed earlier studies reporting high prevalence of NRLS in SB population. The prevalence of atopy in SB is within the range previously reported. It appears that most atopic SB subjects are the children of atopic parents.

Abstracts

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1407

Multi-Center Study of the Biologic Activity of Recombinant Group 5 Mite Allergens. M Chapman, L Vailes,

These results demonstrate that Dp and Bt are important sensitizers in atopics of P Alegre. There is a concordance between skin tests and specific IgE for all age groups. Skin test reaction to Bt was more frequent than to Dp among normal controls. Isolated skin test reaction was elicited by Bt in 4 atopics and in 1 control. Further studies are needed to evaluate the role of Bt in atopic diseases.

S.Bavbek, A Smith, LB Wah, C. Rizzo, C. Naspitz, LK Armda. Charlottesville USA, Sao Paulo Brazil, Manchester UK, and Singapore. Sensitization to Blomia tropicalis is associated with asthma in the tropics, however, standardized B. t extracts are not available for diagnosis or for immunotherapy. To investigate the use of recombinant allergens for diagnostic purposes, we carried out a multi-center study of immediate skin test responses to recombinant Group 5 allergens among mite allergic patients from Brazil (n 33) and Singapore (n=64), who were exposed to both Blomia and Dennatophagoides spp.; and patients from Charlottesville U.S. (n=281 and Manchester U.K. (n=201, exclusively exposed to D.pt. [ntradermal skin tests were carried out using 10 fold dilutions of D.pt. or B.t extracts, or using rBlo t 5 and rDer p 5 produced in pGEX and purified by glutathione chromatography and HPLC. In children, sensitization was assessed by skin prick tests. The prevalence of +ve skin tests to B.t was >90% in patients from Brazil or Singapore, and 60-70% showed reactivity to rGroup 5 allergens. Among "unexposed" patients from the US or UK, 40-60% showed +ve prick tests to B.t, indicating cross-reactivity with D.pt. allergens. The prevalence of +ve skin test to rDer p 5 was 40-50%, however <10% of these patients were skin test +ve to rBlo t 5. In sensitized patients, rBlo t 5 and rDer p 5 gave positive skin tests at 10 5 to 10 1 ixg/ml. Non-allergic controls (n--10) from each study site gave - v c skin tests at 1 ~xg/ml. Serum lgE ab to B.t were detected in >90% of patients from Brazil and Singapore, but in < 10% of patients living in the US or UK. The results show that rGroup 5 allergens have excellent skin test reactivity in B.t sensitized patients and suggest that these allergens, in addition to other recombinant B.t allergens, will be useful for clinical studies of mite allergy and asthma in tropical countries.

1408

S345

1409

IgE Binding Pattern to Dermatophagoides pteronyssinus in Younger and Older Korean Asthmatic Children. SY Lee MD, KE Kim MD Ajou University Hospital Suwon and Yonsei University Hospital Seoul, Korea House dust mites are an important cause of childhood asthma and the positive rate of allergy skin test up to 80% in atopic asthmatic children in Korea. It is widely known that several major IgE-binding proteins of house dust mite such as Group I(25KD), Group II(14-15KD) and Group III(30KD) mite allergens. To identify the IgE binding pattern to Dermatophagoides pteronyssinus (Dp) in Korean atopic children, we conducted IgE immunoblot using Dp extract from culture extract produced in Korea. The sera were collected from 25 atopic asthmatics: Group I, younger children aged 1-3, Group II, older children aged 4-12. In the group I subjects, the 15 KD protein was significantly bound by 69.2% (9/13) of sera and 98 KD, 59KD, 31KD, 25KD components were bound by 8-16% of sera, respectively. In the group II subjects, the 15KD and 25 KD proteins were strongly bound by 92% and 67% of sera, and 18 KD, 94KD by 33%, 210KD, 98KD by 25%, 45KD by 8% of sera, respectively. In conclusion, 15KD protein is the most prevalent IgEbinding antigen in younger and older age groups, thus it is considered as an important component in early IgE response to house dust mite, and 25KD component also major allergenic component in older atopic children in Korea.

1410

The Incidence of House Dust Mite and Common Allergens in Asthma from Infancy to Childhood. KE Kim, SYLee*, HH Lee, KH Park, BJ Jeong, KY Lee Yonsei University, Seoul and *Ajou University, Suwon, Korea There are several studies on the risk factors for the development of atopy, but none are confirmative. To investigate the risk factors inducing sensitization to allergens, we studied the incidence of sensitization to D. pteronyssinus (DP) and D. farinae (DF) and 9 common allergens with MAST in 15(1 patients with asthma from infancy to childhood. The results are as follows; The rate of positive reaction (any one of 11 allergens) was 46.6% (69/150). The rate of positive reaction was increased with age, and the difference in the rate of positive reaction between the groups of 3 years of age and under 2 years of age was significant (p < 0.033). The rate of positive reaction was higher in children with high total IgE level (60.7%) than with normal lgE level (24.5%). The specific IgE antibodies to DP (88.4%) and DF (88.4%) were frequently detected in all ages. The rate of positive reaction to DP and DF allergens were increased with age. In conclusion, DP and DF were the most common and the first inhalant allergens sensitized in children with asthma, and sensitization developed after 3 years old.

Serum specific lgE and skin prick test reactivity to D

pteronyssinus and B tropicalis: a controlled study. S M Spalding, L A G Bernd, V Wald, J P Barbiero, L C Ambrozio. Fac Farmficia UFRGS, FFFCMPA, Fac Veterinfiria UFRGS, Porto Alegre, RS, Brazil. D pteronyssitms (Dp) and B tropicalis (Bt) are the most prevalent domestic mites in our city. We determined serum specific IgE and skin prick test (SPT) reactivity to these mites species in 156 patients (5 to 29 years old) with asthma and/or allergic rhinitis. Control group was constituted by sex and age matched normal individuals. SPT positive (wheal ~ 3 ram) to Dp and Bt were observed in 105 and 120 atopics, respectively, and in 5 and 7 controls (p < 0.05).

Table 1. Skin prick test (mean wheal diameter/positive reactions)

Dp Bt

Atopics

Controls

4.6/105 4.8/ 120

5.7/5 4.6/7

Serum specific IgE was performed in Lab Weinmann P Alegre, using RAST Pharmacia reagents. Results of specific IgE also demonstrated significant difference between atopics and normal controls (p > 0.05). Serum specific IgE is showed in Table 2.

Table 2. Serum Specific IgE to Dp and Bt (IU/ml) Atopics

Controls

Age

Dp

Bt

Dp

Bt

5-9 10-14 15-19 20-24 25-29

22.7 24.0 25.3 17.8 14.2

13.2 15.1 12.1 8.7 5.5

1.8 1.9 1.4 1.2 1.3

3.1 3.7 1.2 1.2 1.2

s JANUARY 1997

1411

Intradermal skin testing with Dermatophagoides pteronyssinus (DP) and Dermatophagoidesfarinae (DF) in a southern United States area where DP is the predominate house dust mite. KS Babe, Jr and SR Marney, Jr, Vanderbilt University, Nashville, TN House dust mite allergens can exacerbate both allergic rhinitis and asthma. Previous data (Babe, et al. JACI, 1996) has demonstrated that DP predominates in Nashville ('IN) bedrooms with an average combined summer and winter dust mite population being 62% DP, 5% DF and 32% immature forms. We did a retrospective review of 100 intradermal skin tests performed on adult patients (age 16 to 76 years) at the Vanderbilt Allergy Center in Nashville from 1994 through 1996. After informed consent was signed, 0.02 cc of antigen (or control) was administered intradermally. A positive histamine control (0.001 mg/ml) and a negative saline control were also obtained. The patients were then tested at increasing concentrations (0.1 AU, 1 AU, 10 AU and 100 AU) of DP and DF antigen (Greer Lab., Inc.) or until a positive reaction. The results were read at fifteen minutes. In some patients the 1 AU test was not performed. Of the 100 patients whose intradermal tests were reviewed, 37 (37%) did not react to either DP or DF, 50% reacted to both DP and DF. However, 9% of the patients tested positive only to DP and 6% of the population reacted to DP at a weaker dilution than DF compared to 4% of the patients who tested positive only to DF and 1% of the population who reacted to DF at a weaker dilution than DP. We conclude that in a region predominated by DP, 15% of those tested intradermally reacted to a weaker dilution of DP than DF or were positive to DP and negative to DF.

1412

1413

The differences and the similarities between Dermatophagoides siboney and other mite species. R. Ferr6ndiz PhD, R. Casas BSc, S. Dreborg MD PhD Link6ping, Sweden; Oslo, Norway Dermatophagoides siboney (Ds) was described as new mite species in 1982. It is found in house dust in the Caribbean. Hereby we present an overview of our studies on the allergenic characterization of Ds. We investigated the pattern of sensitization to this species, in a population of mite sensitive Cuban asthmatic patients, by skin test and specific IgE assay. Major allergens were purified and the crossreactivity between Ds and other mite species was investigated. Up to 97% (n --- 148) of the studied population was sensitized to Ds. Extracts of this mite showed as least 13 allergenic proteins when analyzed by SDS-PAGE and Western blotting. Among these, three major allergens were purified and characterized. They were named Der s 1, Der s 2 and Der s 3. All of three crossreact with the homologous major allergens of Dermatophagoides. The highest crossreactivity of Ds was observed with D. farinae, D. microceras and D. pteronyssinus by IgE inhibition assays, Less crossreactivity was found with L. destructor, A. siro, T. putrescentiae and B. tropicalis. The analysis of the crossreactivity of individual allergens showed that the 65, 62, 37 and 30 kD proteins were always inhibited more than 50% by the different mite species. The 80, 52, 43, 27 and 14 kD were the least crossreacting proteins. No allergenic bands has been found unique for this species.

Storage mite sensitization in German farmers,

H

Mi~sken1, J Th Franze, A Paap3, 0 CromwelP, G Masuch e, K Ch Bergmann I 1Allergy- and Asthma-Clinic, Bad Lippspringe, 2University of Paderborn, 3Allergopharma Joachim Ganzer KG, Reinbek, Germany The storage mite (SM) fauna of German farms includes many species, most of hitherto unknown allergological importance. We performed skin prick tests with SM in 86 farmers (58 male, 28 female, mean age 54.1 years) with rhinitis and/or asthma using extracts from Blomia tjibodas (Btj), Blomia tropicalis (Btr), Blomia kulagini (Bk), Euroglyphus maynei (Era), Glycyphagus domesticus (Gd), Acarus siro (As), Lepidoglyphus destructor (Ld), Tyrophagus

putrescentiae (Tp), Acarus farris (Af), Chortoglyphus arcuatus (Ca), and Thyreophagus entomophagus (Te), In total 51/86 patients (=59.3%) were sensitized to one or several mites. The table shows the rank order of positive reactions.

n %

n %

Btr

Em

Bk

Btj

Gd

Ca

40 46.5

35 40.7

34 39.5

33 38.4

33 38.4

28 32.6

Tp

Ld

As

Te

Af

28 32.6

25 30

23 26.7

18 21

17 19.8

Except for As, Ld and Tp, this is the first time that it has been possible to determine sensitization in Germany using skin prick test. We conclude that German farmers are sensitized to a variety of mites. Our data suggest that several species of SM - especially Blomia species, EM and GD - may be a source of occupational and indoor allergy in Germany.

1414

Skin test reactivity to recombinant Group 5 allergens in mite allergic children living in Sho Paulo, Brazil. LK

Arruda, MC Rizzo, CK Naspitz, LD Vailes and MD Chapman, University of Virginia, Charlottesville VA and Paulista School of Medicine, S~o Paulo, Brazil. Group 5 mite allergens from Blomia tropicalis and D. pteronyssinus have been identified by molecular cloning, and expressed as recombinant proteins. Recombinant Blot 5 (rBlo t 5) produced in E. coil binds IgE antibodies in vitro, and induces positive immediate skin tests in mite allergic children. Serologic analysis revealed that IgE ab to rBlo t 5 and rDer p 5 are detectable in 45% and 55% of mite allergic patients who are exposed to both Dpt and Bt, respectively. To further study the reactivity of recombinant Group 5 mite allergens on skin testing, a group of 33 children with asthma and/or rhinitis living in S~o Paulo, Brazil, aged 7-18 years old, was selected based on a positive skin test to D. pteronyssinus. Skin prick tests were performed using D. pteronyssinus extract (Bayer 10,000 AU/ ml), B. tropicalis extract (2 mg/ml), and rBlo t 5 and rDer p 5 at 5 ~g/ml. In addition, children with negative prick tests under,vent intradermal skin testing with 10-fold increasing concentrations of allergen (10 5 _ 1 Ixg/ml). The results were as follows:

Prick test

Dpt Bt rDer p 5 rBlo t 5

33/33 (100%) 31/33 (93.9%) 20/33 (60.6%) 8/33 (22.2%)

ID test

Allergen concentration

11/33 (33.3%) 23/33 (69.7%)

10,000 AU/ml 2 mg/ml 10-5_10 2 txg/ml 10-5-10 -1 ixg/ml

In keeping with previous studies in Brazil, the prevalence of positive skin tests to Bt among Dpt sensitized children was high (93.9%). The results show that recombinant Group 5 allergens gave positive skin tests in >90% of mite allergic children, at concentrations as low as 10 -5 ~tg/ml. We conclude that rBlo t 5 and other recombinant B. tropicalis allergens will be useful for the evaluation of children with allergic symptoms living in Brazil and other tropical areas of the world.

Abstracts

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1415

Sensitisation to Recombinant BIo t 5 and Der p 5 Allergens in Atopic Subjects and the Evaluation of their Cross Reactivity. Lee BW, Chew FT, Zhang L, *Arruda LK, *Chapman MD. National University of Singapore, Singapore, and *University of Virginia, Charlottesville, VA, USA. Dust mite allergy is highly prevalent amongst the atopic population of Singapore. With the availability of recombinant mite allergens, rBIo t 5 and rDer p 5, which share 40% homology, this study evaluated the sensitisation of these allergens in our atopic populatkm. Seventy-five atopic subjects wcre tested to extracts of D pteronyssinus(Der p) and B tropicalis(Blo t), and recombinant Blo t 5 and Dcr p 5 by skin prick test(SPT), and in 31 of these by intradcrmal tests(ID). Strong SPT reactions(->4• weal) were seen in 59 % and 61 c,f of subjects to Der p and Blo t, rcspectivcly, with 49 % reacting to both. In contrast, although 45 % reacted to rBIo t 5, only 19 % were positive for rDcr p 5 (p <: IL01), with 15 % reacting to both. With the ID tests, there were no difference in the distributkm of Der p and Blot reaction endpoints ( > 8 • weal). However with the recombinant extracts, rBlo t 5 positive reaction cndpoints were clustered at lower allergen concentrations cumpared to rDer p 5 reactions (p < 0.(1111). FAST inhibition studies also showed substantial differences in the inhibition capacity of thc two recombinant extracts. We conclude that rBh~ t 5 is highly allergcnic in our population. Our data suggests that there is little cross reactivity between rBlo t 5 and rDer p 5.

1416

Standardization of a Blomia tropicalis Extract in Brazilian Atopic Children. MC Rizzo, CK Naspitz, D Sold, M Casanovas, E Fermindez-Caldas. UNIFESP-EPM, Brazil, CB.F. Leti, Spain. Blomia tropicalis is onc of the predominant mite species in Brazil. Previous studies have demonstrated a high rate of sensitization to this mite among asthmatic children ( > 90%). The objective of this study was to standardize a B. tropicalis extract in Brazilian atopic children. A fully grown culture was extracted, dialyzed and lyophilizcd. The final product contained 58f/~, of protein. Prick skin tests were performed to determine the amount of lyophilized material required to produce a wheal size equivalent to 10 mg/ml of histaminc HCL The patient population consisted of 28 children, ages 3 to 16 (12 males and 16 females); 24 had asthma (8 mild, 12 moderate and 4 severe) and 4 had allergic rhinitis alone. The extract was diluted in 3 vials containing 5, (I.5 and 0.05 mg/ml. The regression analysis of the bioassay results showed that 1.3 mg/ml are equivalent to 10 Histamine Equivalent Prick units (HEPs). The wheal sizes obtained for each dilution ranged from I to 37.7 mm 2 for 0.05 mg/ml, from 12 to 96 mm ~ for 0.5 mg/ml and from 2(1 to 170 mm z for 5 mg/ml. The reactkms to histamine ranged from 15 to 72mm ~. This is the first B. tropicalis extract standardized in children. Small amounts of lyophilizcd extract are required to elicit a standardized skin test response.

1417

Analysis of the cross-reactivity between BtM and Der p 5, two group 5 recombinant allergens from Blomia tropicalis (Bt) and Dermatophagoides pteronyssinus (Dp). L Cara-

hallo, D Mercado, S Jim~nez, L Moreno, L Puerta, KY Chua. University of Cartagena, Cartagena-Colombia and National Taiwan University, Taipei-Taiwan In tropical climates, sensitization to Bt and Dp is high and directed to species-specific allergens. There is some cross-rcactivity (CR) between extracts of these mites, probably depending on group 5 allergens that have high sequence homology. Knowledge of the extent and specificity of this CR is important to evaluate its clinical role and the IgE response. We used RAST and RAST-inhibition experiments to define the CR between the rccombinant allergens BtM and Der p 5 expressed as Glutathionc S-transferase (GST) fusion proteins, to mapping the epitopes involved and to analyze the importance of this CR. A good correlation among the IgE reactivities of 20 sera from asthmatic patients to the mite extracts was observed (r - 0.6, p = 0.001/. Seventy nine percent of 48 patients sera were RAST

S347

positive to both recombinants, with a strong correlation (r = 0.8, p < 0.0001). BtM inhibited 25 and 21% of IgE-binding to Bt. and Dp extracts respectively and Der p 5 inhibited 22 and 21% of lgE-binding to Dp and Bt. extracts. Furthermore, BtM inhibited 74% of IgE-binding to Der p 5 and Der p 5 inhibited 72% of IgE-binding to BtM. No significant inhibition was achieved with GST or with Bt6~ another recombinant allergen from Bt. RAST inhibition with BtM-derived synthetic peptides showed that P4 (residues 35-50) and P5 (residues 46-61) inhibited 37% and 16% of IgE-binding to BtM while only P5 could inhibit the IgE-binding (32%) to Der p 5. These results show that: 1) There is a high CR between BtM and Der p 5; 2) BtM and Der p 5 account for more than 20% of the IgE-binding capacity of the mite extracts; 3) Sensitization to BtM and Der p 5 occur in 79% of asthmatic patients from this tropical region; 4) The CR between BtM and Der p 5 seems to rely on epitope(s) at the C-terminal segment of these allergens. Supported by Colciencias.

1418

Sensitization to Blomia tropicalis in a city of Argentina and it's relationship with different clinical and immunological parameters. Ardusso LRF, Fern{mdez Caldas E(*), Crisci CD, Ardusso DD, Lock~ RF(*), Procopio N, Mut~oz E,

Galimany J, Marcipar A, Celentano O, Vacirca A1, Bertoya NHI. Rosario Asociation of Allergy and Immunology, Argentina. (*) University of South Florida, Tampa, USA, The aim of the present study was three fold: 1) to evaluate the prevalence of the sensitivity to Blomia tropicalis (BT) in the city of Rosario, 2) to ascertain whether there were differences between the cutaneous responses to whole body and faecal particles of this mite, and 3) to relate Bt sensitization with different clinical and laboratory parameters. Three hundred and fourteen patients, (164 females) aged 5 to 55 years old (~20,8; DS _+ 13.7) were studied. Eighty-seven patients suffered from asthma, 91 from rhinitis and 136 from both pathologies. Skin prick test (SPT) were carried out with 1/50 w/v Bt' whole body (WBE) and Bt' faecal particles (FPE) extracts, as well as with an aeroallergen battery, comparing the wheal obtained from each extract with the one produced by histamine; an a histamine rate >0.5 were regarded as positive. A total of 280 (89.2%) positive SPT was obtained for at least one aeroallergen. From 314 SPT, 224 (71.3%) were positive for Bt WBE and 207 (66%) for Bt FPE. Twenty (7.1%) out of 280 ( + ) SPT responded exclusively to Bt. The prevalence of sensitivity to D pteronyssinus and/or D farinae was 76.1% (239 patients) whereas 38.5% (121 patient) showed positive SPT to other aeroallergens. The comparative analysis demonstrated that the sensitivity to Bt is meaningfully larger in the age group comprised 10 to 20 years (p < 0.ll0l), in patients with a total IgE >300kU/1 (p < II.ll01) and in patients with asthma + rhinitis (p < 0.001 ). Differences in mean size reaction to Bt WBE among groups were also significant (p < 0,001) as well as the levels of total lgE (p < 0.001). In conclusion, sensitivity to Bt in Rosario is as significant as the one to the Pyroglyphids mites, and it is related to age, symptoms and IgE levels.

$348

Abstracts

J ALLERGY CLIN IMMUNOL JANUARY 1997

1419

Allergenicity of the mite Hemisarcoptes cooremani. MA Houck, MS Morgan* LG Arlian*. Texas Tech Univ, Lub-

detect the presence of general proteases, elastase, trypsin, chymotrypsin, lysozyme and amylase. In the carbohydrases group, enzymatic reactions were detected for ~-mannosidase, ~-fucosidase, ~-glucosidase, a-galactosidase, [3-galactosidase, [3-glucosidase, [3-glucoronidase, hexosaminidase, and lysozyme. The proteases detected were trypsin, cysteine proteases, chymotrypsin and elastase. In the phosphatases group, both acid and alkaline activities were seen. Esterase activities for lipase, esterase lipase and nonspecific esterases were also present. Qualitative differences in the enzymatic activity between the extracts analyzed were observed. The analyses of the data showed that the alkaline phosphatase activity was minimal in the fecal particles and similar results were obtained for esterase lipase, trypsin and 13-glucosidase. Cysteine arylmidase and eL-galactosidase activity were detected only in those extracts obtained from the mite bodies while [3-glucosidase was detected in whole mite culture. The analyses of the results clearly demonstrate that B. tropicalis has a large and diverse number of enzyme activities comparable to other domestic mite species reflecting its adaptability to a wide range of environments. In addition, by using enzymatic assays our results also strongly suggest that B. tropical& has allergens belonging to Groups I (cysteine proteases), II (lysozyme), III (trypsin), IV (amylase) and VI (chymotrypsin).

bock, TX and *Wright State Univ, Dayton, OH The astigmatid mite Hemisarcoptes cooremani is commonly found on many species of plants. Since certain plants (e.g., Euonymus) almost always have scales containing H. cooremani, orchard managers, ornamental nurserymen and even lay gardeners may be exposed to this mite. The purpose of this study was to investigate the allergenicity and cross-reactivity between H. cooremani and other astigmatid mites. A 50 year old woman who has worked with H. cooremani cultures for 13 years reported experiencing allergic symptoms (rhinitis, watery eyes). Skin prick tests revealed a 4+ reaction to the house dust mite Dermatophagoides farinae and the patient received immunotherapy. Serum from this subject was tested by lmmunoCAP and was class 1/0 to D. farinae but negative to D. pteronyssinus and Euroglyphus maynei. Total IgE was 66 U/ml. Serum from this patient was used to probe an SDSPAGE immunoblot containing 9 species of astigmatid mites (H. cooremani, Lepidoglyphus destructor, Blomia

tropicalis, Tyrophagus putrescentiae, D. farinae D. pteronyssinus, E. maynei, Sarcoptes scabiei. Psoroptes ovis). Two IgE binding bands of 18 and 20 kD were observed in the lane containing 14. cooremani. An 18 kD IgE binding band was also observed in the lanes containing extracts of the house dust mite E. maynei and the parasitic mites S. scabiei andP. OPiS.

The results of this study indicate that the mite Hemisarcoptes cooremani contains IgE-binding allergens that are similar to allergens present in other astigmatid mites. Additionally, allergy to this mite may develop and should be considered in the diagnosis of allergy in those who garden. 1420

Allergenicity of the Storage Mite, Tyrophagusputrescentiae.

LG Arlian, DL Vyszenski Moher, M van Hage-Hamsten* Wright State University, Dayton, Ohio, USA, and *Karolinska Instituet, Stockholm, Sweden Significant numbers of non-pyroglyphid storage mites are found in habitats associated with farm buildings, grain storage facilities, and with their contents. Storage mites sensitize and elicit allergic reactions in farmers and persons in related fields (grain handlers, bakers, workers in grain storage facilities, etc.) who are exposed to them in the work environment. Therefore, the purpose of this study was to characterize the allergenicity of Tyrophagus putrescentiae (TP) using sera from 24 individuals whose main occupation was farming. All of the farmers were RAST positive (0.9-28 PRU/ml) to TP. Homologous CIE of TP extract electrophoresed into rabbit antiserum built to TP resulted in 20 antigen/antibody precipitates. CRIEs of the homologous TP reactions using sera of individual farmers revealed this population had circulating IgE that recognized 14 of the 20 TP antigens as allergens. The number of precipitates that bound IgE in the individual sera ranged from 5 to 11. Autoradiograms revealed that 38 and 25% of the sera had IgE binding to 6 and 7 of the allergens, respectively. One hundred percent of the farmers' sera recognized 5 of the same allergens (all anodieally migrating). One of the cathodically migrating peaks was recognized by 50% of the subjects. The results of this study showed that farmers who were occupationally exposed to storage mites had serum IgE specific for numerous allergens in Tyrophagus putrescentiae extract. This indicates that persons who are exposed to large numbers of stored product mites in occupational settings may be at risk for allergic reactions. 1421

Enzyme activities in extracts from the domestic mite

Blomia tropicalis. F. Montealegre, C. Qui~ones and S. Ortiz. Ponce, Puerto Rico. The purpose of this study was to detect the enzymatic activities in extracts from purified bodies, fecal particles or whole mite culture from the domestic mite B. tropical&. To measure the enzyme activities present in these extracts, the Apizym kit was used in conjunction with other substrates to

1422

Cockroach Allergy: Relative Sensitivity to American and German Cockroach. P Ben&casa MD, A Wolff MD, L Bielory MD, E Orange VA Medical Center, E Orange, N J. Several studies support the importance of cockroach sensitivity in inner city populations with allergic respiratory diseases. High prevalence of skin test reactivity to cockroach among atopic individuals has been observed. We retrospectively reviewed skin test results from 84 patients in an adult VA allergy clinic to determine exposure to cockroach (CR) and the relative incidence of sensitivity to the two species most commonly associated with the human environment: Blatella Germanica and Periplaneta americana. The study population consisted predominantly of inner city, adult males with allergic rhinitis with or without asthma. Commercially available whole body extracts to American (AWBE) and German CR (GWBE) were used. 22 (26.19%) of 84 patients had a positive skin test to CR. Of these 18 (21.42%) reacted to AWBE and 21 (25%) to GWBE. 17 (20.23%) patients were sensitive to both species while only 5 were sensitive exclusively to one or the other: 1 (1.19%) only to AWBE and 4 (4.76%) only to GWBE. These data support previous reports on the prevalence of CR sensitivity in atopic inner city populations and evidence that there is sufficient exposure to these allergens to develop significant levels of specific IgE. The slightly decreased incidence of CR sensitivity found when compared to prior studies may be due to population characteristics. Ongoing research has identified antigenic/allergenic proteins from both whole body extracts and feces and evidence of shared interspecies allergens has emerged. Positive skin reactivity to AWBE and GWBE in the majority of patients may reflect significant cross-reactivity of antigen rather than exposure to both species and may justify testing only to one: GWBE. In our population this would have produced only 1 false negative result. These data support the inclusion of CR antigen in the routine battery of inhalant skin tests for patients with allergic rhinitis and/or asthma.

J ALLERGY CLIN IMMUNOL

Abstracts

S349

VOLUME 99, NUMBER 1, PART 2

1423

Cockroach skin test reactivity as a marker of bronchial obstruction in children with asthma. SB Sarpong and T Karrison. The University of Chicago, Chicago, IL. Background: Specific lgE response to cockroach and other indoor allergens have been found to relate univariately to children prcsenting to the emergency room with asthma, but their independent relationships are often unclear because they are interrehtted and there are geographic and demographic variations. Objective: The purpose of this study was to present a multivariate analysis of the association between asthma severity as assessed by forced expiratory volume in one second (FEV~) and skin test rcactivity to indoor allergens, demographic and geographical characteristics. Methods: The charts of 159 consecutive children, aged 5 to 18 years, with asthma initially referred to a Pediatric Allergy clinic were reviewed to obtain the results of skin tests to cat, dog, cockroach and dust mite and FEV~. Stepwise multiple regression was used to examine the association between independent variables and low FEV~(Icss than 80<~ predicted). Results: The rate of allergen sensitivities were: cockroach 50%, dust mite 54%, dog 18% and cat 32r In a multivariate analysis of the skin tests only, dog and cockroach sensitivities werc related to low FEV~, whereas cat was negatively related and dust mite showed no relation. The significance of cockroach sensitivity remained after further adjusting for urban residence and financial class. However, cockroach sensitivity was not found to bc a significant independent predictor of low FEV: after adjusting for race. Conclusion: Cockroach sensitivity was positively related to low FEV~, even after adjusting for other skin test rcsults. Cockroach sensitivity may bc interrelated with race.

1424

Manufacture of diafiltered and lyophilized German and American cockroach extracts. L Baldwin, G Plunkett, M Soto-Aguilar, M Amend, and T Kordash. Bayer Corp, Spokane, WA, University of South Alabama, Mobile, AL. Cockroaches are important sources of indoor airborne allergens. Commercial sources of cockroach extracts have been limited to weight/volume (w/v) extractions. The purpose of this study was to 1) analyze cockroach (w/v) extracts and 2) investigate alternative manufacturing schemes. Weight/volume German and American cockroach extracts were produced by extracting whole lyophilizcd and defattcd insects in Coca's or Glycero-Coca's diluent. These extracts were shown to contain high quantities of low molecular weight components that may interfere with skin test results. Some of these compounds intcr|i:rcd with PNU values. These extracts also contained proteases which are detrimental to the stability of the extracts (or extracts mixed with cockroach extracts). SDS-PAGE immunoblot analysis of w/v cockroach extracts showed protein band differcnecs between fresh and stored extracts. For this reason, extracts were produced that were dialyzed and then lyophilizcd. These extracts were analyzed by IgE ELISA inhibition using a scra pool composed of cockroach sensitive individuals and were shown to have comparable potency to w/v extracts at equivalent extraction weight. To achievc extracts of higher potency than w/v extracts, the preparations could be concentrated after the dialysis step and reconstituted to various volumes. This was verified by skin testing and histamine release in 15 cockroach sensitive patients. SDS-PAGE immunoblotting showed lgE reactive proteins of MW 10 kD to 120 kD. Dialyzed anti lyophilized cockroach extracts would, therefore, provide more consistency and stability over w/v extracts. These extracts would also have the advantage of containing less low molecular weight compounds and irritants.

1425

Characterization of group I and 5 pollen allergens from various grass species. H lpsen I, J Arnved 2, M Lombardcro ~, JN Larsen l, MD Span~'ort I, M Wissenhach I, ~ALKABELL(), H,arsholm, Denmark. 2Copenhagen, Denmark ~ALK-ABELL6, Madrid, Spain. The group l and 5 allergens are the most important allergens of grass pollens. Both allergen zroups seem to

exist in numerous isoforms in the wlrious grass species and patients" serum lgE antibodies seem to cross-react extensively with the various isoforms and homologous proteins from other grass species. Purified natural group 1 allergens from four grass species Lol p, Phi p, Dac g and Poa p and purified natural and recombinant Phl p 5 were mapped with regard to reactivity towards murine monochmal antibodies raised against Lol p 1, Cyn d 1, Lol p 5 and Phl p 5. The binding patterns observed with four different group 1 specific monoclonal antibodies indicate that the antibody binding epitopes on the allergens, from various grass species, differ with regard to affinity. IgE from individual grass pollen allergic patients exhibit differences in their reaction with group 1 allergens from individual grass species indicating that the allergens contain both species, or group of species, specific and common antibody binding epitopes. The reaction of four different monochmal antibodies with natural and recombinant Phi p 5 indicate that individual isoforms (rPhl p 5) do not contain the complete spectrum of antibody binding epitopes. Further, natural group 5 allergens from various grass species and recombinant Phi p 5 appear to exist as a dimers. ELISA experiments performed with the same monoclonal antibody as catching and detecting antibody indicates that only one antibody with the same specificity binds to the dimer molecule. The existence of dimers of group 5 allergens complicates the characterization of these allergens since both isoforms, and homo-/hetcro-dimers should be considered.

1426

Characterization of Allergens from 15 PeniciUium Species. AJ Thaker I, P Comtois:, M W De Vouge 1, M Burton l, B Escamilla Garcia-', P Ernst -~, MR Becklake ~ and H M Vijay( ~Life Sciences Div., Bureau of Drug Research, Drugs Directorate, Health Canada, Ottawa, CANADA; Respiratory Health Network Centres of Excellence: "-Department of Geography, Universit6 de Montr6al, Montr6al, CANADA; 3Dept. of Epidemiology & Biostatistics, McGill University, Montrt!al, CANADA. Penicillium is not only one of the most frequently encountered and diverse of mold genera found in Canada's indoor air, it is also one of the most predominant genera whose abundance may be related to the increased incidence of childhood asthma. To determine whether specific Pcnici[hum species may he significant to the study of asthma epidemiology, we investigated the allergenic characteristics of 15 Penicillium species determined to be the most abundant and frequently observed in the indoor air of the Montr6al and Vancouver metropolitan areas. Extracts were investigated by total protein and specific enzyme determinations, isoelectrofocusing, SDS-PAGE and immunoblotting using pooled human atopic IgE. Considerable variation was observed between Penicillium species with respect to protein yield in the extracts, as well as the number of distinct protein bands resolved in IEF. Enzymatic activities in extracts were determined using the Api-Zym diagnostic system: the most common activities observed among the extracts included acid and alkaline phosphatases, phosphodiamidase and [3-glucosaminidase. The number of discrete IgE-reactive bands in immunoblots of Penicillium extracts ranged from ! (P. clwsogenum) to 9 (P. viridicatuml. Certain allergens showed potential for cross-reactivity between species, including 52 and 54 kDa proteins in P. mL~te and P. putpurcscens and 40 kD proteins in several species. These findings support the need to precisely identify resident species in indoor air, and test atopic individuals with known, specific and selected extracts.

S350 Abstracts

1427

Relevance of a Novel Allergen ofAlternaria alternata in the Sensitization of Allergic Patients in Argentina. /4. E.

Neffen, A. Cipolatti, M. C. Lurdt de Calafell, M. Etcheverrigaray. Facultad de Bioquimica y Ciencias Biol6gicas. Universidad Nacional del Litoral, Santa Fe. Provincia de Santa Fe. Argentina. The high incidence of airborne fungal allergens as ethiologic factors of bronchial asthma in this region and the low efficiency in diagnosis and treatment of this type of hipersensitivity with foreign extracts, gave rise to the development of standardized local extracts with defined potency and composition. A local strain of Alternaria alternata was grown in different chemically defined media (Czapeck and Asparragina Broth), without stirring for 28 days. Mycelia and spores were separated from the culture supernatant by filtration, extracted, dialyzed and freezed. Culture supernatant was dialyzed, concentrated and freezed. IgE binding proteins from the allergenic materials were identified by Western-Blot, after SDS-PAGE under reduced conditions, with pooled sera from sensitive patients, by an Avidin-Biotin amplified ELISA. The potency of the crude extracts and the supernatants was established by RAST-Inhibition assay. A high MW protein (108 kD), not previously described in other strains of Alternaria, was the major allergen recognized by patient's sera and several other allergens with different MW were also found, both in mycelia and spore's extracts and in culture supernatants. In contrast, the commercial extracts assayed in parallel showed no bands. Moreover, their potency was lot dependent. These results suggest that the foreign allergens are not specific for the IgE present in our patient's sera and demonstrate the importance of using local allergens in the treatment of this fungal hipersensitivity.

1428

Mushroom Allergens. KA Brander, A Helbfing, Institute of Immunology and Allergy, University Hospital, Bern, Switzerland A recent European multicenter study showed that sensitization to basidiomycetes (common mushroom) occurs in up to 20% of subjects with respiratory allergic diseases. This survey strongly suggests that basidiomycete (-spores) are important aeroallergens which may cause respiratory allergies. In order to further characterize basidiomycete allergens we prepared extracts of common and edible mushrooms: Coprinus comams (Cc), Pleurotus ostreatus (Po) and Boletus edulis (Be). Extracts were routinely used to evaluate sera of basidiomycete skin test positive subjects. These sera were investigated by using dot-blot and SDS-PAGE analysis. Detection with peroxidase-linked anti-IgE and ECL revealed different IgE-reactive proteins in a species-specific pattern. Among the 4 to 9 detected bands of Po and Cc, respectively, some putative common allergens (same size) were found to be 26, 65 and 83 kDa in size. Screening of a hgt 11 expression library of Cc yielded, beside some others, in the isolation of a strongly IgE-binding clone, designated Cop c I. This cDNA is not full length and no homology to any isolated gene could be found. The deduced amino acid sequence showed a positively charged protein with a pI of 11.2. Presently we are determining the 5' end of the gene and are producing the C-terminal part of Cop c I in E. coli as a His-tagged protein of 171 amino acids (18.7 kDa). In order to isolate most of the allergens from Cc and Po we started to synthesize phage display libraries. Via molecular isolations and characterizations of genes, encoding basidiomycete allergens, the contribution of specific allergens in patient's total IgE-reactivity to basidiomycetes will be determined. These recombinant allergens will facilitate the development of reliable diagnostic approaches for patients with basidiomycete-respiratory allergies.

J ALLERGY CLIN IMMUNOL JANUARY 1997

1429

Recombinant Alt a 2 Is A Significant Alternaria Allergen. R Bush, H Sanchez, D Geisler, Wm S Middleton VA Hospital, Madison, WI Alternaria is an important factor in the pathogenesis of asthma. To date, Alternaria allergens have not been fully characterized. We have molecularly cloned, expressed, and determined the cDNA sequence of a recombinant Alternaria allergen, r Alt a 2. In this study, we sought to further characterize and evaluate the significance of r Alt a 2. r Ah a 2 was expressed in Pichia pastoris and the secreted protein purified by Sephadex G-75 gel filtration. IgE binding to r Alt a 2 was found in 16 of 26 (61%) sera fromAlternaria sensitive asthmatics by dot-immunoblotting. SDS-polyacrylamide gel electrophoresis of r Alt a 2 demonstrated a 25 KD band and isoelectric focusing indicated a pI of 7.1 which closely agree with the molecular weight and isoelectric point predicted by the deduced amino acid sequence of the protein. We have produced a recombinant Alternaria protein, r Alt a 2, which appears to be a significant allergen.

1430

Allergy to Ficus benjamina (Fb) and Natural Latex (NL). B Przybilla, F Rufff Dermatologische Klinik und Poliklinik der Ludwig-Maximilians-Universit/it, Munich, Germany. In view of the epidemic of immediate type allergy to NL, cross reactivity between NL and other allergens has found increasing interest. Investigations using sera of NL-allergic patients have demonstrated common allergenic structures in NL and Fb, a common ornamental plant. We treated a 34-year-old female gardener with a two-year history of rhinoconjunctivitis during work and contact urticaria to Fb. For some months she had developed also asthma attacks at her work-place. She had noticed swelling of the lips after blowing up toy balloons, and ingestion of papaya or apples produced oral pruritus. Skin prick tests (SPT) with standard allergens revealed multiple reactions to various pollen and animal allergens, and to NL milk. SPT with sap and homogenized leaves from Fb as well as with homogenized papaya caused immediate skin reactions, which were not seen in 10 controls. Specific IgE-antibodies in the serum were found by CAP-FEIA to NL (class 2) and to Fb (class 5). Nasal challenge tests with sap from Fb (0.001%) induced rhinoconjunctivitis and oral pruritus. Blowing up a NL toy balloon caused swelling of the lips and coughing. Binding of the patient's IgE antibodies to NL in the CAP-FEIA was inhibited with a commercial Fb preparation in a dose-dependent manner up to 100%, whereas no inhibition of binding ofFb-specific IgE antibodies occurred after preincubation with a commercial NL preparation. It is concluded i) that cross reactivity between NL and Fb can be clinically relevant, and ii) that in this patient NL allergy possibly was secondary to Fb sensitization.

Abstracts

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1431

Cross-Reactivity Among Mugwort and Sage Pollens: Evaluation by ELISA Inhibition and Immunoblotting of Nine Artemisia Species. R W Weber, FL Lin, W W Stafford, RA Ledoux, CR Westlev, RK Katial, Walter Reed Army Medical Center, Washington, DC. Background: Plants of the genus Artemisia are a source of fall allergic symptoms, particularly in the western United States. Studies have characterized the allergens in one of the major species (.4. vulgaris) but currently there is no cross-reactivity data on the major United States species, Objective: The purpose of this study was to investigate the in vitro cross-reactivity among nine Artemisia species: A. frigida, A. atmua, A. biennis, A. filiJblia, A. tridentata, A. caliJbnzica, A. gnaphalodes, A. h~doviciana, and A. vulgaris. Methods: The cross-reactivity was demonstrated with the use of enzyme-linked immunosorbent assay inhibitions and immunoblotting techniques utilizing a serum pool from patients allergic to Artemisia species. Results: The enzyme-linked immunosorbent assay inhibitions revealed strong cross-reactivity among all nine species with A. biennis and A. tridentata being two of the strongest inhibitors. The polyacrylamide gel electrophoresis showed a great deal of similarity in the bands among the nine species. The nitrocellulose blots showed similar lgE binding patterns among the Artemisia species with strong inhibition among all 9 extracts. Conclusions: These data all demonstrate very strong in vitro cross-reactivity among the nine Artemisia species studied. Such data has significant clinical relevance for allergy skin testing and formulation of immunotherapy extracts.

1432

Identification of an lgE-binding epitope of a recombinant allergen from the mite Blomia tropicalis (Bt). L Moreno. RG Hamilton, T Rafnar, S Jim&zez, DG Marsh, L Caraballo, University of Cartagena, Cartagena, Colombia and Johns Hopkins Asthma and Allergy Center, Baltimore, MD. Bt is an important source of allergens of clinical significance in tropical and subtropical regions, specially as asthma and rhinitis inducer. BtM is a major, group 5, recombinant allergen from Bt. For evaluating its immunological properties and possible diagnostic and therapeutic usefulness, in this study we analyzed the B-cell allergenic epitopes on BtM, Enzyme immunoassay (EIA) inhibition of IgE antibody binding was done with BtM-deduced overlapping synthetic peptides (P1-P6) and sera from mite allergic patients. BtM was expressed in pGEX-4T-3 as a fusion protein with Glutathione S-transferase (OST-BtM) to be used on solid phase. Thirty-three sera were screened by EIA for IgE antibodies to Bt extract and GST-BtM. Ten were positive to both and all were negative to GST alone. Testing these ten sera using the peptides as inhibitors, P4 (residues 35-50) produced more than 50% of inhibition and a dose response curve in 60% of the sera. With one of these, serum A10I, a complete inhibition (>95%) was obtained. Furthermore, this and another serum (A97) were monospecific to P4. With two sera, P6 induced 34 and 26g{of inhibition. No inhibition was achieved with an irrelevant peptide from Arab a 5 allergen. Our results suggest that the recombinant allergen BtM has one B cell epitope recognized by specific IgE from allergic patients. Based on the patterns of sera reactivity, this allergenic epitopc (residues 35-50) might be linear and immunodominant. The existence of another epitope, probably conformational, is suggested by the reactivity of two sera with P6. Supported by grants HDP/HDR/RG/COL/I105 from PAHO and 131-92 from COLCIENCIAS.

1433

Monoclonal Antibodies Induced by Cupressus arizonica Pollen Extract Recognize Also Allergenic Components Of The Closely Related Species Cupressus sempervirens. G Di Felice, B Barletta, A Mari, C Afferni, R Tinghino, C Pini, Istituto Superiore di Sanith, Rome, Italy. Plants belonging to the Cupressaceae family are recognized as an important cause of respiratory allergies in North America, South Africa, Australia and in the Mediterranean area. Monoclonal antibodies (MoAbs) specific for allergenic

$351

components of C. arizonica were produced by injecting mice with the whole pollen extract (CaE) or with the major allergenic component separated by SDS-PAGE and transferred onto nitrocellulose (NC). Their reactivity was investigated by ELISA and immunoblotting after SDS-PAGE. To determine if they recognized carbohydrate or protein epitopes, mild periodate oxidation at acid pH was applied to CaE bound to ELISA wells or to NC sheets. Three MoAbs were selected on the basis of their differential pattern of reactivity against CaE. MoAbs 4A6 and 2D5 recognized periodate-resistant epitopes whereas the MoAb 5E6 reacted with a periodate-sensitive determinant, as detected by direct ELISA. Immunoblotting analysis confirmed these results and evidentiated that the three MoAbs recognized epitopes represented on the CaE major allergen (a couple of bands with MW of 43 and 41 kDa) but also shared by other components, with different degrees of heterogeneity between the MoAbs reactivity pattern. When the three MoAbs were tested against C. sempervirens pollen extract (CsE), restricted patterns of reactivity were obtained with the two "'protein" specific MoAbs (4A6 and 2D5), which bound few components in the MW range 50-42 kDa, whereas the "carbohydrate" specific MoAb 5E6 maintained a spread reactivity with poorly defined bands in the 100-70 and 36 kDa regions. These MoAbs will be useful for the fine characterization of the nature of allergenic components of cypress pollen, for the standardization of pollen extracts and to study the relationship between allergens of related species belonging to the same family.

1434

Demonstration of conformational IgE epitopes in ash (Fraxinus excelsior) pollen allergens by western blot. W Hemmer, M Focke, R Bracun, F Wantke, M Gbtz, R Jarisch, Dermatologic & Pediatric Allergy Centre, Vienna, Austria. In a previous study on ash pollen allergens we observed that many patients shown to be allergic to ash by skin prick test and RAST turn out negative in the western blot. We therefore investigated whether this could be due to the presence of conformational allergen epitopes in ash pollen destroyed by the denaturing conditions of routine SDSPAGE. Sera from 25 patients with positive skin prick test and positive RAST to ash but negative immunoblot results were reinvestigated by western blot following non-denaturing/ non-reducing gel electrophoresis. In 18 patients (72%) immunoblots became positive showing IgE binding predominantly to proteins of 17 and 20kD, presumably corresponding to the ash major allergen Fra e 1 double band. Additional IgE binding occurred at 23 and approximately 40kD. In 7 patients immunoblots remained negative. Blots with reference sera indicate that electrophoretic migration of denatured and non-denatured allergens does not differ significantly. In correspondence with recent reports about conformational IgG epitopes in the homologous olive protein Ole e 1, the present findings strongly indicate the presence of conformational IgE specific epitopes on Fra e 1 and other ash allergens. Future immunoblot studies on sensitization patterns to Oleaceae pollen allergens have to be better carried out employing non-denaturing gel elecrophoresis.

S352

1435

Abstracts

Identification of profilin in oilseed rape (Brassica napus) pollen. M Focke, W Hemmer, R Bracun, F Wantke, M GOtz, R Jarisch, Dermatologic & Pediatric Allergy Centre, Vienna, Austria. Allergens from oilseed rape (OSR) pollen have been recently shown to lie between 6/8 and 70 kD. We investigated the potential profilin-like character of OSR proteins, particularly a 12/14 kD double band, by PLP-affinity chromatography, SDS-PAGE and western blots. OSR pollen extracts were incubated overnight at 4~ with poly-(L-prolin) coupled to BrCN activated Sepharose 4B. After washing, profilin was eluted with 6M urea. The non-bound and the eluted fraction as well as the original extract were separated by SDS-PAGE and either stained with silver or transferred to nitrocellulose for immunoblotting with sera of OSR and birch allergic patients. Inhibition experiments were carried out with an OSR-positive serum pool using various concentrations of birch pollen extract and recombinant birch profilin. All extracts were also investigated for their affinity to a mouse monoclonal antiBet v 2 antibody. Only a 14 kD protein showed affinity to the PLP column as revealed by total protein staining of gels, however, on western blots with OSR-positive sera also weak binding to a 6/8 kD double band was seen. IgE binding of birch allergic sera to OSR proteins occurred only to the 14 kD protein and was only positive in patients with known Bet v 2 sensitivity. The 14 kD protein was strongly recognized by the a-Bet v 2 moAb. Preincubation of the OSR-pool with birch extract or rBet v 2 revealed complete inhibition of IgE binding at 14 kD. Binding to the 6/8 kD double band was slightly quenched. The results show that a profilin of 14 kD represents an important allergen in OSR pollen. This allergen is recognized by more than 50% of OSR-allergic patients. The 6/8 kD double band seems to be partially profilin-like.

1436

Comparative Analysis of IgE-Binding Epitopes of Fruit and Pollen Extracts of the Date Palm (Phoenix dactylifera L.). AAA Kwaasi, RS Parhar, FA AI-Mohanna, KS Collison, S Saleh, ST Al-Sedairy, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia The date palm pollen is a potent allergen that possesses 6 major allergens Pho d 1-Pho d V1. Since date fruit, rather than pollen extract has been commercially available for skin prick testing (SPT) for several years we decided to investigate how the IgE reactivities of local fruit extracts compare with pollen. IgE ELISA of 25 individual sera from date allergic patients revealed weaker reactivities with fruits than the pollen (p = <0.05). Western blotting of IgE binding components using pooled allergic sera gave 10 distinct bands with pollen extracts compared to 6 with fruit extracts. Out of the six known major Pho d allergens of date pollen only Pho d I & II (12kD and 14.4 kD) bands bound IgE in the fruit extract. However, strong fruit extractspecific bands of 18-20kD, 23-25kD, 26-28kD and 66-69kD which are distinctly different from those of the pollen bound IgE in patients' sera. Results of radioallergosorbent assay (RAST) using disks prepared in our laboratory with these extracts gave higher pollen-specific IgE values than were obtained for fruit extracts (p < 0.02). These results indicate that date palm pollen is more allergenic than the fruit and the decreased allergenicity of the fruit may be due to the lack of the full complement of the major allergens. The role of the 4 fruit-specific IgE binding peptides that are absent from pollen warrant further elucidation.

1437

Isoallergens in exposure and response. J.N. Larsen, C. Cvitanich, H. [psen, R.J.J. van Neerven, M. Spangfort, C. Schou. ALK-Abell6, H~rsholm, Denmark. The present study addresses differences in composition of the sensitizing allergen, and the differential reactivity of individual patients' T- and B-cells towards isoallergens. Major allergens are composed of mixtures of isoallergens differing in amino acid sequence. The heterogeneity between individual Birch trees was studied by Southern blotting showing from 3 to 11 bands of varying mobility, indicating differences in both the number and identity of

J ALLERGY CLIN IMMUNOL JANUARY 1997

genes per tree. These results were supported by analyses of single stranded conformation polymorphism and 2-D PAGE immunoblotting of pollen from individual trees showing a comparable number of spots. Gene cloning and sequencing showed up to 38 nucleotide substitutions comparing sequences derived from different trees and up to 35 substitutions comparing sequences from the same tree. Thus, the substitution frequency between isoallergens from the same tree is comparable to that of different trees. When analyses of 55 Bet v 1 sequences were combined with the recently determined 3-D structure, conserved residues exposed on the surface are shown to form areas large enough to accomodate cross-reactive B-cell epitopes. Non-conserved residues suggest a complex pattern of epitopes shared by various subsets of isoallergens. This model is supported by quantitative differences in IgE binding using purified recombinant allergens. Also the specificity patterns of Bet v 1 specific T-cell clones were found to differ. One T-cell clone which was mapped using synthetic peptides to react with an epitope within aminoacid residues 19-33, was found not to be reactive with an isoallergen carrying a D to G substitution in position 25. In conclusion, both IgE and T-cells show differential reactivity towards purified recombinant isoallergens of Bet v 1.

1438

Purification, Sequencing, and Biological Characterization of Two New Allergens of Olea europaea Pollen. L Boluda, C Alonso, E Ferndndez-Caldas, Universidad Autdnoma and C.B.F. LETI, Madrid, Spain The inhalation of olive tree (Olea europaea) pollen is an important cause of allergic respiratory diseases in southern Europe and California. Three allergens (Ole e 1, Ole e 2, and Ole e 3) have been previously described. The aims of this study were to characterize the allergenic composition of this pollen and to purify two previously unrecognized allergens. One hundred grams of O. europaea pollen, collected in California (Biopol, Spokane, WA), were extracted in ammonium bicarbonate, dialyzed in 10,000 kDa cut-off membranes and lyophilized. Allergens were isolated by different chromatographic procedures, including gel filtration (Sephadex G75), ion exchange (DEAE cellulose), and hydrophobic interaction (Phenylsepharose). Two main allergens, Ole e 4 and Ole e 5, with an IgE binding frequency by immunoblot of greater than 70% and 30%, respectively, were isolated in two different fractions. Ole e 4 has an apparent molecular weight, under reducing conditions, of 32 kDa. The analysis of the aminoacid sequence of an internal region revealed no homology with other known proteins. Ole e 5 has a molecular weight of 16 kDa. The aminoacid sequence of the amino terminal showed a very high degree of homology with the enzyme superoxide dismutase. Olive pollen extracts have a heterogeneous composition, with several important allergens, one of which is a superoxide dismutase.

J ALLERGY CLIN tMMUNOL VOLUME 99, NUMBER 1, PART 2

1439

Ole e 3 allergen from olive pollen can be considered one of the responsible molecule for the cross-reactivity among pollens, M Villalba, E Batanero, A Ledesma, RI Monsah'e,

MA Gonz(dez, E Gonz~lez, R. Rodriguez, Departamento Bioquimica, Facultad Quimicas, UCM, Madrid Studies were carried out in order to establish the allergenic cross-reactivity of Olc e 3, a new allergen isolated from olive tree pollen, with pollen from different species. This protein exhibits a molecular mass of 9.3 kDa, a pI of 4.2 and a prevalence of 20% of patients allergic to olive pollen. The presence of homologous proteins was tested by ELISA inhibition of the IgE binding to Ole e 3 with different pollen extracts as inhibitors, by using individual sera from patients with IgE specific to this allergen. Highest inhibition was observed with pollen from Oleaceae and Grammeae members while pollen from other trees (birch) and weeds (mugwort and rose) appeared as less active. Besides these angiosperm pollens, pine and cypress as gymnosperm cxamples were also used, owning a significant inhibition capacity. Cross-reactivity is restricted exclusively to pollen because extracts from fruits (apple), seeds (yellow mustard) and insect (Ceratitis capitata) did not bind to the specific-Ole e 3 lgE antibodies. Immunoblotting analyses of pollen extracts with a pool of sera revealed IgE binding to a band around the expected molecular weight in all the species tested. A partial or total disappearance of this Ole e 3 band from the olive pollen extract was observed when every extract was used as inhibitor. In summary, Ole e 3-like allergen must be one of the molecules involved in the clinical cross-reactivity among pollens from different species and seems to be highly conserved in phmt kingdom. 1441

Molecular characterization of the major 22-kD Aedes communis mosquito saliva allergen. H Brummer-l~rvenkon-

tio*, N Kalkkinenr T Palosuo*, T Reunala~2, *National Public Health Institute. ?Institute of Biotechnology and :}:Department of Dermatology, University of Helsinki, Finland Almost all subjects arc sensitized to mosquito bites in childhood. Subsequent bites cause wheals and delayed bite papules which are most intense at thc onset of mosquito season. Whealing is mediated by saliva specific IgE anti-

$353

bodies. We have previously shown by immunoblotting that saliva of Aedes comnzunis, a ubiquitous North-European mosquito species, contains three important saliva allergens; the 22-, 30- and 36-kD proteins. Intense IgE binding to 22-kD allergen is seen in subjects experiencing large wheals from A. communis bites. In the present study A. comrnunis saliva was run in SDS-PAGE, blotted to PVDF-membrane and stained with Coomassie brilliant blue. The 22-kD protein band was cut off and subjected to N-terminal amino acid sequencing. The search in protein databases of the 16 amino acid sequence obtained did not show significant homology to any anticoagulant or other proteins involved e.g. in blood clotting. The 22-kD A. comrnunis allergen is thus a unique protein the function of which is at present unknown.

Investigations on the lgE binding capacity of the carbohydrate structure on Phi p 1: functional relevance? A

Petersen, W-M Becker, H Haas, M Schtaak, Forschtmgszentrum Borstel, Borstel, Germany Although many allergens are glycoproteins, the influence of the carbohydrate structure for allergenicity has been poorly studied. We investigated the carbohydrate moiety of the major allergen Phi p 1 of timothy grass pollen (Phleum pratense). Phl p 1 bears one N-glycosylation site. The carbohydrate composition consists of arabinose, xylose, fucose, mannose, and N-acetylglucosamine. An ed,3-fucosylation site attached to the terminal N-acetylglucosamine was identified to bind IgE. This configuration is frequently found in plants (e.g. Cry j 1, horseradish peroxidase, bromelain) and arthropodes (e.g., phosholipase A2 of bee venom). The xylose and arabinose moieties in Phi p 1 might also serve as binding sites, since both pentoses only exist in plants. We screened 50 patient sera recognizing the Phi p 1 allergen for their ability to detect carbohydrate structures. While 2 sera (4%,) exclusively bound glycans, 16 (32%) recognized carbohydrate and protein moieties. 32 sera (64%) revealed only lgE reactivity to the protein structure. Interestingly, those sera that recognized only glycans were from polysensitized patients, while most of the other sera were taken from patients just suffering from rhinitis. Furthermore, we know from immunoblotting studies that Phi p 1 allergen partly forms dimers. After dimerization two similar glycans are exposed on one molecule. We expect that such Phl p 1 dimer can cross-link IgE antibodies on mast cells and basophils and elicit mediator release, To confirm this hypothesis we currently perform histamine release tests. 1440

Abstracts

1442

Exposure to Aspergillus spores elicits allergic responses similar to soluble antigens in a model of allergic bronchopulmonary aspergillosis. PS Murali, K Thompson, KI

Kelly, JN Fink and VP Kurup. Medical College of Wisconsin, VAMC, Milwaukee, WI Aspergillus fimzigatus (Af), a ubiquitous fungus, causes allergic bronchopulmonary aspergillosis (ABPA). To study the immune mechanism in ABPA we have developed models by exposing mice to soluble Af antigen intranasally (I.N.) or intraperitoneally (I.P.). These mice exhibit elevated serum IgE, Af specific IgG1 and eosinophilia in blood, lungs and bone marrow (BM). The present study was conducted to simulate a more natural exposure of Af, by giving mice Af spores (10 • 106/mouse, 1 dose) either I.P. or I.N. and comparing their immune responses with the model developed with soluble antigens. Mice were sacririced and studied on different days after Af spores, In the I.P. group eosinophils (cos) increased in blood and BM (peak Day 7) followed by an increase in eosinophil peroxidase (EPO) levels (peak Day 13). IgE and M-specific IgG1 showed maximum levels on Day 13 in sera, lung lavage fluid and spleen cell supernatants, while mRNA transcripts for IFN~/was detected in spleen cells on Days 13 and 15. A similar course of events (with levels lower than in the I.P. group) were observed with cos, EPO, cytokine mRNA and immunoglobulins in the I.N. group. Lungs and femur from the I.P. group showed growth of Af on Saboraud's agar plate and this coincided with the eosinophilia in these organs. Femur from I.N. group on Saboraud's agar plates did not exhibit growth of Af. This could indicate a more efficient handling of spores by the lungs in this group which could also contribute to lower values of the other parameters observed in the I.N. group. We conclude that an I.P. exposure of Af spores in mice produce characteristic immunologic features of ABPA as previously observed with soluble Af antigen exposure.

S354

Abstracts

J ALLERGY CLIN IMMUNOL JANUARY 1997

1443

1444

Pleural recruitment of eosinophils and down regulation of Thl response by Aspergillus fumigatus antigen in an IL4 knockout murine model of Allergic Aspergillosis. T Kelly, PS Murali, VP Kurup, KJ Kelly, and JN Fink, Medical College of Wisconsin, VAMC, Milwaukee, WI Allergic bronchopulmonary aspergillosis (ABPA) is an allergic response to Aspergillus fumigatus and is characterized by peripheral blood eosinophilia and elevated serum IgE, typical of a Th2 type response. We have described models of ABPA exhibiting these features, by exposing mice to soluble A. fumigatus antigen (Af). IIA knockout ( I L 4 - / - ) mice have facilitated the study of the eosinophil response to Af in the absence of IgE. In the present study I L 4 - / - mice (C57BL/6) received Af intranasally (I.N.) and were divided into 2 groups. The test group received 1 dose of Af intrapleurally (I.P1. mice) while the controls received PBS carrier. Mice were sacrificed (72h after I.P1. exposure) and their eosinophil and antibody responses were compared. Results indicate that eosinophils (%) were elevated in I.P1 mice (29.8 -+ 7, P < 0.01) compared to control mice (2.5 _+ 0.4) in peripheral blood and in the pleural fluid (10.4 _+ 2.9 vs. 0.6 _+ 0.4, P < 0.02). Pooled cells from the pleural fluid, and cells from individual spleens were cultured and tested for cytokines (pg/ml) and Af specific IgG1 (Af-lgG1, net O.D.). No ILA or IL5 was detected in cultures of pleural exudate cells from either mice groups while IFN~ was lower in the I.P1. group (502 vs. 757 in control mice). Pleural cultures showed elevated Af-IgG1 in I.P1 mice (0.042) compared to control mice (0.021). IFN~/was undetectable in spleen cell cultures from I.P1. mice but was seen in 3 of 5 control mice (777 _+ 512). Af-IgG1 was also elevated in these cultures from I.P1. mice (0.07 _+ 0.02 vs. 0.02 § 0.002, P < 0.05). In conclusion, intrapleural exposure of Af in IL4 knockout mice previously sensitized with Af showed an influx of eosinophils and Af-IgG1 into the pleural cavity and a decrease in IFN% a Thl cytokine.

Eosinophil Degranulation is induced by Respiratory Syncytial Virus-infected Airway Epithelial Cells. B OlszewskaPazdrak, PL Ogra, RP Garofalo, University of Texas Medical Branch, Department of Pediatrics, Galveston, TX Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in infants and young children. High levels of eosinophil cationic protein (ECP) in the airways of RSV-infected infants have been shown to correlate with the severity of disease. Recent studies suggest that adhesion plays an important role in eosinophil degranulation. The mechanisms inducing eosinophil degranulation in viral bronchiolitis are not known. Objectives. In this study we have investigated whether adhesion of eosinophils to respiratory epithelial cells infected with RSV induces eosinophil degranulation. Methods. Monolayers of lung type II alveolar epithelial cells (A549) were infected with sucrose-gradient purified preparations of RSV at multiplicity of infection of 1. Blood eosinophils were isolated from healthy donors by discontinuous Percoll gradients and immunomagnetic selection. Eosinophils (1 • 105) were added to 24-hr RSV-infected or control epithelial cell monolayers. Supernatants from the cocultures were collected after 12 hr and ECP was measured by a radioimmunoassay (Pharmacia, Sweden). Expression of CD11b/CD18 by eosinophils was determined by flow cytometry analysis. Results. ECP levels in supernatants were significantly higher when eosinophils were cocultured with RSV-infected epithelial cells than when cocultured with noninfected epithelial cells (62.7 -+ 6.5 vs 14.7 +- 2.7 ng/ml, respectively, mean _+ SD, p < 0.01). Expression of C D l l b / CD18 was significantly upregulated on eosinophils exposed to RSV-infected epithelial cell supernatant. Addition of neutralizing Ab anti-CD18 to cocultures of eosinophils and RSV-infected cells partially inhibited the release of ECP. Coneluslous. Our results demonstrate for the first time that RSV-infected lung epithelial cells induce eosinophil degranulation by a CD18-dependent mechanism. This process may play a central role in the pathogenesis of airway mucosa inflammation following viral infection.

1445

Processing of the Proform of Eosinophil Granule Major Basic Protein (MBP) to the Mature Form by an Eosinophil-Like Cell Line. IG Ovsyannikova, CC Paul, DA

Loegering, JL Checkel, PD Popken-Harris and GJ Gleich, Mayo Clinic, Rochester, MN and Dayton's VA Hospital, Dayton, OH. Previous studies have shown that the AML14.3D10 tumor cell spontaneously differentiates to eosinophils in the absence of cytokines and contains eosinophil granule MBP (Paul CC et al, Blood 86:3737, 1995). Using antibodies with preferential activity for specific forms of proMBP (33 kDa) and MBP (14 kDa), cells and lysates were examined by immunofluorescence (IF), immunoelectron microscopy (IEM), RIA and Western blotting (WB). To determine how the intracellular processing of proMBP to MBP occurs, pulse-chase experiments were performed in a primary culture of AML14.3D10 ceils labeled for 3 hr with [35S]cysteine (500 ~Ci) and chased for different periods, up to 9 hr. Immunoprecipitation was performed by incubation of reduced and alkylated lysates with mAb to MBP 0146B6) and proMBP (J163-15E10). Conversion of metabolically labeled proMBP to lower molecular weight forms was detected by SDS-PAGE and autoradiography. MBP was readily detectable in AML14.3D10 by RIA (337 ng/106 cells), IF and WB. IEM showed that MBP was localized to cytoplasmic granules. ProMBP was detected by IF and WB. By pulse-chase experiments, proMBP was first synthesized after 3 hr of labeling as a precursor with a molecular weight ->33 kDa. After 0.5 hr chase, proMBP was processed to an intermediate form of proMBP at 21 kDa; at 1 hr and 1.5 hr, proMBP was processed to both the intermediate form and the 14 kDa MBP. Complete conversion of proMBP to 14 kDa MBP was achieved after a 3 hr chase. Thus, AML 14.3D10 cells synthesize MBP as a predominantly 33 kDa precursor that is then processed to the mature 14 kDa MBP form via a 21 kDa intermediate. This conversion of proMBP to 14 kDa MBP evidently occurs in a 2-step process.

1446

Expression of LTC 4 synthase during the development of eosinophils in vitro from cord blood progenitors. JA

Boyce, BK Lam, JF Penrose, DS Friend, S Parsons, WF Owen, and KF Austen, Departments of Medicine and Pathology, Harvard Medical School, and the Division of Rheumatology and Immunology and the Department of Pathology, Brigham and Women's Hospital, Boston, MA Leukotriene C4 synthase (LTC4S) expression was examined during the IL-3 and IL-5-driven development of eosinophils in vitro. At 7 days, mixed cord blood mononuclear cells contained mRNA and SDS-PAGE immunoblot signals for cytosolic phospholipase A 2 (cPLA2), 5-1ipoxygenase (5-LO), and 5-1ipoxygenase activating protein (FLAP), but lacked LTC4S and did not generate LTC 4 when stimulated with 20 p~M calcium ionophore. At 14 days, 94% of the cells were of eosinophil lineage, most having coexistent eosinophilic and basophilic granules (hybrid granulocytes), possessed both LTC4S mRNA and protein, and generated 23.9 _+ 6.0 pmol cysteinyl leukotrienes/106 eosinophil lineage cells. By 28 days, there was progressive eosinophil maturation and further increases in 5-LO, FLAP and LTC4S proteins, with the production of 94.6 + 9.0 pmol cysteinyl leukotrienes/106 eosinophil lineage cells. Purified CD34+ progenitors lacked all 5-LO/ LTC4 S pathway proteins, acquired cPLA 2 and 5-LO by 3 days of culture, FLAP by 7 days, and LTC4S by 10 days. Thus eosinophilopoiesis in vitro is accompanied by early expression of cPLA2, 5-LO and FLAP, and later expression of LTC4S. Once the lineage is established, IL-3 and IL-5 mediate further upregulation of FLAP and LTC4S, members of a newly recognized gene family, as well as 5-LO, during ongoing cell maturation. (Supported by NIH grants HL-36110, AI-31599, DK45656, AI-22531, AI-23483, AR-36308, and AI-07306, by a grant from Jannsen Pharmaceutica, and by a grant from the Charles H. Hood Foundation.)

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1447

1448

Bidirectional Regulation of Eosinophil Survival and Apoptosis by F%R. J-T Kim. GJ Gleich and H Kita. Mayo Clinic, Rochester, MN. We have previously shown that eosinophil interaction with immobilized IgG through F%RII causes cellular activation. In this study, we investigated whether FcvR regulates eosinophil survival. F%RII (CD32) was highly expressed on the surfaces of eosinophils from normal individuals. Two of five monoclonal antibody (mAb) clones showed that FcvRIII (CDI6) was also detectable on the same eosinophils. When cultured without cytokine, most eosinuphils died within 4 days. In contrast, eosinophils cultured with soluble anti-F%Rll mAb (clone IV.3) or anti-F%RIlI mAb (clone 3G8) showed enhanced survival. The dose-response curve with these antibodies was bellshaped; the highest survival was observed with 2.5 ixg/ml of mAb, and >5.0 po~ml of mAb slightly inhibited the viability. Eosinophils incubated for 4 days with 100 pg/ml of IL-5 had 80% survival. This IL 5-mediated survival was inhibited by ->5.0 ixg/ml of anti-F%Rll or anti-F%Rlll mAb. Because survival enhanced by mAb for FcyR was blocked by anti-GM-CSF mAb, but not by anti-IL-3 or anti-fL-5 mAb, the low concentrations of mAb for F%R likely stimulated the autocrine production of GM-CSF. GM-CSF mRNA was also detected in eosinophil lysates by RT-PCR. As examined by Hoechst 33342 dye staining and flow cytometry, the inhibited survival induced by high concentrations of mAb was due to apoptosis, but not due to necrosis. Eosinophils cultured with soluble heat aggregated human lgG, instead of antireceptor mAb, also showed similar results. These findings suggest that F%R modulates eosinophil survival bidirectionally; the receptor either enhances survival via GM-CSF production, or suppresses the survival via activation of an apoptotic pathway. Thus, by regulating homeostasis of eosinophils in tissues, FcyR may be important in regulating allergic inflammation.

The Effect of Transendothelial Migration on Eosinophil (EOS) Survival. H Yamamoto, JB Sedgm,ick. WW Busse, University of Wisconsin, Madison, WI EOS migration from the peripheral circulation is a key step and factor in the development of allergic inflammation. Many factors influence and determine this process including adhesion molecules, cytokines, chemokines and matrix proteins. Furthermore, it is our hypothesis that this migratory process not only causes directed cell migration but also change in the EOS phenotype and inflammatory capacity. To determine the effect of EOS transmigration on cell function, 3 btM pore polycarbonate filters were coated with human microvascular lung endothelial cells (HMVEC-L) and used as transwell inserts in tissue culture wells. Isolated human EOS were placed in the top well and migration to the bottom well was determined after 3 hr. Treatment of HMVEC by IL-l[3 or TNF-c~ significantly enhanced EOS migration (control 4.3 _+ 0.9%, IL-l[3 = 18.1 _+ 2.5%, p < 0.005 andTNF-o~ - 7.0 - 1.5%, p < 0.05). EOS migration was most effectively blocked by a combination of anti-CD l la and anti-CDl8 mAb. Furthermore, when in vitro survival at 48 hrs of transmigrated EOS were compared to unmigrated cells, there was significantly greater survival (IL-II3:32.2 _+ 4.4% vs 16.2 + 2.6% respectively, p < 0.001; TNF-cc 26.1 + 5.6% vs 13.5 + 3.4% respectively, p < 0.001). The enhanced survival of EOS which migrated through IL-I[3 treated HMVEC was inhibited if cultured with anti-GM-CSF antibody. These observations indicate that transendothelial migration of EOS is associated with an anti-apoptotic effect (prolonged survival), and this upregulation is associated with generation of GM-CSF. Thus, EOS migration not only directs cell movement to sites of inflammation but also enhances this cell's inflammatory capacity.

Abstracts

S355

1449

Eosinophil granule proteins induce IL8 and MCP-1 production by peripheral blood mononuclear cells (PBMC). S Koppula, L L Thomas, G J Gleich, and J N Moy Rush-Presbyterian-St. Luke's Medical Center and Cook County Children's Hospital, Chicago IL and Mayo Clinic, Rochester, MN Eosinophil granule proteins and in particular major basic protein (MBP) are implicated in the pathogenesis of asthma and other disorders characterized by eosinophilia. Based on the ability of MBP to induce IL-8 production by eosinophils, we examined the potential for MBP to stimulate CXC and CC chemokine production by PBMC. Incubating l(P PBMC with 0.1 IxM to 3.0 IxM MBP in 500 btl RPMI-1640 (containing 5% heat-inactivated autologous serum) for 48 hr in 5% CO2 resulted in a concentrationdependent production of IL-8 (up to 79,800 pg/ml) and MCP-I (up to 10,700 pg/ml). Absolute levels of IL-8 and MCP-1 production, however, varied widely among different donors. Time course measurements demonstrated that IL-8 production continued to increase for 48 to 72 hr in response to stimulation by 1.0 ixm MBP. In contrast, MCP-I production reached a maximum after 24 hr of incubation. No IL-8 or MCP-1 production was detected after 4 hr of incubation with 1_0 Ixm MBP. Evaluating eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) for similar activity at 1.0 txM and 3.0 IxM concentrations revealed that EDN and ECP are equipotent and of equal effectiveness to MBP in stimulating IL-8 production. EDN, however, is less effective as a stimulus for MCP-I production. These results demonstrate that MBP as well as ECP and EDN may contribute to eosinophil-associated inflammation through the induction of CXC and CC chemokines.

1450

The expression of adhesion molecules on eosinophils during antigen challenge. DA Wong MD and PM O'Byrne MD Virginia Commonwealth University, Richmond, VA and McMaster University, Hamilton, ON, CANADA Adhesion molecules play a role in the selective migration of leukocytes and their regulation. To study which adhesion molecules are involved in the eosinophil we examined the changes in adhesion molecule expression on eosinophils during allergen challenge. Depolarized light was used to identify eosinophils by flow cytometry allowing the quantitative study of cell surface proteins on circulating cells. Blood from 7 subjects with mild asthma, not on regular treatment were collected before and then 1, 4, and 24 hrs after allergen or diluent challenge. Leukocytes were then stained with a panel of adhesion antibodies. Circulating eosinophils were found to express ~4[31 (CD49d/CD29), 0~4~7 (CD49d/~37), e~lq32 (CDlla/CD18). L-selectin (CD62L), and ICAM-3 (CD50). A comparison of adhesion molecule expression on eosinophils 1 and 4 hrs after allergen challenge suggested a decrease in c~L[32expressed on circulating eosinophils at one hour and a increased expression at 4 hours with a return to baseline levels at 24 hours. A possible explanation of these results is the recruitment of eosinophils which express high levels of c~cl3~ into the lung in the first hour after antigen challenge. The increase in overall e~1132expression by 4 hrs. could be due signals from the lungs as the reaction develops. Our studies suggest a role for ~c[32 in the recruitment of eosinophils in human allergen challenge.

S356

1451

1452

Abstracts

Phosphodiesterase (PDE) Inhibitors Can Suppress Prolonged Survival of Eosinophils with Interleukin-5 (IL-5). K Ohta, S Kubota, M Nakajima, S Sawamoto, T Mliyasaka, Y Nakajima, K Sekine, C Sakamaki, J Nakano, K Mano, H Miyashita, Teikyo University School of Medicine, Tokyo, Japan. As we reported previously, theophylline can inhibit prolonged survival of eosinophils with IL-5 in vitro via induction of apoptosis. Since theophylline has action as a PDE inhibitor, we attempted to study if PDE isoenzyme inhibitors could downregulate prolonged survival of eosinophils with IL-5. Human eosinophils were isolated from heparinized blood obtained from eosinophilic patients by Percoll gradient centrifugation and magnetic cell sorting. We cultured the cells with or without IL-5 for 4 days by adding a PDE inhibitor to some cultures. The cell viability was assessed by trypan-blue dye exclusion, and apoptosis was detected by DNA fragmentation in DNA extracted from cultured eosinophils at day 2. Inasmuch as eosinophils have been found to possess PDE IV, we have expected that only PDE IV inhibitors will decrease %survival of eosinophils calculated by assuming the viable cell number in the culture of IL-5 alone as 100%. However, we observed that cilostasol and amrinon, PDE III inhibitors, significantly suppressed eosinophil survival with IL-5 (6.7 _+ 1.7 and 60.7 _+ 7.6, respectively at 0.1 mM as mean _+ SEM of %survival, p < 0.05) similarly to Ro-20-1724 and roliprarn, PDE IV inhibitors (60.7 _+ 7.6 and 68.0 _+ 1.8, respectively, p < 0.05). Zaprinast, a PDE V inhibitor, did not show any significant suppression in %survival. Electrophoresis of DNA revealed that the inhibition with the PDE III and IV inhibitors was expressed via apoptosis in the eosinophils activated by IL-5. Our results will raise a question if eosinophils have PDE III as well as PDE IV, and if not, how PDE III inhibitors can affect survival of eosinophils. In conclusion, some PDE III and IV inhibitors could be new anti-asthma drugs capable of suppressing eosinophilic inflammation. Nerve Growth Factor (NGF) Activates Peripheral Blood Eosinophils of Vernal Keratoconjunctivitis (VKC) Patients. A Solomon, L Aloe, J Pe'er, J Frucht-Pery, St Bonini, F Levi-Schaffer. The Hebrew University-Hadassah Medical School Jerusalem Israel, CNR and Tor Vergata University Rome Italy. NGF seems to have an important role in allergy. In VKC, high serum levels of NGF positively correlates with mast cell numbers in the conjunctiva while inversely correlates with the number of circulating eosinophils (EOs). In this study we aimed to define the effects of NGF on peripheral blood EOs from patients with VKC in vitro. EOs were isolated and purified by negative immunoselection (MACS purity>98%) from 5 male VKC patients (10-20 yr.), 2 matched pts with seasonal allergic conjunctivitis and 3 volunteers with mild blood eosinophilia. EOs were incubated with different concentrations of NGF (501000 ng/ml) in RPMI containing 5% FCS and supernatants collected for Eosinophil Peroxidase (EPO, 20 min, colorimetric/enzymatic assay) and IL-6 (12 hrs, ELISA kit) determinations. Viable EOs were evaluated by Trypan blue exclusion test (day 2,3,4). NGF caused a dose-dependent release of EPO which was highly significant when the effect of 100 ng/ml NGF on EOs was compared with that of medium alone (increase of 120%, n=7, p=0.013). NGF induced a significant EPO release also from EOs isolated from the non-VKC pts. To assess whether NGF also induces the release of cytokines, IL-6 levels were evaluated in the culture medium of EOs from 3 pts with VKC. IL-6 released from EOs of 1 out of 3 VKC pts incubated with 500 ng/ml NGF was noticeably higher than the IL-6 spontaneously released into culture medium alone. The number of viable EOs incubated in medium in the absence or presence of NGF at the different time points was similar. In summary NGF activates peripheral blood EOs from VKC and non-VKC volunteers to release EPO but does not influence their in vitro survival. The capacity of NGF to cause a secretory response in EOs indicates an additional role for NGF in allergic conditions associated with eosinophilia.

J ALLERGY CLIN IMMUNOL JANUARY 1997

1453

Signal Transduction Induced by PAF, IL-5 and Secretory IgA Conjugated to Sepharose Beads (sIgA-beads) in Cultured Eosinophils. H Ohashi, 1 M Takei, 1 H Kita, 2 GJ Gleich, 2 H Fukamachi, 1 lKirin Brewery, Takasaki, Japan, ZMayo Clinic and Foundation, Rochester, MN We examined signal transduction induced by PAF, IL-5 and sIgA-beads in cultured eosinophils. Cultured eosinophils were obtained from umbilical cord blood mononuclear cells as described by MK Bach et al. (Int Arch Allergy Appl Immunol 93:323 1990). We investigated the time course of tyrosine phosphorylation (Tyr-P) induced by PAF, IL-5 and sIgA-beads using anti-phosphotyrosine antibody (4G10). We also examine EDN degranulation induced by these stimulus. Although each stimulus induced Tyr-P of common many proteins such as ERK-2, 150kDa and 120kDa proteins, the time course of their Tyr-P and the levels of Tyr-P were different. PAF strongly induced Tyr-P of ERK-2, 150kDa and 120kDa protein in 30 seconds, while IL-5 induced Tyr-P of ERK-2 and 150kDa protein in 3-5 minutes and the level of Tyr-P of 120kDa protein was very low. sIgA-beads induced Tyr-P of 150kDa and 120kDa protein in 3 minutes and indused Tyr-P of ERK-2 in 5-10 minutes. PAF and sIgA-beads induced EDN degranulation in 30 minutes from these cultured eosinophils, however IL-5 did not. These results suggest signal transduction for degranulation is different from that for growth, and Tyr-P of 120kDa protein is an important signal to degranulate in eosinophils.

1454

Phosphatidylinositoi 3-kinase Signaling Pathway Is Involved in the Blockade of Spontaneous Apoptosis in Human Eosinophils, But Not in the Prevention of Apoptosis by Granulocyte-macrophage Colony-stimulating Factor. S Miike, KKurasawa, MHiraguri, YSaito, llwamoto, Chiba University, Chiba, Japan It has been reported that phosphatidylinositol-3 kinase (PI3-kinase) plays an important role in the prevention of apoptosis by nerve growth factor in rat PC-12 cells. However, the role of PI3-kinase signaling pathway in the survival of eosinophils has not been elucidated. To determine whether PI3-kinase is involved in the survival of eosinophils induced by granulocyte-macrophage colonystimulating factor (GM-CSF), we examined the effects of wortmannin (a specific inhibitor of PI-3 kinase) on the survival of human eosinophils induced by GM-CSF. Human normal eosinophils were purified by anti-CD16 negative selection and then cultured in RPMI1640 with 10% FCS in the presence of GM-CSF or wortmannin. We assessed the apoptosis of eosinophils by trypan blue dye exclusion, Hoechst 33342, and cell cycle analysis. We found that, when eosinophils were cultured without GM-CSF, more than 90% of the cells were lead to apoptosis in 5 to 7 days. Addition of wortmannin into the culture significantly enhanced the apoptosis of eosinophils. On the other hand, when eosinophils were cultured in the presence of GM-CSF, they survived more than 7 days. However, wortmannin failed to induce the apoptosis of eosinophils in the presence of GM-CSF. These results indicate that PI3kinase signaling pathway is involved in the blockade of spontaneous apoptosis in human eosinophils, but not in the prevention of apoptosis by GM-CSF.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1455

Effect of Phosphodiesterase IV Inhibitor or Dibutyryl cyclic AMP on TNF.ot induced CD4 Expression of Human Eosinophils. Y Okubo, T Momose, S Horie, M Hossain, M Sekiguchi, Shinshu University, Matsumoto, Japan. Theophylline and procaterol (~-stimulator) are useful for the treatment of bronchial asthma. Their effects have recently been suggested to be anti-inflammatory. On the other hand, CD4 on eosinophils was observed in hypereosinophilic syndrome, parasite infection and eosinophilic pneumonia. Furthermore, CD4 on human eosinophils was newly expressed by tumor necrosis factor (TNF)-c~ stimulation in vitro. To analyze the mechanism of CD4 expression, theophylline, procaterol, phosphodiesterase (PDE) IV inhibitor or dibutyryl cyclic AMP (DB-cAMP) were used for TNF-u induced CD4 expression. Human eosinophils (sg > 1.082, purity >98%) were obtained by magnetic cell separation using anti-CDl6 monoclonal antibody. Eosinophils were cultured with TNF-e~ (10 ng/ml) in the presence of various concentrations of theophylline, proeaterol, PDE IV inhibitor or DB-cAMP at 37~ C, 5% CO 2 for 24 hr. The mean fluorescence intensity (MFI) of CD4 expression on eosinophils was examined by flow cytometric analysis. TNF-c~ induced CD4 expression was down-regulated by theophyllin (10 5M-10 ~M) and procaterol (l(l ~M-1t) 7M) in a dose dependent fashion. TNF-o~ induced CD4 expression by the combination (theophylline: 10 aM and procaterol: 10-1'~M-10 7M) was clearly downregulated in a dose dependent fashion. TNF-c~--induced CD4 was significantly inhibited by PDE IV inhibitor (10 ~M-10 ~'M) andDB-cAMP(lll "M-10 3M).Itissuggested that cyclic AMP through phosphodiesterase IV inhibition suppress CD4 expression on eosinophils and CD4+ eosinophils may play an important role in allergic inflammation.

1456

Effect of Phosphodiesterase IV Inhibitor on Degranula. tion, CDIIb Expression and Survival of Human Eosino. phils. T Momose, Y Okubo, S Horie, M Sekiguchi, Shinshu University, Matsumoto, Japan. Eosinophils play an important role in the pathogenesis of bronchial asthma. Eosinophils are found in large numbers in the air ways and air way interstitium. The eosinophiI major basic protein causes bronchial hyperresponsiveness and is capable of damaging air way epithelium. Theophylline has been used for the treatment of bronchial asthma, and the effect of theophylline is suggested to be through phosphodiesterasc (PDE) inhibition. However, the effect of theophylline on eosinophil biological function is not clear. In the present study, we examined effect of theophylline or PDE IV inhibitor on eosinophil degranulation, surface antigen expression and survival. Human eosinophils from peripheral blood of normal subjects and patients with mild allergic rhinitis were purified by Percoll density gradient centrifugation and CD16 negative selection. We examined the effects of theophylline or PDE IV inhibitor on human eosinophil degranulation as well as C D I l b expression by granulocyte/macrophage-colony stimulating factor (GM-CSF) (10 ng/ml) or platelet activating factor (PAF) (10 ~M) stimulation. Furthermore, the effects of theophylline or PDE IV inhibitor on eosinophil survival in the presence of human recombinant interleukin-5 (100 pg/ml) were examined. Eosinophil degranulation induced by GM-CSF was inhibited by theophylline (ll) 5M-10 ~M) and PDE IV inhibitor (10 "M-10 5M). GM-CSF- or PAFstimulated CD1 lb up-regulation on eosinophils was inhibited by theophylline (10-aM-10 3M, 10 4M-10 3M) and PDE IV inhibitor (10-SM-10-3M, 10 5M-10 3M).Eosinophi[ survival was inhibited by theophylline (10 3M) and PDE IV inhibitor (10-4M-10-3M). These results suggest that PDE IV inhibitor may play a key role in the antiinflammatory action of theophylline.

1457

Effect of Herbal Medicine (Sho-seiryu-to and Ryo-kan-kyomi-shin-ge-nin-to) on Human Eosinophil Biological Function. S Takashi, Y Okubo, M Hossain, S Horie, T Momose, M Sekiguchi, Shinshu University, Matsumoto, Japan Herbal medicine is used in various diseases, including bronchial asthma and allergic rhinitis. It is reported that

Abstracts

S357

eosinophils play an important role in allergic diseases. The effect of Sho-seiryu-to and Ryo-kan-kyo-mi-shin-ge-nin-to on human eosinophil biological function such as degranulation, expression of adhesion molecules and viability was examined. Human eosinophils (sg > 1.082, purity > 98%) were obtained by magnetic cell separation using anti-CD16 monoclonal antibody. Eosinophils were stimulated by platelet activating factor (PAF) (10-t'M) or granulocyte/ macrophage-colony stimulating factor (GM-CSF) (10 ng/ ml) for 4 hr, and effects of various concentrations of Sho-seiryu-to and Ryo-kan-kyo-mi-shin-ge-nin-to on degranulation were examined. Eosinophil degranulation induced by PAF or GM-CSF was significantly inhibited by Sho-seiryu-to and Ryo-kan-kyo-mi-shin-ge-nin-to in a dose dependent fashion. Effects of Sho-seiryu-to or Ryo-kankyo-mi-shin-ge-nin-to on CD1 lb/CD18 of PAF- or GMCSF-stimulated eosinophils was examined using flow cytometry after 2 hr culture. The mean fluorescence intensity (MFI) of CD1 lb/CD18 on eosinophils which are triggering molecules for eosinophil degranulation, was augmented by GM-CSF or PAF stimulation. The increased CD 1lb/CD 18 expression on eosinophils was significantly down-regulated by Sho-seiryu-to, but not by Ryo-kan-kyo-mi-shin-ge-ninto. Eosinophil survival in the presence of recombinant human interleukin-5 (100 pg/ml) after 4 day culture was significantly inhibited by Sho-seiryu-to and Ryo-kan-kyomi-shin-ge-nin-to. These data showed inhibitory effects of herbal medicine on eosinophil function, suggesting that Sho-seiryu-to and Ryo-kan-kyo-mi-shin-ge-nin-to are useful in the treatment of allergic diseases.

1458

Eosinophils in peripheral blood in infants in relation to atopic disease at six years of age. MP Borres & B BjOrkst~n. Department of Pediatrics, Link6ping University Hospital, Sweden Eosinophi[ counts were studied prospectively in peripheral blood in 67 infants with and without a family history of atopic disease. They were followed by venous blood sampling, skin prick and lungfunction testing at 3, 6, 9, 18 months and 6 years. Eosinophilia (>4 • 10~ cells/l) was associated with simultaneous presence or subsequent development of atopic disease at 3, 9 and 18 months of age, but not significantly so at 6 months and 6 years. Eosinophilia in cord blood had no significant relation to atopic disease. Infants with eosinophilia at three months of age (n = 19) continued to have elevated eosinophil counts at 6 years (mean 5.8 x IIIs cells/l) compared to infants without eosinophilia (3.4 • 10~ cells/l, p < 0.05) and were more likely to be atopic (p < 0.01). We conclude that elevated eosinophil counts in peripheral blood of apparently healthy infants at three months of age is associated with a subsequent diagnosis of atopic disease.

$358

1459

1460

1461

Abstracts

Attenuation of Eosinophil Adhesion to Airway Epithelial Cells by Terfenadine. D K Agrawal, C Ishikawa, T Chatham, A Kiboneka, R G Townl~ Allergic Disease Center, Creighton University, Omaha, NE. There is increasing evidence for the contribution of eosinophils in the pathogenesis of allergic asthma. Eosinophils adhere to and transmigrate through epithelial and endothelial cells in the airways. Little is known about the eosinophil-epithelial cell interaction. In this report, we examined the effect of second-generation antihistamine, Terfenadine, on eosinophil adhesion to human lung epithelial ccll line (A549). Eosinophils were purified (>98% purity and viability) from allergic donors. The A549 cells were treated with TNF, (100 U/ml) and eosinophils were treated with PAF (1 ~xM) or IL-5 for four hours in the presence or absence of Terfenadine. There was a significant increase in eosinophi[ adhesion following TNF, and either PAF or IL-5. Terfenadine (10-:M - 10-SM) inhibited eosinophil adhesion only when eosinophils were treated with PAF but not with IL-5; the degree of inhibition ranged 17-22% (P < 0.05). The dot-blot analysis revealed that Terfenadine attenuated the expression of ICAM-1 in TNF~-treated A549 cells. These data suggest that apart from a strong antihistamine effect, the therapeutic effect of Terfenadine in allergic asthma may at least in part, be due to an inhibition of eosinophil adhesion possibly via ICAM-1 expression in the airway epithelium. Oxidative Burst of Eosinophils and IL-2 Effect. A Conesa, J De Sanctis. H Rivera, NE Bianco, 0 Aldrey. Institute of Immunology, Faculty of Medicine, Central University of Venezuela. Caracas, Venezuela. Eosinophils are associated with late phase reaction in asthma. They have the capability to secrete a number of potent mediators, including superoxide and peroxide. This may make eosinophils responsible for much of the tissue damage seen in asthma. The purpose of our study was to analyse the oxidative burst of normodense eosinophils in asthmatic patients, investigating also the effect of IL-2 on such function. Eosinophils were separated from whole blood of healthy donors and asthmatic patients by 6% dextran followed by Percoll gradients. The purified cell population (patients: eosinophils > 92,5% and CD16(-) >93%; controls: eosinophils >80,7% and CD16(-) >90%) were labeled as described previously by Davis (1993) and stimulated with 1L-2 for a time kinetics. Cell suspensions were incubated with anti- Tac prior to the oxidative burst analysis, under the same conditions. No differences were observed on oxidative burst function of non-stimulated controls and patients normodense eosinophils. Addition of IL-2 (1000 IU/ml) increased significantly (p < 0.01) H=O= production in asthmatic patients eosinophils, while in healthy donors decreased significantly (p < 0.05). Moreover, anti Tac inhibited by 50% the IL-2 dependent increase. No statistic difference was found on CD25 and CD122 surface expression between controls and asthmatic eosinophils. However, asthmatics eosinophils expressed very low percentages of CD23 (1.1% + 0.5), with a statistical significant difference when compared to control eosinophils (34.5% • 9.6). Davis B. Oxidative brust methods. In Robinson J.P. editor. Handbook of Flow Cytometry Methods. New York, 1993: 146-54. Synergistic effect of procaterol and theophylline on eosinophii degranulation. T Fujisawa, A Terada, K Iguchi, H Kamiya, Mie National Hospital, Tsu, Japan Inhibition of release of toxic granule proteins from eosinophils is a possible target for treatment of allergic inflammation. This study was performed to determine whether procaterol and theophylline, commonly used bronchodilators, have anti-inflammatory effect through inhibition of eosinophil degranulation induced by IL-5. Purified eosinophils from atopic donors were incubated with IL-5 for 24 hrs in the presence of dexamethasone, theophylline and/or procaterol. EDN in the supernatant was measured by RIA. IL-5 at 10 ng/ml induced EDN release up to 30% of its total content. Theophylline signif-

J ALLERGY CLIN IMMUNOL JANUARY 1997

icantly inhibited the EDN release in a dose dependent manner. EDS0 was 2 • 10 -5 M, which was comparable to therapeutic concentrations. Dexamethasone at 10 ~L10-6 M was also shown to be highly inhibitory. Procaterol inhibited degranulation only at high concentrations. However, procaterol at 10 -') M, a therapeutic concentration, together with theophylline at a suboptimal concentration inhibited the degranulation by 44%, a finding that indicate that combination of the two drugs has a synergistic effect. These results suggest that theophylline and procaterol may have anti-inflammatory effect in the treatment of asthma as well as dexamethasone.

1462

Fc~ Receptor CD32 Is Linked to Apoptotic Pathways in Murine Granulocyte Precursors and Mature Eosinophils. B. de Andres, A. Blum ~, J. Weinstock~, S. Verbeek*, M. Sandor-, A. Mueller and R.G. Lynch. Departments of Pathology and Internal Medicine A, University of Iowa, Iowa City, Iowa, *Department of Immunology, University of Utrecht, The Netherlands, and - D e p a r t m e n t of Pathology, University of Wisconsin, Madison, WI. Fc~ receptors (CD16/Fc~RIII and CD32/Fc-/RII) are expressed early in myeloid and lymphoid cell development. To determine whether Fc',/R can influence eosinophil development, normal adult bone marrow was cultured with a combination of cytokines (GM-CSF, IL-3, IL-5) that generates large numbers of eosinophils, and the effect of anti-Fc-!R Ab was determined. Cultures treated with 2.4G2, a rat mAb that binds to CD16 and CD32, but not cultures treated with normal rat Ig, showed high levels of granulocyte (dr-l+) apoptosis evidenced by Annexin-V binding, expression of J~,s, electron microscopy, release of eosinophil perioxidase, and the absence of significant numbers of eosinophils. Apoptosis was also induced by 2.4(12 in mature eosinophils purified from hepatic granulomas of S. mansoni-infected mice. Apoptosis induced by 2.4G2 is: 1) dependent on fas since it did not occur in bone marrow cultures established from fas mutant mice (MRL lpr/lpr), and 2) it is mediated through CD32 since it was observed in bone marrow cultures established from CD16 k.o. mice. These studies have identified a new function for CD32 (Fc-/RII) on murine eosinophils.

1463

Skin Chamber Inflammatory Cell Responses After IgEMediated and Opiate-Induced Mast Cell Activation. B Zweiman, C yon Allmen, M David, Hospital of the University of PA, Philadelphia, PA Both IgE-mediated reactions and opiates induce prominent immediate mast cell activation in the skin but only the former is followed by indurated late phase reactions peaking at 6-12 hours. To explore mechanisms underlying these differences in LPR reactivity, we induced skin blisters by heat/suction, unroofed them, irrigated the blister bases and appended collection chambers to the forearms of 7 humans with previously demonstrated LPR to pollen antigens (Ag). To individual sites were added either: a) Ag, 100 PNU/ml; b) codeine, 8 mg/ml (Cod); c) buffer diluent (B). Fluids were removed after one hour (Hr) and replaced with like solutions for an additional 4 Hrs. There was similar, prominent histamine release at Ag vs Cod sites in the first Hr (111 • 25 vs 91 +_ 33 ng/ml, p = NS). Both of these were significantly greater than at B sites (4 _+ 2). However, during the 2nd-5th Hrs there were greater levels at the Ag than Cod sites in: a) exuding leukocytes (4.9 • 0.2 vs 1.9 • 0.8 • 105, p < 0.02); b) lactoferrin, released from neutrophils (15 _+ 22 vs 4 • 1.5 p,g/ml, p < 0.02); c) eosinophilic cationic protein (13 • 2.0 vs 3 _+ 0.1 ng/ml, p < 0.05); d) neutrophil superoxide production (75 • 5 vs 10 _+ 5% NBT * cells, p < 0.01); e) IL8 (5.7 _+ 2.5 vs 1.5 -+ 0.1 ng/ml). The increased IL-8 levels may be possibly responsible for the greater neutrophil exudation in Ag sites. However, skin chamber albumin levels (a marker of vascular permeability) were similar in Ag and Cod sites. Conclusion--LPR may reflect inflammatory cell responses which occur following mast cell activation induced by IgE-mediated reactions but not after mast cell activation induced by opiates.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1464

Inflammatory Cells in Skin Immunological Infiltrations.

Abstracts

1466

Inhibition of Eosinophil Infiltration Into Peritoneal Cavities of Allergen-Challenged Mice by Lipopolysaccharide (LPS). A Schimming, GJ Gleich, and H Kita, Mayo Clinic, Rochester, MN. Eosinophils, but not neutrophils, preferentially infiltrate into the sites of chronic allergic inflammation. In contrast, neutrophils are abundant at sites of bacterial infection. Therefore, we hypothesized that distinctive tissue environments are created to selectively recruit these different granulocytes into sites of inflammation. To test this hypothesis, we sensitized BALB/c mice with short ragweed pollen extracts (SRW). Mice were challenged by intraperitoneal injection of either saline, LPS, SRW, or SRW+LPS. Mice challenged with LPS showed neutrophil, but not eosinophil, infiltration into the peritoneal cavity after 6 hours. In contrast, by 48 hours, mice challenged with SRW showed marked infiltration of eosinophils. However, eosinophil infiltration was abolished when mice were challenged with SRW+LPS. Because no blood eosinopenia was seen at any time, this suppression of eosinophil infiltration was not likely due to endogenous steroids. Furthermore, the serum levels of corticosterone were not increased in LPS mice. By ELISA and semiquantitative RT-PCR, IL-5 was detected in the peritoneal lavage of both the SRW and S R W + L P S groups, but not in the other groups. Interestingly, despite the marked suppression of eosinophil infiltration, the levels of IL-5 in the S R W + L P S group were higher than in the SRW group. No differences in the expression of other cytokines, such as IL-4, IFN-3,, and TNF-c~, were observed between the two groups. These findings suggest that eosinophil infiltration into sites of allergic inflammation is inhibited by LPS, and that this inhibitory effect is independent of IL-5.

1467

Nerve Growth Factor (NGF) enhances allergic immune responses in a murine model for allergic disease. A

J Wadniewski, R Matusiewicz, First Department of Internal Diseases, Grochdw Hospital, Warszawa, Poland The clinical ewduation of neutrophils participation in the cffector phase of allergic reactions as well as function measurement results of these cells obtained by various researchers is not univocal. Different opinions also concern other morphotic components of the inflammatory infiltration in allergic patients. The cell composition was tested in 64 patients with atopic asthma as well as in 62 healthy patients after inducing an inflammatory infiltration by grass pollens allergen, PAF-acether, tuberculin, DNCB, and mechanical injury in 0.5, 3, 6, 12, 48, and 72 ~a hour after evoking the infiltrations. The ewduation was done according to the Southam method [21], Matusiewicz et al. [22], and through skin biopsy. Certain regularities concerning the cell composition were observed and were common to all the agents evoking the inflammation and dependent on the duration of the inflammatory reaction. Neutrophils dominate in inflammatory cell compositions in the first hours, in the 6 Th hour the number of eosinophils and basophils rises, in the 12th hour the number of macrophages increases. After 24 hours, both in the allergic reaction and after mechanical injury, the celt percentage reversed to the initial state. As the inflammation continues, the number of lymphocytes does not show any significant differences. Similarities of cell composition in inflammatory infiltrations at the observed time intervals are significant and seem to be independent of the mechanisms evoking the inflammatory reaction. Our own investigations do not confirm other researchers observations concerning the significant increase of basophils in allergic reactions.

1465

Comparison of Humoral and Cellular Inflammatory Skin Responses in IgE-Mediated Late Phase Reactions and Delayed Hypersensitivity in the Same Individual. C yon Alhnen, B Zweiman, A R Moskovitz, Hospital of the University of PA, Philadelphia, PA lgE-mediated late phase reactions (LPR) developing hours after intradermal pollen antigen (Ag) injection in some subjects may look grossly similar to typical developing delayed hypersensitivity (DH) reactions to microbial antigens. To explore underlying mechanisms in these two immunotogically distinct responses, we carried out skin chamber studies in 6 humans with previously demonstrated indurated reactions of similar size to pollen Ag and Candida (Ca) 24 hours after intradermal injection. Skin blisters were induced by heat/suction, unroofed, the bases irrigated, and collection chamber appended. Replicate individual chambers were filled with either: a) pollen Ag 100 PNU/ml; b) candida solution (Ca); c) buffer diluent (B). Fluids were removed after one hour and replaced with fresh like solutions for the next 4 hours. After removal of those fluids, cover glasses were then appended to the blister bases (at the dermal-epidermal junction) for 15' for cytologic study. Histamine levels after the 1st hour of challenge were much higher (p < = 0.002) at Ag sites (70 _+ 10 ng/ml) than at Ca sites (2 + 1). The latter was not significantly different from that at B sites (2 + 2). Levels of lactoferrin (released from neutrophils) were also higher at Ag sites (13 _+ 3.7 ~gm/ml) than at Ca sites (3.8 + 1.6), p = (I.02. Total numbers of exuding inflammatory cells (>neutrophils) were also higher in Ag vs Ca sites (6 _+ 2.1 vs 0.7 + 0.1 • 105, p 0.005) as well as the % activated cells forming superoxide (NBT ~) Ag - 60 + 11% vs Ca - 25 _+ 6% (p = 0.004). However, surprisingly, the % eosinophils were not significantly different in Ag vs Ca sites (19 + 9 vs 12 • 5). Conclusions--different inflammatory patterns occur in early developing skin LPR and DH.

S359

Braun, I E Appeld R Baruch, 2 U He~, l C Brodie,'- and H Renz, I Wirchow Klinikum of the Humboldt University, Berlin, Germany; 2Departement of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Allergic diseases are characterised by development of a neuro-immunological inflammatory response. Since N G F is known as a potent mediator acting in development and differentiation of both neuronal and immune cells, the role of NGF was examined in an animal model for allergic disorders. We found that splenic mononuctear cells expressed trk A on both the mRNA and protein level, that was further enhanced by mitogenic stimuli. In addition these cells expressed mRNA for NGF and secreted detectable levels of this protein. Moreover, intracellular staining indicated that NGF is mainly produced by T cells. The synthesis of NGF was further increased by stimulation with Con A, LPS, substance P and Noreadrenalin. In order to assess the effects of NGF, splenic mononuclear cells from OVA sensitized Balb/c were stimulated in vitro with NGF and OVA. After costimulation with N G F together with a suboptimal concentration of OVA (5 l~g/ml) a dose dependent increase of the TH2 cytokines, IL-4 and IL-5, and the corresponding immunoglobulins IgG1 and IgE was observed. In contrast, the TH1 cytokine IFN-y and the associated immunoglobulin lgG2a levels remained unaffected. These effects were NGF specific, since they could be blocked by an anti- NGF-antibody. These results suggest that NGF produced by mouse lymphocytes acts as a positive immunomodulatory factor in the development of the allergic immune response.

$360

Abstracts

1468

Ciliary Beat Frequency of Bronchial Epithelial Cells is Reversibly Inhibited by Platelet-Activating Factor (PAF). U. Klettke, W. Luck, B. Niggemann, K. Paul, U. Wahn. Children's Hospital, Virchow-Clinic, Humboldt-University, Berlin, Germany Several inflammatory mediators like histamine and leucotrienes have been shown not only to play a role in initiating and maintaining bronchial hyperresponsiveness but also to influence mucociliary clearance. The aim of our study was to investigate the effect of platelet-activating factor on bronchial ciliary beat frequency (CBF) and the reversebility of this effect by the PAF-antagonist WEB 2086. Brush biopsies were obtained from eight children (mean age 3.8 years) who underwent bronchoscopy for clinical reasons (e.g. for foreign body aspiration). Immediate measurement of CBF by phase-contrast microscopy and photoelectric technique was performed: (1.) on native samples, (2.) after adding PAF (10 -5 M), (3.) after adding the antagonist WEB 2086 (10 -5 M) and (4.) after adding both, respectively. The concentrations of PAF and WEB 2086 were chosen following previously published data in human bronchial-alveolar lavage and in the sheep trachea. CBF maintained without any significant change for two hours in native samples. Addition of PAF reduced median CBF by 9% to 30% (median 19%, p<0.05). The inhibitory effect of PAF was completely reversed by the addition of the PAF-antagonist WEB 2086, while WEB 2086 alone did not significantly change CBF compared to native samples. Conclusion: Our data indicate that PAF during airway inflammation contributes to a decrease in mucociliary clearance in humans.

1469

Histological changes of the nasal mucosa after allergen challenge in seasonal allergic rhinitis. G Rasp, P Ostertag, E Pfrogner, ENT department, Ludwig-Maximilians University Munich, Germany Allergic rhinitis is associated with mucosal inflammation, which is characterized by an accumulation of eosinophils, mast cells, and T-lymphocytes. Once activated, the cells release a variety of mediators such as Eosinophil cationic protein (ECP) from eosinophils and Tryptase from mast cells. In this study we examined 20 patients with a proved grass pollen allergy. All patients underwent nasal surgery with conchotomia. The patients were randomized and challenged either 4 (_+2) or 24 (_+2) hours before operation outside the grass pollen season. Antigen challenge was performed under direct vision on only one side by application of 1000 BE of grass pollen extract with a pipette on the anterior portion of the inferior turbinate. A control solution was given to the opposite side. Nasal mucosa was snap frozen postoperatively. Immunostaining was performed on cryostat sections by the ABC method using the monoclonal antibody EG for ECP, AA1 for Tryptasc and CD4 for T-helper-lymphocytes. The presented design enabled a direct comparison of allergen and control challenge in one patient. Furthermore the time course of the cellular activation could be investigated by performing conchotomia either 4 or 24 hours after allergen challenge. Already 4 hours after allergen contact, an increase of ECP and Tryptase could be found in the challenged side compared to the control side. 14 hours after provocation the number and activation of especially eosinophils but also mast cells was even more pronounced. No substantial increase of T-helper-lymphocytes was seen. In this study we could demonstrate an activation of eosinophils and mast cells by unilateral allergen challenge in allergic rhinitis. The non challenged side could herein work as direct control. The histological changes were most distinct after 24 hours.

J ALLERGY CLIN IMMUNOL JANUARY 1997

1470

The airway inflammatory response induced by intratracheal injection of antigen pulsed dendritic cells is modified by systemic adjuvant. Lambrecht BN, Peleman t~4, Bullock GR, Pauwels RA. Department of Respiratory Diseases, University Hospital, Ghent, Belgium. Dendritic Cells (DC) are the major antigen presenting cells of the airways, involved in priming naive T lymphocytes to respond to inhaled antigen. Their precise role in the induction of allergic sensitization is unexplored. We have developed an experimental system in which injection of only 2.104 antigen pulsed DC into the trachea of rats, primes the immune system for a secondary T cell response to inhaled antigen, without inducing airway eosinophilia or antigen specific IgE formation. To test the hypothesis that DC can induce allergic sensitization under circumstances of immune activation, we passively transfered 5.104 ovalbumin-pulsed DC (OVADC) into the trachea of unimmunized Brown Norway rats (n = 8 per group). These had received IP injection of adjuvant (heat killed Bordetella pertussis in AI(OH)3 ) immediately prior to transfer. Control animals received either OVA-DC or IP adjuvant only. Ten days later, animals were exposed daily to 30 rain OVA aerosol for 7 d. Total and differential cell counts and flow cytometry were performed on BAL cells 24 h after the last aerosol dose. In animals injected with DC/adjuvant, the recovery of BAL T-lymphocytes after 7 days of aerosol was respectively 2.2 and 7 times higher than the recovery in animals injected with DC or adjuvant only. BAL eosinophilia (3.2% of total cells) was present in the group that received OVA-DC/ adjuvant and absent in the control groups. OVA specific IgE was significantly higher after the challenge period in the DC/adjuvant group (39U/ml), compared with the control groups (2U/ml for DC and 5U/ml for adjuvant IP). We therefore conclude that Bordetella pertussis adjuvant shifts the type of airway inflammatory response induced by DC towards a T-helper-2 type response, characterized by the presence of specific T cells, eosinophils and antigen specific IgE formation.

1471

Topical Glucocorticoid Treatment of Ragweed Hay Fever. I. Effects on IgE and IgA Antibody Production. Jorgensen, GJ Gleich, H Kita and CE Reed, Mayo Clinic, Rochester, MN. Topical glucocorticoids are an effective treatment for allergic rhinitis, acting, in part, by inhibiting TH2 cytokine production and suppressing eosinophils, IL-3, IL-5, and GM-CSF in nasal secretions. Seasonal exposure to ragweed induces a rise in IgE antibody in serum and IgA antibody in nasal lavage fluid. In this study, we determined whether topical budesonide suppresses the production of ragweed specific IgE in serum, and secretory IgA and the TH2 cytokines in nasal secretions. Patients with ragweed hay fever and a strongly positive serologic test for ragweed IgE antibody received budesonide nasal spray (256 meg/day) or placebo in a randomized parallel double-blind study. The treatment began 2 weeks before and continued for 2 weeks after the pollination period. IL-4, IL-5, and ragweed specific lgA antibody were measured immunohistochemically in the nasal lavage fluid, and ragweed specific IgE antibody was measured in the serum. Nasal symptom scores were strikingly lower in the budesonide group compared to the placebo group (p < 0.01). A 2-fold increase in serum ragweed IgE antibody was seen by week 11, but no significant difference between the two groups (p = 0.796) existed. Similarly, a 3-fold increase in IgA antibody in nasal secretions was present at week 8 and also was unaffected by budesonide treatment (p = 0.683). Contrary to the hypothesis, IL-4 increased during the season in both groups (p = 0.436), whereas nasal lavage fluid IL-5 was significantly lower in the budesonide treated patients (p = 0.041). We conclude that in hay fever topical glucocorticoids are not effective at preventing a rise in IL-4 production or antibody responses, including production of IgE and secretory IgA antibodies, whereas they do suppress the rise in production of IL-5.

S274

1116

1117

Abstracts

Co-stimulatory Molecule Expression in the Cornea: The Role in Graft Rejection. BM Gebhardt, LSU Medical Center/LSU Eye Center, New Orleans, LA The presence of cells expressing the B7 co-stimulatory molecule in the corneas of mice and rabbits and the capacity of these B7-expressing cells to promote corneal allograft immune responses were investigated. Rabbit and mouse corneas were collected and analyzed for the presence of B7-expressing cells by immunohistochemical and molecular biological techniques. The presence of B7-expressing cells in the rabbit cornea was correlated with the capacity of the cornea to be rejected following transplantation into an allogeneic recipient. Corneas treated with ultraviolet irradiation to eliminate B7-expressing cells were compared with unirradiated corneas. Immunohistochemical staining showed that the peripheral one-third of both the rabbit and mouse corneas contained cells that stained for B7 co-stimulatory molecules. The morphology and distribution of these cells suggested that they represent the corneal Langerhans cell population. Reverse transcriptionpolymerase chain reaction analysis of corneal mRNAs from mouse and rabbit corneas and subpopulations of corneal cells (epithelium, keratocytes, endothelium) indicated that there was a constitutive expression of B7 transcripts in these corneas. Orthotopic transplantation of irradiated and unirradiated rabbit corneas revealed that the capacity of the irradiated cornea to induce an allograft immune reaction was significantly reduced. Approximately 55% of the irradiated corneal allografts survived, whereas only 28% of the unirradiated corneal allografts survived the 60-day observation period. Heterotopic transplantation of irradiated and unirradiated allogeneic corneas in mice (H2 a to H2 b) yielded similar results. The results of this study indicate that the cornea, an immunologically privileged site, has cells which constitutively express the B7 co-stimulatory molecule and that cells expressing this molecule are important in immune recognition and corneal allograft rejection.

Regulation of Allogeneic Cellular Immune Responses by Human Donor Bone Marrow Cells: Possible Role of Precursor T Cells. JM Mathew, M Carreno, T. Vellone, L Fuller, C Ricordi, V Esquenazi and J Miller. Dept. Surgery, Div. Transplantation, U. Miami and V. A. Hosp., Miami, FL. In order to evaluate the immunoregulatory mechanisms brought about by human cadaveric vertebral-body Donor Bone Marrow Cell (DBMC) infusions accompanying organ transplantation, we have established in vitro culture systems analogous to the transplant model. Previously we had reported that DBMC inhibited both MLC and CML responses of allogeneic cells to irradiated donor spleen cells. We had also characterized the experimental conditions as well as analyzed the underlying mechanisms of this immunoregulation. Currently, we are attempting to identify the cell population(s) responsible for this immunoregulatory effects of DBMC. It was observed that addition of DBMC precultured with responder cells even on day 4 after the initiation of the cultures (as opposed to 2 days by uncultured DBMC) inhibited CML and MLC, thus suggesting that "differentiated" DBMC might be the regulatory cells. Both CD34 + and negative DBMC were able to inhibit MLC and CML of allogenic cells, thus indicating that a cell population that developed from the CD34 + progenitors in culture might be the responsible for the regulatory activity. When DBMC and purified CD34 + cells were stimulated with irradiated allogeneic cells, the major population that developed within the 7 days in culture were CD38 + cells. Furthermore, DBMC population(s) rich in CD38 + cells, separated on discontinuous gradients between 47.5 - 55.0% percoll (three fractions) gave the maximal inhibitory activity. Similarly, donor bone marrow T cells depleted of both CD4 and CD8 cells inhibited MLC and CML responses to donor antigens. These results indicated that precursor T cells present in donor bone marrow, or cell population(s) that developed from them in culture might be the regulatory cells in donor reactive allo-immune responses.

J ALLERGY CLIN IMMUNOL JANUARY 1997

1118

TCR diversity in Gamma/Delta hybridomas derived from mice with prolonged graft survival after portal vein immunization. R.M. Gorczynski, YSun, Z.Chen & S.Chung. The Toronto Hospital, Transplant Research Division Non-vascularized (skin) and vascularized (kidney, small intestine) grafts in mice and rats survive longer if animals receive pre- or peri-transplant donor specific immunization via the portal vein. We have shown that this increased graft survival is associated with oligoclonal expansion of gamma/ delta TCR+ cells in the liver and mucosal lymphoid tissue of the recipient animals. Furthermore, these gamma/delta TCR+ cells preferentially produce IL-10 on antigen-specific restimulation in vitro, and they can adoptively transfer increased graft survival to naive recipients in a manner which is inhibitable by anti-IL-10 Mab. The current study was designed to explore sequence diversity (VJ junctional sequences) in TCR+ hybridomas derived from mice receiving iv or pv immunization in a number of different strain combinations. We found that some 50% of gamma/ delta hybridomas derived from C3H mice immunized with B10.BR or BALB.K, or from C3H.SW immunized with C57BL/6, used identical VJ TCR junctional sequences. Nearly 90% of hybridomas derived from iv immunized mice used the same sequence. The remainder of the hybridomas derived from pv immunized mice used sequences unique to different strain combinations. Only hybridoma cells of the latter type produced cytokines after hsp stimulation in vitro. The relative functional activity of these different hybridoma cells in conferring adoptive transfer of graft survival is under investigation.

1119

Pre-treatment with Cyclosporin A (CYA) Facilitates Induction of Mixed Chimerism and Central Tolerance. B Nikolic and M Sykes. Massachusetts General Hospital/ Harvard Medical School, Boston MA The treatment of mice with anti-CD4/CD8 mAbs on day -5, plus 3 Gy whole body irradiation (WBI) and 7 Gy thymic irradiation (TI) on day 0 allows allo-BM engraftment and the induction of tolerance. TI can be replaced with a second anti-T cell mAb injection on day -1 before BMT, in order to deplete or inactivate residual host thymocytes which are otherwise capable of causing the intrathymic rejection of donor hematopoietic cells, even when peripheral engraftment is achieved. To further reduce the toxicity of this regimen, we have attempted to eliminate TI and additional mAbs injection by treating C57BL/6 recipient mice with CYA (20 mg/kg/day s.c.) for 12 days prior to BMT. CYA blocks the development of CD4+CD8 + thymocytes, resulting in a 10-fold and a 4-fold reduction in CD4+CD8 and CD4-CD8 + cells, respectively. We hypothesized that CYA pre-treatment would enhance donor thymic repopulation, allowing permanent tolerance without a second mAb injection or TI. Engraftment of fully allogeneic hematopoietic stem cells was observed in mice treated from day -15 to -3 with CYA, a single anti-CD4/CD8 mAb injection on day -5, followed by 3 Gy WBI and injection of 15 x 10 6 B10.A BMC on day 0. High levels of donor multilineage repopulation (51-63% donor WBC), including high early T cell chimerism (2452% after 6 weeks) were observed. Animals receiving the same treatment without CYA pre-treatment showed only transient chimerism, with very low early donor T cell population. The administration of corticosteroids on day 0 abolished the engraftment-promoting effects of CYA. No sign of GVHD was observed in any group. Skin grafting was performed at 9 weeks post-BMT, and all third party grafts were rejected (MST = 12 days). Animals receiving CYA pretreatment accepted donor skin grafts ( > 100 days), whereas animals not receiving CYA or treated with CYA plus corticosteroids rejected donor skin grafts, Pre-BMT CYA treatment therefore permits allo-BM engraftment with minimal conditioning, by creating thymic "space" or overcoming intrathymic alloresistance.

S362

1476

1477

Abstracts

J ALLERGY CLIN IMMUNOL JANUARY 1997

Expression of Eosinophil Cationic Protein (ECP), Soluble Adhesion Molecules (ICAM-I, VCAM-1) of Tears, Sera in children with Allergic Conjunctivitis. Jae-Won Oh MD, Ha-Baik Lee MD, Jung-chal Shin MD, Seoul KOREA The eye is a common target organ of the allergy and allergic conjunctivitis is among the most common of eye diseases. The presence of conjunctival eosinophilia may be considered to be a diagnostic indicator of allergic conjunctivitis. Intercellular adhesion molecule-1 (ICAM-1), Vascular Cellular Adhesion Molecule-1 (VCAM-1) have been detected on inflammatory site. The objective of this study is to measure ECP levels and ICAM-I and VCAM-1 levels of tears and sera in normal subjects and in patients with active allergic conjunctivitis and to assess the correlation of these mediators with the severity of the disease. Seventeen subjects were selected on the basis of clinical manifestation, history, skin prick test, IgE. A microcapillary tube was used to collect the tears from the inner canthus, conjunctival epithelia were obtained by scraping the upper tarsal conjunctiva. Serum IgE and eosinophil count were increased in 10 patients, allergic skin prick test were positive in 11 (D.P: 9, D.f: 8), eosinophilia in tears were present in 11 (4 patients: >3/HPF, 7 patients: 1-3/HPF). ECP in tears were increased in patients significantly (12.6 vs 4.03 ng/ml, p < 0.007), but not in serum. ICAM-1 in serum and tears was no difference between both groups, however, VCAM-1 in serum was elevated in patients significantly (1916.45 vs 1315.25 ng/ml, p < 0.009). In conclusion, eosinophil and its granule proteins as ECP in tears and VCAM-1 in serum may be very important role in allergic conjunctivitis. We will further evaluate and compare ICAM-1, VCAM-1 on conjunctival epithelium between same patients and controls by immunohistochemistry using monoclonal antibody.

Alterations in total collagen and fibroneetin expression after allergen inhalation in ovalbumin-immunized mice. GP Anderson, A M Campbell, C Marry, G Bullock, J Bousquet INSERM U454 Montpellier, France; CHU, Nimes, France; University Hospital, Ghent, Belgium; Ciba-Geigy Ltd, Basle, Switzerland. The immunization of mice with ovalbumin (OA) has been validated as a model for allergic lung disease such as asthma. Asthma is associated with changes in lung matrix structure which lead to permanent alterations in lung function. The aim of this study was to investigate whether alterations in collagen levels and fibronectin were observed in a mouse model following sub-chronic allergen exposure. Mice were immunized by parenteral injections of OA (or sham immunized) and were then challenged 21 days later with either OA or PBS for 5 days before being sacrificed on day 29 and the lungs fixed in formol and embedded in paratfin. Sections were stained with either Sirius red as a measurement of total collagen or with a monoclonal antibody to fibronectin. The levels of expression were assessed using the Leica CAS (cell analytical system) which allows analysis of the percentage positive tissue over the entire section excluding the outside edge as this gives high control values regardless of the treatment given.

Immunized

Challenge

Total collagen

Fibronectin

OA sham sham

OA OA PBS

23 (8-24) 25 (14-39) 16 (8-20)*

33 (16-59) 27 (11-51)*** 28 (15-88)**

Data are given as median (range) of percentage of positive tissue *p < 0.05 compared with OA/OA and Sham/OA groups **p < 0.01 compared with OA/OA group ***p < 0.001 compared with OA/OA group This study shows that OA immunization and challenge leads to alterations in the extracellular matrix of the lungs. and would thus appear to mimic at least some of the

changes associated with asthma, this could be of importance in the development of new therapeutic treatments.

1478

Allergen Specific Nasal Challenge: Response Kinetic to Rechailenge. G W Canonica, V Ricca, L Fregonese, M Mincarini, A Bongiovanni, G Passalacqua, M Bagnasco, G Ciprandi, Allergy & Clin Imm Service, Genoa University, Genoa, Italy Allergen specific nasal challenge is an optimal issue to study the pathophysiologic mechanisms leading to the allergic inflammation. Nasal challenge induces an immediate clinical response in sensitised subjects with a concomitant appearence of an inflammatory infiltrate. The mucosal inflammation may persist up to 48-72 hours after allergen exposure. If the subjects are rechallenged within this period the response is more pronounced: the well known priming effect. Aim of the study was to evaluate the effects of nasal rechallenge, performed at different intervals: 3 days, 1, 2 and 4 weeks after the first challenge. Forty allergic subjects underwent two nasal challenge: at baseline and after a period as above mentioned (10 per group). Studied parameters were clinical and inflammatory (number of eosinophils and neutrophils recovered by nasal brushing). Three day interval shows a hyperreactive response (priming effect), 1 and 4 week interval show a response equal to baseline, 2 week interval shows a hyporeactive response ("tolerogenic effect"). The last phenomenon may be related to a possible immunologic response similar to that achievable during specific immunotherapy.

1479

Effect of 2 novel compounds on mediator release from human nasal polyp cells. B Lebel, A M Campbell, A M Orloff,, A M Bonnard, C Vergnes, J Bousquet, INSERM U454 Montpellier and Pierre Fabre, Paris, France. Nasal polyp cells were used as a model for the study of anti-inflammatory drugs since cells obtained from this tissue and already in a primed state and therefore this may represent airways inflammatory disease more closely than other in vitro systems. The aim of this study was to investigate the effect of 2 novel compounds, L0042 and L0066 on the release of eicosanoids and cytokines from dispersed nasal polyp cells. Disaggregation of the polyps was obtained by enzymatic digestion using protease, collagenase and hyaluronidase. For eicosanoid measurements, cells were stimulated with anti-IgE for 45 min and for cytokine measurements they were incubated at 37~ for 24 h in the absence of stimulating agents.

L0042 0.I ~M 1 gM 10 I~M L0066 0.1 IzM 1 ~M 10 IxM

LTB4

LTC4

TNFa

GM-CSF

IL-8

(n:7) 60 26 9 (n=6) 47 31 59

(n= 12) 0 6 38 (n=12) 0 21 52

(n= 14) 25 37 48 (n= 9) 33 19 42

(n= 13) 7 16 41 (n= 8) 47 22 99

(n:8) 0 0 0 (n=8) 0 0 16

Results are shown as median percent inhibition. These results show that both compounds inhibit leukotriene release, TNFa and GM-CSF release but have no effect on IL-8 release. These drugs may be of therapeutic use in the treatment of inflammatory airways disease but an in vivo study is required to confirm these observations.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1480

Superantigen Triggers Allergic Inflammation of the Airways. U Herz, ~ R Riickert, t N Schnov,: and H Renzfl Departments of ~Clinical Chemistry and Biochemistry and 2Pathology, Virchow-Klinikum, Humboldt-University, Berlin, FRG. Bronchial asthma is characterized by a Th-2 type immune response plus chronic inflammation of the airways. When C57BL/6 mice were sensitized to allergens they developed allergic immune responses, but did not show the inflammatory component. Therefore, we postulated that stimulation of airway inflammation requires additional trigger factors. It was analyzed whether local superantigen exposure would enhance the allergic immune response. Mice were sensitized to ovalbumin (OVA) followed by intranasal SEB treatment. SEB-treatment resulted in: (1) influx of lymphocytes und eosinophils in broncho-alveolar lavage. (2) Synergistic enhancement of allergen induced increased airway responsiveness. We conclude that bacterial superantigens are potent factors for triggering and enhancement of allergic airway-inflammation together with increased airway responsiveness.

1481

Evaluation of the Pruritogenic Activity of lnterleukin-2 IIL-2) and TNF-alpha at the Dermal-epidermal Junction Level. U Darsow. i E Scharein,: B Bromm, 2 J Ring, l tDept" of Dermatology and Allergology Biederstein, Technical University Munich, and 2Institute of Physiology, University Hospital EppendorL Hamburg, Germany The pathogenesis of itch in atopic eczema and other non-whealing pruritic dermatoses is still unknown. Since nonsedating antihistamines do not relieve the itch in atopic eczema significantly and T-lymphocytic infiltrate is prominent at the dermal-epidermal junction level, where the itch receptors (free endings of unmyelinated C-fibers) are located, cytokines have been proposed as histamine-independent itch mediators. To investigate this hypothesis, single doses of IL-2 (10 MU/ml) and TNF-a (10 gg/ml) were delivered to the epidermis of 10 healthy volunteers with a controlled skin-prick-model which was previously validated with other pruritic and antipruritic substances. 1% histamine and solvent controls were included in a double-blind, randomized cross-over design. Itch ratings were obtained every 20 sec for 15 rain with a computerized visual analog scale (VAS) and cutaneous reactions (wheal and flare diameters, temperature) were measured. Reactions were also recorded after 2, 24 and 48 hrs. Mean itch ratings (15 rain, % VAS) were: Histamine 35.5*; IL-2 3.3*; TNF-a 1.6: and solvent control 1.75 (*p < 0.01 difference compared to solvent control). Wheal and flare reactions occurred only with histamine. In 2 volunteers, an inflammatory papule with transient pruritus developed at the site of IL-2 puncture after 12-18 hrs. Conclusion: Administered at the receptor level, IL-2 shows a very weak but significant pruritogenic effect which may be followed by a late-phase inflammatory response. TNF-a has no itch-inducing or histamine-releasing capacity in this setting. Skin prick testing with appropriate doses of potential pruritogens provides a safe and sensitive model for further chemoreceptor studies.

1482

GM-CSF Regulation by Differentiating Eosinophils After Allergen Challenge. GM Gauvreau, PM O'Byrne, RM Watson, and JA Denburg. Asthma Research Group, McMaster University, Hamilton, Ontario, CANADA. Increases in eosinophil/basophil colony forming units (Eo/B CFU), and GM-CSF localizatkm in the Eo/B colony cells have both been demonstratcd in the blood of atopic subjects when compared to nonatopic subjects. These observations suggested that Eo/B blood progenitor cells may aid in their own differentiation. Since allergen inhalation by atopic asthmatics is associated with acute increases of eosinophils, basophils and Eo/B CFU in blood, the current study used immunocytochemical methods to examine whether allergen inhalation induces a change in cytokine localization in Eo/B colony cells grown for 14 days in methylcellulose culture. Twenty-five non-smoking, atopic asthmatics with a documented late bronchoconstrictor response to inhaled allergen were studied before and 24

Abstracts

$363

hours after allergen inhalation. Blood was collected for absolute eosinophil counts, and non-adherent mononuclear cells (NAMC) were stimulated with GM-CSF or a combination of SCF and IL-3, and grown in methylcellulose culture for 14 days. Cytospins were prepared for immunocytochemical analysis of colony cells grown with SCF and IL-3. All subjects demonstrated a dual response to inhaled allergen. The maximal late response to allergen was a fall in FEV 1 of - 2 8 . l % _+ 2.9. The number of circulating eosinophils rose from a baseline of 37.0• ml _+ 3.8 to 56.7• 104/ml _+ 5.4 at 24 hours after allergen inhalation (p=0.0001). The number of circulating Eo/B CFU's stimulated with GM-CSF and enumerated after 14 days in methylcellulose culture also increased from 10.2/106 NAMC +_ 1.5 to 13.9/10 ~' NAMC _+ 1.8 (p=0.02). The percentage of colony cells localizing GM-CSF increased from 15.4% _+ 1.3 to 29.9% _+ 2.9 (p 0.0009), however, the percentage of colony cells localizing IL-5 remained unchanged from 3.9% -+0.8 to 5.7% + 0.8 (p=0.12). These results indicate that GM-CSF localization to Eo/B colony cells may provide an autocrine growth factor from developing eosinophils to enhance Eo/B progenitor differentiation after allergen challenge. Supported by MRC, Canada.

1483

Is There a Strain Difference in Susceptibility of Asthmatic Inflammation in Guinea Pigs? B C Kang, D Zhou, G Chen, J Kim. Lexington, KY. Genetic susceptibility of development of allergy and asthma has been well recognized in humans, but little is known whether a similar difference exists in guinea pigs. To examine the strain differences in the susceptibility of guinea pigs toward allergic sensitization and asthmatic inflammation, we studied cockroach allergic guinea pigs. Three strains, English Short Hair (ESH), Hartley (H), and NC/21, of guinea pigs were sensitized with cockroach allergen (CRa), 5 rag, 2• 5 days/week for 4 weeks and compared with their own control sensitization (C). Anaphy[actic antibody was measured by skin test at the end of sensitization. After one week rest, guinea pigs were challenged with CRa and specific airway resistance (SRaw) was measured continuously for 8 hours and again at 24 hour using a plethysmograph in conscious state. Bronchoalveolar lavage fluid (BALF) was collected 24 hours post CRa challenge. Skin tests revealed similar results in all three strains. CRa challenge induced a significant increase in SRaw in H (p<0.0001) a slight increase in ESH (p<0.01), but no increase in NC/21, as compared with control. All three strains of the sensitized guinea pigs showed leukocytosis in BALF as compared with controls: (total leukocytes: CRa-sens-H 19.5• ~' vs. C 5.7• p<0.001; CRa-sensESH 14.4• 10~'vs C 5.3• 10~', p<0.05; and CRa-sens-NC/21 15.9XllP vs. C 8.8x10", p<0.05). However, each strain showed different cells increased: macrophages and neutrophils in ESH; lymphocytes and macrophages in NC/21; and neutrophils and eosinophils in H. Thus, results showed that CRa exposure induced anaphylactic antibody and leucocytosis in BALF in all three strains of guinea pigs. Yet, only H strain showed the "asthma-like" lung function changes and leucocytosis with eosinophilia in BALF. The conclusion is that there is a strain difference only in expression of asthmatic inflammation, but not in antibody production among 3 strains of guinea pigs studied.

$364

1484

1485

Abstracts

Effect of cigarette smoke on house dust mite allergen (DER Pl)-induced increase in human bronchial epithelial cell permeability in vitro. C Rusznak, JL Devalia, RJ Sapsford, *AJ Wood, RI Davies and S Lozewicz. Departments of Respiratory Medicine and *Cardiothoracic Surgery, St Bartholomews and the Royal London School of Medicine and Dentistry, Royal Hospitals NHS Trust, London, UK. Although studies have suggested that exposure to cigarette smoke may be associated with the development of atopy, the underlying mechanisms are not clearly understood. It has been proposed that cigarette smoke impairs the barrier function of the airway epithelium leading to ready access f allergens such as Der pl, with subsequent sensitisation of the airways. In order to test this hypothesis we have culture human bronchial epithelial cell cultures (HBEC) in cell culture inserts, as explant cultures from surgical tissue, and exposed these for 20 minutes to cigarette smoke in the absence or presence of 300ng/ml Der pl, over a period of 24hours. The cultures were assessed for changes in i) electrical resistance and ii) movement of 14C-BSA and/or Der pl across the epithelial cell culture. Exposure of HBEC for 20 minutes to cigarette smoke did not significantly alter either the electrical resistance or movement of 14C-BSA across HBEC layer, when compared with exposure to air. In contrast, incubation of HBEC with Der pl led to a significant decrease in electrical resistance over a period of 6 hours and an increase in the movement of 14C-BSA over a period of 24 hours. Exposure of the cells for 20 min to cigarette smoke significantly enhanced these effects. The passage of Der pl itself progressively increased with time during incubation. Movement of Der pl was also increased by exposure to cigarette smoke. These results suggest that although short term exposure to cigarette smoke does not increase non-specific epithelial permeability, it may render the epithelium more susceptible to adverse effects of allergens such as Der pl.

Inhaled Allergen-Induced Changes in Bone Marrow Eosinophil/Basophil Progenitors in Mild Asthmatic Subjects. L.J. Wood, M.D. lnman, R.M. Watson, R. Foley, s Denburg & P.M. O'Byrne Asthma Research Group, McMaster University, Hamilton, Ontario, Canada. Increases in inflammatory cell progenitors, particularly eosinophil/basophil colony forming cells, (Eo/B-CFU) have been demonstrated in the peripheral blood of atopic, compared with non-atopic, subjects as well as following allergen provocation in allergic asthmatic subjects. Also, increases in bone marrow (BM) granulocyte-macrophage colony forming cells (GM-CFU) have been demonstrated in a canine model of allergen-induced airway hyperresponsiveness. The purpose of this study was to examine the effect of inhaled allergen challenge on changes in human BM progenitor cell growth. A group of 15 mild, stable asthmatic subjects, 8 dual (DR) and 7 isolated early responders (ER), was challenged with inhaled allergen (Ag). Bone marrow aspirates were taken before and 24h post Ag challenge. Low density non-adherent mononuclear cells (NAMNC) were harvested by centrifugation of BM over 65% Percoll density gradients. Progenitors were enumerated by a 14 day methylcellulose colony forming assay in the presence or absence of hemopoietic cytokines. The number of Eo/B-CFU increased in both groups after allergen challenge (rmANOVA time effect; p<0.0001). In the DR group, the increases were significant for BM incubated with granulocyte-macrophage colony stimulating factor (GM-CSF 10 ng/ml) and Interleukin-5 (IL-5 1 ng/ml) but not Interleukin 3 (IL-3 1 ng/ml). In the ER group, the increases were significant for all three cytokines tested. There were no significant differences in the magnitude of the Eo/B response between groups. However, at suboptimal concentrations of IL-5 (0.1 ng/ml), there was a significant increase in the number of Eo/B-CFU following allergen in DR (5.3-+1.2 to 9.7-+2.1 p<0.01) but not in ER (7.8_+3.0 to 7.8+-2.6 p=0.94). In addition, there was a significant difference in the magnitude of the Eo/B-CFU response between the two groups at this concentration of IL-5 (p<0.05). These data suggest that BM cells of DR are more responsive to IL-5 following allergen challenge,

J ALLERGY CLIN IMMUNOL JANUARY 1997

which may reflect a population of more committed, IL-5 responsive eosinophil/basophil progenitors. MRC Canada, Astra Draco and Rhone-Poulenc Rorer

1486

Eotaxin is Increased in the Airways and Bronchoalveolar Lavage of Asthmatic Patients. P. Renzi e, B. Lamkhioued 2, S. Allakhverdi 2, M. Rothenberg ~, A. Luster ~, D.Y.M. Leung4, and Q. Harold 1. 1McGill University, 2Notre Dame Hospital, Montreal, Canada, 3Harvard Medical School, Boston, MA, and 4National Jewish Center for Immunology and Respiratory Medicine, Denver, CO. Asthma is associated with eosinophilic inflammation of the bronchial mucosa, however the mechanisms underlying preferential eosinophil accumulation within the airways remain to be determined. Eotaxin is a novel eosinophilspecific chemoattractant which may be important for eosinophil recruitment in disease states. The aim of this study was to determine the expression of eotaxin-immunoreactivity and mRNA in bronchial biopsies and bronchoalveolar lavage (BAL) cells from asthmatic (n = 10) and normal (n = 9) individuals. Using immunocytochemistry and in situ hybridization, eotaxin-immunoreactivity and mRNA were localised to the bronchial epithelium and mucosa of asthmatic patients. Eotaxin transcripts and protein were significantly increased in the BAL (p < 0.01) and biopsies of asthmatics (p < 0.01) compared to normal subjects. Biopsy sections from normal controls exhibited weak positive signals, predominantly in the epithelium. These quantitative differences were confirmed by performing semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) with oligonucleotides encoding human eotaxin on RNA extracted from biopsies. Furthermore, we colocalized eotaxin mRNA to cells expressing protein markers for epithelial cells, macrophages, lymphocytes, and eosinophils. These results provide the first evidence for the increased expression of eotaxin in asthmatic airways and suggest that eotaxin is involved in the selective recruitment of eosinophils into the airways of patients with asthma.

1487

ICAM-I (CD54), Fas (CD95), CD40 Expression and Glutathione Peroxidase (GPx) Activity by Human Bronchial Epithelial Cells (HBEC) in vitro: Effect of Biostim. C Amsellem, F Gormand, C Bloy*, Y Pach~co, Laboratoire d'Immunoallergologie respiratoire, Pierre-B6nite and *Laboratoires Cassenne, Paris, France Apart from its physical barrier function, epithelium can contribute to inflammatory and immunological reactions in the lung. The aim of the present work was to study the CD95, CD40, and CD54 expression on HBEC and the GPx enzymatic activity of these cells. The influence of an immunostimulating compound, Biostim (0.1, 1 and 10 ~g/ml) was also investigated on HBEC (ECCAC WI26VA4 cell line) treated or not for 48 h with TNFc~ (100 U/ml) or IFNy (1000 U/ml). Surface antigen expression was determined using immunofluorescence staining and analysed with a FACScan flow cytometer. Antioxidant GPx activity was measured using spectrophotometric methods. Our results confirmed that ICAM-1 is expressed in basal conditions and increases under IFN-y stimulation, CD40 appears only after 1FNy treatment. Biostim has no direct effect on these antigen expressions and does not modify cytokine action. GPx activity and Fas expression are detected in both conditions. Expression of ICAM-1 and CD40 antigens are important for neutrophils (ICAM-1) and activated T lymphocytes (CD40) mobilization for bronchial defence. In bronchial cell cultures treated with Biostim, we observed a significant decrease of GPx activity (for all concentrations), an overexpression of Fas antigen (0.1 and 1 p,g/ml) and a trend toward increase in CD40 expression. As in other cellular models Fas upregulation and GPx decreased activity have been correlated with apoptosis, we hypothesize a possible effect of this immunostimulant compound in HBEC apoptosis. This effect could be relevant in viral infections.

Abstracts

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1488

Measurement of Tryptase in Human Bronchoalveolar Lavage Samples Following Antigen Challenge. A L Niles and M Haak-Frendscho. Promega Corporation, Madison, Wl Increased mast cell (MC) numbers are a prominent clinical feature in a number of chronic lung disorders. The precise role of the MC in airway injury repair, inflammation and maintenance is largely unknown. Increasing evidence suggests that the MC-specific serine protease tryptase may exert a broad range of effects, from promoting fibroblast mitogcnesis to airway hyperreactivity. A major limitation in defining the biological significance of tryptase in chronic airway pathogenesis has been low assay sensitivity. We now report the ability to quantitate tryptase in both pre- and post-allergen (AG) challenged human bronchoalveolar lawlge (BALl fluid using an improved two-site immunoassay. Prior to the bronchoscopy, an inhaled AG challenge was performed in two subjects with allergic rhinitis to determine the AG PD,, (the AG dose required to reduce FEVI by 20%). Segmental bronchoprow)cation (SBP) and BAL was performed, then BAL cells were removed by centrifugation, and sample supernatant frozen until analyzed. Basal levels of tryptase, as low as 39 pg/ml tryptase and below the sensitivity of other assay systems, were detected in the saline challenged control samples. In samples collected immediately after local instillation of AG with 5% or 10% AG PD,,, up to a 65-fold increase in tryptase concentration was detected. These findings suggest that this new immunoassay may provide the necessary sensitivity for establishing basal levels of tryptase. By defining pre-challenged tryptase content in BAL, insight may be gained into the amplitude of sin allergic airway response and for the evaluation of potential treatment modalities.

1489

E-selectin Preferentially Supports Neutrophil but not Eosinophil Rolling Under Conditions of Flow in vitro and in vivo. P Srirarnarao*, CR Norton.~, P Borgstrom*, RG DiScipio*, BA Wolitzlgv.{and DH Broide$ *La Jolla Institute for Experimental Medicine, La Jolla, CA; w Roche, Nutley, N J; +University of California San Diego, La Jolla, CA. E-selectin is an inducible vascular adhesion molecule that supports leukocyte rolling in vivo. However, its ability to support eosinophil rolling under conditions of flow in vitro and in viw~ has not been examined. Using function blocking mAbs (8B9 and 8G9) raised against rabbit Eselectin, we have determined whether E-selectin supports human eosinophil rolling in IL-1 stimulated rabbit mesenteric venules utilizing intravital microscopy. Anti-rabbit E-selectin mAbs and mAb 8B9 F(ab)2 fragments were found to inhibit neutrophil but not eosinophil rolling on venular endothclium. In support of these in vivo observations, significant numbers of neutrophils but not cosinophils were found to avidly roll on monolayers of E-selectin transfectants under physiologic conditions of flow in a laminar flow assay. In contrast, under subphysiologic conditions of shear (0.17-(t.5 dyn/cm2), human eosinophils rolled on E-selectin, albeit in lower numbers (3-7 fold) compared to neutrophils. In addition the rolling velocity of eosinophils was significantly higher compared to neutrophils on E-selectin transfectants. These and other studies suggest that at physiological shear rates, VCAM-I and P-selectin are likely to subserve as rolling receptors for eosinophils, while E-selectin is likely to function as a vascular adhesion receptor in mediating neutrophil but not eosinophil rolling in inflamed postcapillary venules.

1490

The Thl/Th2 cell may not exists in vivo: paradigm challenged by double-color cytokine ELISA spot assay. Alexs Y. Karulin & Paul K I~ehmann, Dept. Of Pathology, Case Western Reserve University, School of Medicine, Cleveland OH 44106 USA. Most cloned lines of murine CD4+ T cells could be classified as either proinflammatory Thl or anti-inflammatory Th2 based on the cytokines they produced. Thl cells have been defined by their production of IL-2, IFN-y and TNF-[3, and Th2 cells are defined by their production of IL-4, IL-5, IL-6, IL-10 and IL-13. Clones which express

$365

both Thl and Th2 cytokines have been classified as Th0 cells and proposed as the precursors of Thl and Th2 cells; however, by the time they are established and can be tested by standard techniques for Thl/Th2 characterisation, T cell clones have been chronically restimulated with antigen in vitro making it unclear how representative they are of the memory cells generated during the immune response in vivo. If the classic definition of Th0, Thl and Th2 cells is correct, any given memory population should consist of individual cells that simultaneously produce the set of cytokines by which it is defined. We have successfully established a double-color ELISA spot assay that can simultaneously detect two different cytokines secreted by a single memory T cell. In combination with computer image analysis, our new assay clearly detects cells producing a single cytokine as well as those producing both. We have shown that the majority of freshly isolated memory T cells secrete only one cytokine; there are almost no doublepositive cells. While it is beyond doubt that Thl and Th2 type immunity exists on the population level, the Thl or Th2 cell that would fit the classic Th0/Thl/Th2 definition cannot be detected in vivo.

1491

Immunostimulatory DNA Sequences (ISS) Are A Thl Promoting Adjuvant. E. Raz, M. Roman, E.M. Orozco, D.A. Carson, University of California San Diego, La Jolla, CA. Vaccination with naked plasmid (p) DNA elicits Thl type immune response as manifested by: 1) induction of IgG2a and IFNy synthesis 2) inhibition of IgE and IL-4 production to the gene product and 3) abrogation of eosinophil recruitment into the airways after bronchial antigen challenge despite subsequent sensitization with antigen in alum. Furthermore, pDNA inhibits an on-going Th2 response i.e. down-regulates IgE and IL-5 synthesis and promotes the production of IFNy. The immunogenicity of pDNA requires specific short palindromic ISS which contain CpG motifs (Science, 273:352, 1996). Based on these data, we hypothesized that co-administration of an ISS enriched pDNA backbone with a protein antigen will bias the subsequent immune response toward Thl. Balb/c mice were immunized with [3-galactosidase ([3-gal) or coimmunized with [3-gal mixed with ISS enriched or deficient pDNAs. Injection of [3-gal/ISS enriched pDNA elicited high levels of lgG2a anti-13-gal and led to IFNy production by antigen stimulated CD4+ T cells. In contrast, [3-gal/ISS deficient pDNA or [3-gal alone induced the production of lgG1 and IL-4. Transfection with ISS, but not with ISSdeficient pDNAs or oligonucleotides induced the transcription of 1FNu, IFN{3, IL-12 and IL-18 (IGIF) (cytokines which induce IFNy and Thl difl'erentiation) from human (h) macrophages and IFN~, from h lymphocytes. These data suggest that: 1) ISS elicit multiple signals for IFNy synthesis 2) APCs, by delivering the appropriate cytokine milieu control the differentiation of naive T cells toward Thl and 3) ISS enriched pDNA can be used as a Thl promoting adjuvant to any given protein antigen and can be useful for vaccination of allergic and certain infectious diseases.

S366

Abstracts

J ALLERGYCLIN IMMUNOL JANUARY 1997

1492

The Capacity of Measured Ligand Density to Switch Thl/Th2 Immunity. J. S. Murray, J. P. Kasselman, and T. Schountz, Division of Biology, Kansas State University, Manhattan, KS 66502. A quantitative mechanism for the differentiation of CD4 T cells into recognized subsets of Thl and Th2 effectors is controversial. In a model system designed to examine Thl/Th2 differentiation, we have defined antigen dose more precisely to the density of a minimal immunogenic peptide presented on the surface of a specific APC type. Thl- and Th2-responder MHC genotypes differ by as much as an order of magnitude in the density of this peptide displayed on B7-2 + B cells. We asked whether such B cells presenting a low ligand density primed Th2 effectors in an MHC genotype with predisposed high density presentation and Thl-type immunity; and whether high ligand density B cells primed Thl effectors in an MHC genotype which normally presents a low density and the Th2 phenotype. While low ligand density had the capacity to switch phenotype in the Thl-responder, high density presentation did not alter genetically determined Th2-responder status. Further analyses utilized a panel of Th clones derived from each MHC genotype, and we observed that Thl-genotype clones proliferate more extensively than those derived from the Th2-genotype, even at the same input ligand density. Together, these data suggest that TCR affinity may govern the capacity of certain MHC genotypes to mount Thl versus Th2 responses against the same peptide.

1493

Th2 Cells Promote the Growth of Resting or Activated Thl Cells in vitro: Evidence for Two Types of Positive Crossregulation. TB Oriss, SA McCarthy, BF Morel, MAK Campana, PA Morel, University of Pittsburgh and Carnegie Mellon University, Pittsburgh, PA The two subsets of T-helper (Th) lymphocytes, Thl and Th2, regulate each other by production of both stimulatory and inhibitory cytokines. Thl and Th2 stimulate themselves and each other by production of IL-2 and IL-4, respectively, Th2-derived IL-10 inhibits Thl proliferation, and Thl-derived IFN-7 inhibits Th2. We have generated routine Th cell lines and clones with different antigenic specificities, and used them to study interactions between Thl and Th2 in two mixed culture systems. In the frst system, Thl and Th2 were placed together in a single culture vessel and were identified at the end of the culture period by virtue of different TCR VI3 usage. The second system utilized physical separation of the two cell populations using transwell culture dishes with a membrane that is permeable to soluble factors but not to cells. In each case, distinct antigenic specificities allowed us to stimulate either Thl or Th2 alone, or both simultaneously. Thl tended to predominate when both cell types were stimulated together, and proliferated better than when stimulated in the absence of Th2. Resting Thl also proliferated in the presence of stimulated Th2 in both culture systems, suggesting that the effect was mediated by cytokines. We found that resting Thl responded to a combination of IL-4 and IL-12. We also present evidence that this cytokine combination may be at least partially responsible for the Thf proliferation we observe in mixed cultures when only Th2 is antigenically stimulated. These results demonstrate a positive crossregulatory effect of Th2 on both resting and stimulated Thl. Our mixed culture systems should allow future studies to address other aspects of Thl/Th2 crossregulation with the intention of eventually modifying unwanted Th responses.

1494

Type 1 and Type 2 T Cell Subsets: Acquisition and Regulation of Effector Functions. Laura L. Carter, Richard IV.. Dutton and Susan L. Swain. Trudeau Institute, Saranac Lake, NY Type 1 (T1) and Type 2 (T2) subsets of CD4 and CD8 T cells have been defined based on secretion of polarized patterns of cytokines and it is clear that these different subsets are critical in distinct immunological situations. Utilizing naive cells from TCR transgenic mice, we have developed in vitro models for the generation of T1 and T2 effector cells which produce IFN7 or IL-4/5 and mediate

effector functions such as killing and B cell help. Here we examine the timing with which naive CD4 and CD8 T cells develop effector functions, modulate expression of surface markers and become susceptible to activation-induced cell death (AICD) when stimulated under polarizing conditions. CD8 T cells grown under either TI or T2 conditions acquire the ability to lyse antigen-specific targets and secrete high levels of IFN7 within 24 hrs of primary stimulation. In contrast, T cells grown under T2 conditions become capable of IL-4/5 production approximately 40 hrs after initial antigen encounter. Stimulation under T1 versus T2 conditions also results in differential modulation of surface markers such as LFA-1 and ICAM-I: CD4 T cells stimulated under polarizing conditions do not produce high levels of IFN'y after 24 hrs, nor do they become lytic. Moreover, only T1 CD4 T cells become sensitive to rapid Fas-mediated AICD several days after antigen encounter. The distinct timing with which effector functions are acquired, surface markers are expressed and AICD occurs amongst T cell subsets has important implications for how, when and where the subsets will function in immune responses.

1495

Kinetics of Commitment and Cytokine Production During TH1 and TH2 Cell Differentiation From Naive Precursors. PS Akai, TR Mosmann. University of Alberta CD441ow, CD62L + T helper(TH) spleen cells from C57BL/6 mice differentiated into interferon (IFN)7-producing (TH1) or interleukin (IL)4-producing (TH2) cells when alloactivated by the B cell hybridoma M 12.4.1 in the presence of ILl2 or IL4 respectively. During TH2 differentiation several intermediate stages were identified and characterized. Up to one day following activation (D1) was an undifferentiated stage defined by undetectable 1FN7 and IL4 production. If TH2 differentiation conditions (IL4, anti-IFN7 Ab and IL2) were replaced by TGFI3, anti-IL4 Ab, anti-IFN~/ Ab and IL2 (conditions previously established to prevent precursor cell differentiation), only undifferentiated cells arose after five additional days. Thus at D1, TH2 cell differentiation is still dependant on continued presence of TH2 differentiation conditions and invariable TH2 commitment has not yet occurred. Between D1 and D3, low levels of IIA become detectable in culture supernatants; if Th2 conditions are now replaced as above, IL4 detection remains low following restimulation five days later. Following D3, a stage of covert differentiation and full commitment develops. IL4 production is not constitutive but high levels are now inducible by restimulation, and replacement of TH2 conditions does not prevent subsequent TH2 cell development with characteristic high IL4 levels. Results of similar experiments describing intermediate stages of TH1 differentiation will also be presented. Characterization of TH cell differentiation intermediates provides valuable information for developing therapeutic strategies to inhibit or alter inappropriate TH cell responses that form the pathogenic basis of several allergic, autoimmune and infectious diseases.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1496

1497

1498

IL-12 induces Thl responses but does not prevent vaccineenhanced illness during viral challenge. P Openshaw, U Khan, T Hussell. Resp. Med., Imperial College, St Mary's Hospital, London, W2 IPG, UK. In humans and mice, sensitisation to respiratory syncytial virus (RSV) antigens can result in severe inflammatory lung disease during subsequent infection with RSV. Specific antiviral T cells are responsible for this augmentation, but the role of different functional subsets in protection and disease is incompletely defined. BALB/c mice sensitiscd to the major surface glycoprotein (G) of RSV expressed by recombinant vaccinia virus develop Th2-driven lung cosinophitia after intranasal challenge with RSV. To modi~ this response, we treated mice with intraperitoneal IL-12 during vaccination. Treatment reduced vaccine-induced lung eosinophilia but increased local CD4+ cell infiltration during challenge. Weight loss and illness was not eliminated and, in some experiments, was increased by IL-12 treatment. Analysis of intracellular cytokines by flow cytometry showed that IFN-y production was increased and IL-4 and IL-5 reduced by IL-12 treatment. In control sensitiesd challenged mice, 40-509~ of bronchoalveolar lymphoid cells were B cells. Treatment reduced this figure to approximately 1.5~4. Previous studies indicate that overinduction of CTL or Th2 immunity causes vaccinc enhanccd disease; the present studies show that exuberant Thl immunity can also be responsible for w~ccinc-augmcnted illness. Anti-lnterleukin 10 Antibody Treatment Prevents Burn Injury Induced T-Cell Anergy. JL Kelly, CC Soberg, A Lyons, Z4 Mannick, and JA Lederer. Department of Surgery (Immunology), Brigham and Women's Hospital and Harvard Medical School, Boston, MA Burn injury induces a delayed state of immune suppression that is associated with decreased mitogcn-induced T-cell proliferation and IL-2 production. The mechanisms responsible for altered T-cell function after burn injury arc not well understood. Moreover, it is not known what triggers this type of immune suppression. This study examines the effect of burn injury on the outcome of a T-cell dependent immune response and addresses whether IL-1[) acts to induce burn injury effects on T-cell function in viw~. Sham and burn injured mice were immunized with TNPOVA in CFA subcutaneously and were given anti-IL-10 antibody or control Ig. Ten days later, serum TNP-specific antibodies, TNP-OVA-stimulated spleen and lymph node cell proliferation, and antigen-induced IL-2, IFN-g, IL-4 and IL-I(I production were determined. The results demonstrated that burn injury caused a reduction in serum TNP-specific lgG2a antibody isotype formation, while TNP-specific lgM, IgG1, and lgE were affected to a lesser extent. TNP-OVA-specific proliferation of splenocytes, CD4-cnriched splenocytes and lymph node cells was significantly reduced in burn injured mice. Antigen-specific IL-2, IFN-g, and 1L-10 was also decreased. All of these observed effects of burn injury on T-cell-dependent immune responses were prevented by anti-lL-10 treatment. Thus, we conclude that IL-10 contributes to burn injury induced cffects on T-cells which includes the down regulation of Thl cells and T cell ancrgy. Antigen Specific Inhibition of IL-2 Mediated T Cell Proliferation in Neonatal Primed Mice: Implications for Anergy Mechanisms of Tolerance. EH Field, Q Gao, N X C/ten, K Kazme~zak. University of Iowa College of Medicine and VA Medical Center, Iowa City, IA Priming neonatal BALB/c mice (Neo I ) with semi-allogcneie CAF~ spleen cells results in A/J specific tolerance, and adult mice accept A/J skin grafts beyond 60 days. Thcsc neonatal tolerant mice demonstrate negative MLR responses against A/J cells, but enhanced Th2 CD4 responses in vitro. We examined whether IL-2 restored MLR proliferation to A/J to determine whether Nen ~ demonstrate in vitro "ancrgy". Exogenous 1L-2 significantly increased MLR responses to A/J, although proliIcration remained lower than controls. Surprisingly, cells from Nco ~ proliferated more to IL-2 in the absence of A/J cells, prompting us

Abstracts

S367

to examine the in vitro effect of IL-2 alone. Culture with IL-2 alone induced in CD4 and CD8 cells from both Neo ~ and naive mice similar incorporation of BrdU. All the B r d U + cells expressed surface IL-2Re~, suggesting that IL-2 expands recently in vivo activated cells. Addition of A/J or B6 cells to 1L-2 treated cultures increased proliferation and BrdU incorporation of CD4 and CD8 cells from either naive or adult primed mice. In contrast, adding A/J cells into cultures of Neo' cells consistently decreased the proliferative and BrdU responses to IL-2, whereas third party B6 cells augmented the lL-2 induced responses. Therefore, Neo I cells exhibit antigen specific inhibition of IL-2 mediated proliferation. The data suggest that maintaining tolerance in this model may involve regulatory cells that block IL-2-dependent T cell proliferation. Moreover, the results underscore thc problems with attributing tolerance to in viw) ~'ancrgy" mechanisms.

1499

Novel Genetic Regulation of Thl/Th2 Cytokine Production and Encephalitogenicity in Inbred Mouse Strains. IM Conboy I, RH DeKmvJq'-, KM Tate I, ZA Cao I, TA Moore 3, D T Umetsu 2, PP Jones 1, ~Stanford University, Stanford, CA; 2Stanford University School of Medicine Stanford, CA; 3DNAX Research Institute, Palo Alto, CA This study presents evidence for genetic difference(s) between inbred mouse strains BI0.A and BI0.BR that most likely act(s) in APC to control Thl/Th2 cytokine profiles and the encephatogenicity of myelin basic protein (MBP)specific T helper cells. MBP Acl-16-specific, Ak-restricted T cell lines from B10.BR are Thl whereas those from BI0.A are Th2. Genetic difference(s) between these strains appears to control the production or activity of a novel B l0.A-dcrived soluble cytokine regulatory factor that influences Thl/Th2 commitment and down-regulates productinn of IFN-~/and TNF-c~ by mature Thl cells. The pro-Th2 phenotype of B10.A cells is dominant in (BI0.A • B10.BR)FI cells. The regulatory effects of this factor are not antigen- or MHC restriction-specific, and can act by affecting individual clonal populations of mature Thl cells rather than by expanding uncommitted precursors or Th0 cells. The BI0.A APC-derived factor appears to be made by macrophagcs and does not depend on IL-4, IL-I(I, IL-13, 11-12p40, IFN-% TGF-[3, TNF-c~, and PGE2. This factor also can be induced in activated B10.A T cells by BI0.A APC.

1500

Neutralizing Anti IL-10 Antibodies Upregulate the Induction and Expression of Delayed Type Hypersensitivity. HC Maguire, KA Ketcha, EC Lattime. Thomas Jefferson University, Philadelphia, PA. 1L-10 is associated with inhibition of delayed type hypersensitivity (DTH) reactions. We have tested the proposition that neutralization of endogenous IL-10 would increase DTH. We used two different rat antimurine 1L-10 monoclonal antibodies as well as a rat monoclonal with specificity only for human IL-10. A series of experiments were performed in mice with allergic contact dermatitis as a model for DTH. In a typical experiment, one group of BALB/c female mice were given 1 mg of anti-IL-10 antibody (JES5-A8 2A5) intraperitoneally. A few hours later they, and a positive control group, were sensitized on the flank with 5% oxazt~lone. Both groups were ear challenged seven days later with 0.2% oxazolone: the induced rcactions were significantly larger in the anti-IL-l[) treated group. The histology was typical of delayed type hypersensitivity. Further, in routinely sensitized mice, when antiIL-10 antibody was given at the time of challenge the reactions were substantially increased. Wc conclude that anti-lL-l() antibody boosts DTH by inactivating otherwise down regulating endogenous IL-10. Neuralizing endogenous IL-10 is likely to be a useful pharmacological approach for modulating DTH as well as other inflammatory reactions.

S368

1501

Abstracts

IL-12 and IFN-T Regulation In the Context of Endogenous IFN-od[5. LP Cousens, HC Su, MC Ruzek, GC Pien, and CA Biron. Brown University, Providence, RI Roles for IFN-cd[3 in cytokine regulation during viral infections are being identified. Effects are being examined with mouse studies in culture, after stimulation of splenic leukocytes with fixed Staphylococcus aureus Cowan strain (SAC), and in vivo, after infections with lymphocytic choriomeningitis virus (LCMV) or murine cytomegalovirus (MCMV) and after treatment with the IFN-[3-inducer polyinosinic-polycytidylic acid (polyI:C). Our experiments have demonstrated that MCMV, but not LCMV, induces IL-12 p70 and NK cell IFN-~/ production. We also have shown that IL-12 expression and the resulting NK cell IFN-~ response are regulated by IFN-~/13: 1) IFN-a or IFN-[3 inhibits in vitro responses to SAC, 2) IFN-~/[3 neutralization enhances or reveals in vivo responses during MCMV and LCMV infections, respectively, and 3) LCMVelicited IFN-a/[3 blocks lipopolysaccharide induction of IL-12 in vivo. Recent studies have extended these observation to further characterize mechanisms for inhibition. Effects were not to due to general decreases in protein synthesis, as IFN-~ did not significantly alter TNF, and enhanced IL-6, production by SAC-stimulation. Lack of detectable IL-12 and IFN-~/ in LCMV, as compared to MCMV, infection was accompanied by greater and extended IFN-a/[3 expression. Surprisingly, polyI:C treatment induced rapid and substantial production of IL-12 p40 but little detectable IFN-~/. Splenic leukocytes isolated from normal, LCMV-infected, MCMV-infected, or polyI:Ctreated mice all maintained responsiveness to SAC for IL-12 and IFN-3, induction. However, after in vivo treatment with the IFN-a/[3 inducers, cells developed unresponsiveness to direct IL-12 stimulation of NK cell IFN-~/ production. Results demonstrate novel pathways for early cytokine regulation during viral infections. Furthermore, they identify changes in NK cell responsiveness to IL-12 and requirements for IFN-~/expression after in vivo IFN~/[3 exposure. Supported by NlH grants RO1-CA41268 and T32-ES07272, and HHMI predoctoral fellowship.

1502

Local expression of TGF[3 in islets of Langerhans leads to alteration of the pancreatic architecture and prevents the onset of diabetes. Iqbal S. Grewal, Kate D. Grewal. F. Susan Wong. Dominic Picarella, Charles A. Janeway Jr. and Richard A. Flavell. HHMI and Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06510 Transforming growth factor(TGF)-[3 is involved in various physiological and pathological processes and has been the focus of studies of autoimmunity; its role in the progression of autoimmune diabetes is, however is still unclear. To analyze the effect of TGF[3 in insulin dependent diabetes mellitus, we generated non obese diabetic transgenic mice expressing the TGF[3 gene under the control of rat insulin II promoter. This resulted in massive fibrosis and disruption of normal tissue pattern which was accompanied with infiltration of mononuclear cells. The islets were replaced with clusters of microislets and fibroblastic extracellular matrix. Expression of TGF[3, however, inhibited the development of diabetes in these mice. Effects of local expression of TGF[3 was also assessed on allograft rejection. Expression of TGF[3 in the donor allogeneic islet did not delay or prevent their rejection. Taken together, our results suggest that expression of TGF[3 causes disorganization of pancreatic architecture, prevents onset of diabetes and does not serve as an immuno-suppressive agent for allograft rejection. Mechanism of TGF[3 action will be discussed.

1503

Systemic administration of IL-12 protects mice from experimental autoimmune uveitis (EAU) through a mechanism involving IFN-T. R.R. Carpi, P.B. Silver, R. Agarwal, L.V. Rizzo, C.C. Chan, and T.K. Tarrant 1. Lab. Immunol. NEI, and IHHMI/NIH Research Scholars Program, Bethesda, MD. IL-12 was shown to promote Thl-type responses. Because pathogenic effector T cells in uveitis are Thl-like, we theorized that IL-12 administration would abrogate resis-

J ALLERGY CLIN IMMUNOL JANUARY 1997

tance to EAU in mouse strains that may be generating a low Thl response. Mice were immunized with a uveitogenie regimen of the retinal antigen IRBP and received 100 ng of IL-12 daily for 5 days either early (days 0-4) or late (days 7-11) after immunization. Serum IFN-~ levels during treatment were assayed by ELISA. EAU was scored by histopathology on day 21, and Ag-specific responses were assessed by DTH, lymphocyte proliferation and IFN-~/ production to IRBP. Unexpectedly, administration of IL-12 decreased the severity of EAU in several mouse strains. Subsequent experiments using C57BL/6 mice showed that only the early treatment was consistently protective. High levels of serum IFN-~/ were present in IL-12-treated mice throughout the treatment. IRBP-specific IFN-~/ production by draining lymph node cells was suppressed in the protected group (early treatment), but was enhanced in the unprotected group (late treatment). Unlike wild-type animals, IFN-',/ gene-disrupted ('knockout') mice were not protected from EAU by IL-12 treatment. We conclude that administration of IL-12 protects from EAU and suppresses the IRBP-specific IFN-~ response through a timeqimited mechanism that involves systemic induction of IFN-~. We hypothesize that overproduction of IFN-'y at the time that uveitogenic effector T cells are being generated, but not later, diverts their differentiation from the Thl pathway by a process of negative feedback.

1504

The Orientation and Selection of a T Cell Receptor on its Peptide: MHC Ligands in Intrathymic Positive Selection and Peripheral Activation. C.//. Janeway, Jr., D.B. Sant'Angelo, S. Hong, P.G. Waterbury, J. Goverman, T. Brabb, A.C. Hayday, R. Medzhitov, C. Viret, A.V. Chervonsky. Section of Immunobiology, Yale University School of Medicine, New Haven, CT. We have used site-directed mutagenesis of an cq3 T cell receptor (TCR) to map out contacts on MHC molecules and the antigenic peptide it responds to, as well as its response to allogeneic stimulators. We have mapped five or the six putative CDR loops onto the peptide:MHC ligand. This allowed us to orient this TCR over the MHC:peptide ligand without having to introduce strained bonds. This structure was recently confirmed by X-ray crystallographic analysis, at least for a TCR binding to a peptide:MHC class I molecule ligand. This leads us to ask if all TCRs recognize their ligands in the same orientation. We have approached this question by measuring the development of several TCRs by PCR-RFLP analysis on various subsets of thymocytes and peripheral T cells of 13chain transgenic mice. We find that different [3 chains select very differently on their own, but all tend to prefer a TCR with the same or a closely related specificity, as determined by sequencing the c~chain of the TCR. We confirmed the preference for these a chains by examining positive selection by the same [3 chain with the parental c~ chain, c~[3 TCRs that select strongly were associated with strong selection of the parental e~ chain, while ~[3 TCRs that selected weakly were associated with weaker selection of the parental ~ chain. These results taken together suggest that the TCR is oriented in the same way with all of its ligands, which may be due in part to the existence of a binding site for the co-receptor molecules CD4 or CD8 on the c~chain V region, and that the TCR is selected on self peptides. Funded in part by the Howard Hughes Medical Institute and NIH grant AI-14579.

J ALLERGYCLIN IMMUNOL

Abstracts

$369

VOLUME 99, NUMBER 1, PART 2

1505

Identification of Large Protein Fragments as a Substrate for Antigen Capture by MHC Class II Molecules. F Castellino, F Zappacosta and R N Germain, Lymphocyte Biology Section, Laboratory of Immunology, NIAID, NIH Bethesda, MD/USA Although their binding sites can accommodate longer ligands, peptides of 15-20 residues are the primary form of processed antigen recovered from major histocompatibility complex (MHC) class II molecules isolated from living cells. Whether generation of these short peptides precedes association with class II, or whether class II initially binds to unfolded proteins or large protein fragments, followed by degradation of the unprotected regions, remains unknown. Using a combination of SDS-PAGE and mass spectrometry approaches, we have now identified an SDS stable, long-lived 12(}kD complex composed of a large fragment of hen egg lysozyme (HEL) bound simultaneously to two different class I1 molecules. This complex is produced within the cndocytic pathway by the interaction of exogenously acquired f I EL with newly synthesized Mt IC class I1 moleculcs and constitutes more than 5q4, of the total class I1 pool loaded with HEL-derived antigen fragmcnts. Largc fragmcnts of other proteins can also bc found associated with class 11 molecules in normal B lymphoblasts. These data suggest that a major pathway of antigen processing inw)lves the initial binding of class II to large protein substrates upon exposure of regions with suitable motifs, followed by clcavagc and/or trimming of the cxposed protein around this bound region.

1506

Amino-Terminal Trimming of MHC Class II Associated Peptides. (Twistopher A. Nelson, llan VidavskT, Michael L. Gross, Nicholas J. Viner & Emil R. Unanue. Washington Univ. Sch. of Med., St. Louis, MO 63110, USA. Binding by MHC class I1 protects peptides from proteolysis during intraccllular processing; but this protection extends only to the peptide core residues that are in contact with the class I1 molecule. The pcptidc ends, because they extend beyond the binding site, are susceptible to trimming by exopeptidase enzymes. We have examined the amino-terminal ends of naturally processed peptides for evidence of trimming. A set of closely related hen-egg lysozyme (HEL) molecules, engineered by site directed mutagencsis of a HEL eDNA, were expressed as individual stable transfcctants in the B-lymphoma line MI2.C3.F6. The mutant HEL proteins were processed and presented on the class II I-A k molecules of these transfected cells. Wc were able to purify, the resulting naturally processed HEL peptidcs for analysis by MALDImass spectrometry. As expected, amino acid substitutions ot the HEL proteins allcred the profile of peptides produced. For example, the most prominent pcptide presented from the wild-type HEL protein spans residues 48-62, DGSTDYGILQINSRW. Replacement of the aspartic acid at position 48 of HEL with prolinc caused most of the HEL peptides to be I residue longer (_>99%, TPGSTDYGILQINSRW). Similarly, almost all of the peptidcs presented after protinc substitution at position 47 were Iwo residues hmger (96%, NPDGSTDYGILQINSRW). Clearly, proline acted as a stop signal for aminopeptidase trimming in these experiments. The ability of proline to protect the amino terminus decreased as the distance from the class II molecule increased. Only 47% of the peptidcs recovered from fIEL with a proline at position 46 contained the introduced prolinc, (RPTDGST...), only 11% from proline at position 45, (NPNTDGST...), and none from HEL with a proline at position 44. Although additional work will bc needed to identity, the enzymes involved, the ability to detect small changes in naturally processed peptides should help to forward our understanding of MHC class II antigen processing.

1507

Two patterns of T cell response after peptide immunization: a challenge to the concept of crypticity. NJ Viner, CV Nelson, ER Unanue. Washington University, St. Louis, MO, USA T cell hybridomas were isolated after immunization of CBA/J mice with peptides representing naturally-processed

forms (48-61, 48-62) of the immunodominant, I-Ak-restricted epitope of hen egg-white lysozyme (HEL). The majority of hybridomas demonstrated a >2 logs more sensitive response to peptide compared with native HEL, regardless of the type of APC used. (These were termed "type B" hybridomas in contrast to "'type A" hybridomas which had comparable responses to peptide and native HEL). To show that the peptides recognized by the two types of T cell were identical, HPLC-fractionated peptides elated from l-Ak molecules of APCs expressing a HEL fusion protein were used to stimulate sensitive Type A and Type B hybridomas. Both responded equally well despite their respective native HEL dose-response curves being >3 logs apart. Antigen conformation was critical in determining the formation of type A complexes from native HEL since reduced HEL was recognized equally well by both types of hybridoma, even when presented by fixed APC. Moreover, partially folded HEL was recognized better than native HEL by type B hybridomas whilst still requiring processing (i.e. live APC). Thus, T cells activated by peptide immunization might not be the same as those that would be activated by naturally-processed antigen (i.e. type B rather than type A). These data have a number of important implications, sounding a cautionary note for the therapeutic potential of peptide vaccination as well as suggesting an alternative interpretation tor many in vitro studies based on peptide immunization.

1508

Collagen induced arthritis (CIA) in HLA-DR, DQ double transgenic mice: implications for extended haplotypes in rheumatoid arthritis. Veena Taneja, J. Hanson, M. Smart, H. S. Luthra, C. S. David. Mayo Clinic, Rochester, MN Predisposition to rheumatoid arthritis (RA) has been linked to 'Shared cpitope" mapped to hypervariable region III (67-74) of the DRB1 chain. HLA-DRBI*I502 (DR2) has been shown to be associated with protection in RA. To study the role of DR and DQ molecules in RA, double transgenic mice were generated, tILA DRBI*I502 and DRBI*0301 genes were introduced in CIA susceptible Abo. DQA1 "0301, DQBI*0302 (DQS) mice. The DR2.DQ8 and DR3.DQ8 mice were immunized with bovine type II collagen and monitored for onset and progression of the disease. Around 75% of the Abo.DQ8 mice were susceptible to CIA. Introduction of the DRBI* 1502 led to a significant decrease in incidence (20%) as well as severity of the disease in Abo.DQ8 mice. On the other hand, there was only a marginal decrease in incidence in DR3.DQ8 mice. In vitro studies using draining lymph nodes from the immunized mice showed a higher proliferative response to BII in DR3.DQ8 compared with DR2.DQ8 mice. Inhibition studies using anti DQ and CD4 antibodies showed response to BII to be DQ and CD4 restricted in both double transgenics. Higher levels of lgG3 were observed in arthritic mice. DR2.DQ8 arthritic mice showed higher levels of IgG2b compared to protected mice. DR2.DQ8 mice showed a lower IFNy level when challenged with BII compared to DR3.DQ8 mice. The results show that while susceptibility to arthritis is determined by DQ locus, polymorphism in DR may modulate the disease by shaping the T cell repertoire.

S370

1509

Abstracts

Location of a Cis-acting Element that Promotes the Transcription of the Gene that Encodes Mouse Mast Cell Protease 5 (mMCP-5). M.F. Gurish, T. Fernandez, K.F. Austen, and R.L. Stevens. Brigham and Women's Hosp. and Harvard Med. Sch., Boston, MA mMCP-5 is a chymase that is selectively expressed by cutaneous mast cells and certain other populations of mast cells. Because it is the first chymase expressed during the in vitro differentiation of bone marrow progenitors into mast cells, experiments were carried out to deduce how the transcription of the mMCP-5 gene is regulated. DNase-I hypersensitivity analysis revealed three potential cis-acting elements 1.6, 2, and 3 kb upstream of exon 1. No DNase-I hypersensitivity sites were found within the gene or its 3' flanking region. To confirm and extend these findings, varied amounts of the 5' flanking sequence were subcloned into an expression vector that contained or lacked the thymidine kinase (TK) promoter. The resulting constructs were then transfected into mouse mast cells that constitutively express mMCP-5. Deletional analysis revealed that the 124-bp sequence residing 3.5 kb upstream of exon l is required for the transcription of the reporter gene by the endogenous promoter. The ability to increase the activity of the TK promoter by >10 fold indicated that the region functions as a strong enhancer. As assessed by gel-mobilityshift analyses, mMCP-5 expressing cells contain a transacting factor that interacts with this positive regulatory element. These studies begin to address at the molecular level, how the transcription of this and other chymase genes are regulated in mouse mast cells. [Supported by NIH grants AI-23483, AI-22531, AI-31599, AR-07530, AR-36308, HL-48958, and HL-36110.]

1510

Regulation of Expression of Dog and Human Mast Cell at-Chymase. GH Caughey, JL Blount, UCSF, San Francisco, CA Chymases are chymotrypsin-like serine proteinases secreted by mast cells. Their actions include degradation of matrix proteins, activation of matrix metalloproteinases, and stimulation of gland secretion, c~-Chymases differ from 13-chymases in structure, function, and species- as well as mast cell subset-specific expression. Dog and human chymases belong to the c~ group. To understand the basis of subset-specific expression, we explored chymase expression in dog BR mastocytoma cells. RNA blots reveal high, steady-state levels of chymase mRNA in BR cells; mRNA levels drop dramatically in cells incubated with phorbol ester or dexamethasone, without affecting levels of tryptase mRNA. Nuclear run-off studies suggest a transcriptional mechanism of regulation. DNA blots reveal that the dog c~-chymase gene, as in humans, may be the sole ehymase in the genome. The dog gene and flanks were sequenced and compared with those of the human gene. Multiple regions of potential regulatory importance are shared by the 5' flanks of the dog and human genes, few of which are present in those of known [3-chymase genes. Chimeric DNA containing portions of the human gene's 5' flank drives expression of a reporter gene when transfected into BR cells and defines regions of the human flank containing promoter activity. In summary, BR cells exhibit high-level expression of dog c~-chymase regulated independently of tryptase and they support transcription using human a-chymase promoters; thus, BR cells are useful in probing mechanisms governing expression of c~ehymases, including the human enzyme. (Supported by NIH HL-54774 and by an ALA Career Investigator Award).

1511

Mast cell tryptase activates p42-44 map kinase in human lung fibroblasts. JA Cairns, AF Walls, Southampton General Hospital, Southampton, UK Tryptase is a serine protease of 32-34 kDa molecular size found exclusively in mast cell granules. It is released during mast cell degranulation and has been shown to stimulate epithelial cell growth, IL-8 release and to up-regulate ICAM-1 expression [J Immunol 156:275-283(1996)]. We have investigated the ability of tryptase to stimulate cell proliferation and to activate p42-44 mitogen activated protein (MAP) kinase in MRC-5 human lung fibroblasts.

J ALLERGY CLIN IMMUNOL JANUARY 1997

Tryptase at 25 mU/ml stimulated a 2.5 fold increase in cell proliferation, assessed by the incorporation of 3H-thymidine into cellular DNA. This effect of tryptase was reduced in the presence of the protease inhibitor leupeptin suggesting involvement of the catalytic site. Immunoblotting with an anti-phosphotyrosine antibody using lysates of fibroblasts treated with 25 mU/ml tryptase revealed enhanced tyrosine phosphorylation of a protein doublet at 42-44 kDa. Tyrosine phosphorylation of p42-44 peaked after 5 min exposure to tryptase and returned to basal level by 15 rain. Enhanced tyrosine phosphorylation of p42-44 by tryptase was also dose dependent. Peak tyrosine phosphorylation occurred between 12.5 and 25 mU/ml tryptase with a return to basal at 50 mU/ml. After a 5 rain exposure to tryptase, p42-44 also showed increased phosphorylation on threonine but not on serine residues. The p42-44 protein was confirmed as map kinase by immunoblotting with a monoclonal antibody specific for map kinase. Incubation of myelin basic protein (MBP) with 32p-ATP in vitro in the presence of map kinase immuno-precipitated from cell lysates revealed increased phosphorylation of MBP in cells treated with tryptase compared to untreated cells indicating that tryptase stimulates map kinase activity. These results suggest that the intracellular signalling mechanism of tryptase may be mediated via activation of p42-44 map kinase.

1512

Fibrinogen is the Preferred Substrate of the Tryptase, Mouse Mast Cell Protease 7 (mMCP-7). C. Huang, G. Wong, Andrej Sali, N. Ghildyal, M.F. Gurish, and R.L. Stevens. Brigham and Women's Hosp. and Harvard Med. Sch., Boston, MA; and Rockefeller Univ., New York, NY mMCP-7 is a tryptase of unknown function expressed by a subpopulation of mast cells that reside in numerous connective tissue sites. Because mMCP-7 is specifically released into the plasma of V3 mastocytosis mice undergoing passive systemic anaphylaxis, this in vivo model system was used to determine the physiologic substrate of the tryptase. Plasma samples taken from V3 mastocytosis mice that had been sensitized with IgE and challenged with antigen were found to contain substantial amounts of four 34- to 55-kDa peptides, all of which were derived from fibrinogen. To determine the substrate specificity of mMCP-7, a phage display peptide library specific for tryptases was constructed and screened with recombinant mMCP-7. One phage clone was obtained repeatedly that possessed a susceptible amino acid sequence nearly identical to a sequence in the middle of the alpha chain of rat fibrinogen. Subsequent in vitro studies revealed that recombinant mMCP-7 is a potent anticoagulant due to its ability to degrade mouse fibrinogen in a sequential manner. Because fibrinogen is the preferred substrate of mMCP-7, this tryptase probably functions in vivo to inhibit fibrin clot formation during mast cell-mediated inflammatory reactions so that circulating immune cells can make their way more easily into tissues. [Supported by NIH grants AI23483 and HL-36110.]

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1513

MAb B12 Directs Human Lung Tryptase to Produce Anticoagulant Fragment D from Fibrinogen. S Ren, A E Lawson, M Carr Jr, C Baumgarten, LB Schwartz. Virginia Commonwealth University, Richmond, VA B12, a murine mAb made against active human lung tryptase, was examined for its ability to modulate tryptase activities. The tripeptide, tosyl-Gly-Pro-Lys-p- nitroanilide (TGPL) and human fibrinogen were used as substrates. BI2 inhibited tryptase-catalyzed cleavage of TGPL at its neutral pH (7./5) optimum in a noncompetitive manner, with a K' of 0./5 nM. At pH 6./5 the ability of B12 to alter the already reduced rate of TGPL cleavage was minimal. Fibrinogenolytic activity of tryptase stabilized either with Dextran sulfate or heparin was optimal at pH 6-6./5. B12 inhibited fibrinogenolytic activity at neutral pH. Surprisingly B12 enhanced the rate of fibrinogenolysis at least 10-fold at acidic pH, and yielded distinct cleavage fragments corresponding to the p[asmin-elcavage product called fibrinogen fragment D. Because this activity was not inhibited by SBT1, and was replicated with rh[3-tryptase, tryptase was likely responsible. A 90 kDa fragment under nonreducing conditions, and 43 and 38 kDa fragments under reducing conditions were found corresponding to Lys63-Ala-lle-Glu-Leu-Thr-Tyr (~/ chain) and Lys134-AsnGlu-Asn-Vat-Val ({3 chain), respectively. Whereas fibrinogenolytic products of the Bl2-tryptase complex showed anticoagulant activity, those of tryptase alone did not. Thus, tryptase activity and specificity can be modulated in a biologically significant manner.

1514

Release and Activation of Matrix Metalloproteinase-9 in the Late-Phase of Human Cutaneous Allergic Reactions. L Zeng, E,/Goetzl and B Zweiman, * University of California Medical Center, San Francisco, CA and University of Pennsylvania School of Medicine,* Philadelphia, PA Challenge of epidermally denuded and chamber-sealed skin sites of allergic subjects with antigen or buffer alone (control) for 6 hours leads to peak release of histamine and tryptase within 1 hour, of leukotrienes and prostaglandin E2 by 2 to 4 hours, and of phospholipid platelet-activating factor at 5 to 6 hours. Matrix metalloproteinases (MMPs) are implicated in immunity by localization in immune cells, involvement in T cell migration through basement membranes, and cleavage of adhesive proteins and cytokines. MMPs in cell-free cutaneous chamber fluids collected at 5 hours were identified by gelatin-non-reducing SDS 10% polyacry[amide get zymography and quantified by densitomctry. MMP-9, the 92kDa gelatinase B derived from granulocytes, macrophages and mast cells, was the principal activity in cutaneous fluids with only minor amounts of MMP-2 and no MMP-I or -3. MMP-9 activity ranged from 1.6- to 9.4-fold higher at antigen than buffer sites (mean + SEM = 3.3 + 0.9) in 8 of lhc 9 subjects studied and was 50% lower in 1 subject (overall mean + SEM = 3.0 _+ 0.9, p < (}.05, n = 9). The ratio of MMP-9 activity in the smaller activated form relative to the precursor was a mean (+_ S.D.) of 0.54 + (I.30 for buffer sites and 0.54 _+ 0.29 for antigen-challenged sites, that were not significantly different. The increases in MMP-9 associated with a cutaneous late-phase allergic reaction suggest a role in leukocyte recruitment and effector functions. (Supported by NIH Grants U01AI 34570 IEJG} and AI 14332 (BZ})

1515

A possible role for eosinophils and neutrophils in airway remodelling in asthma. J.A. Warner, J.K. Shute*, P. Lackie*. P. Juliusw W. Luttmannw and C. Kroegelw School of Biological Sciences and University Medicine*, University of Southampton, Southampton, UK and Pneumology, Dept IV, Medical Clinic IVw Friedrich-Schiller-University, Jena, Germany. Matrix metalloproteinases (MMPs) are involved in tissue remodelling as well as leukocyte recruitment. We measured MMPs in bronchoalveolar lavage (BALl of 22 patients with mild, atopic asthma 10 rains and 18 hrs after antigen or sham challenge. MMP activity was low 10 rains after challenge but substantial amounts were found in both antigen and saline challenged sites after 18 hrs. Levels of

Abstracts

S371

MMPs were 3-4 fold higher in the BAL from the antigen challenge site and MMP levels correlated with the numbers of eosinophils (rho 0.52, P < (1.001) and neutrophils (rho = 0.58, P < 0.001) infiltrating the airways. In BAL from 10 patients we measured a range of mediators and cytokines and found that MMP activity correlated strongly with the presence of the cosinophil granule protein, ECP (rho = 0.80, P < 0.001) but not with histamine release. MMP activity also correlated with the presence of the eosinophil attractants, IL-5 (rho 0.50, P < 0.05) and GM-CSF (rho = 0.59, P < 0.0i) but not with other Th2 cytokines such as IL-4 or Thl cytokines such as IFN-% There was also a strong correlation between MMP activity and the levels of the multifunctional cytokine IL-8 (rho 0.612, P < 0.01) which can act as a chemoattractant for both eosinophils and neutrophils. In summary, the MMP activi~ in the BAL is strongly correlated with the number of eosinophils and neutrophils recovered from the BAL as well as the levels of IL-5, GM-CSF and IL-8.

1516

Silencer Elements Controlling the Murine B29 (lg-[3) Minimal Promoter. CS Mahme l, SA Omori t, K Buchanan 2, C Webb'~, and R Wall I, tUCLA School of Medicine, Los Angeles, CA ~'Oklahoma Medical Research Foundation, Oklahoma City, OK Because B cell development is dependent on the expression of B29, we have investigated the features controlling the transcriptional activity of the murine B29 promoter. The B29 minimal promoter is negatively affected by adjacent 5' sequences suggesting that this region contains silencer elements. DNAscl footprint analyses over the 5' negative regulatory regions 2 regions of protection that correspond to at least 3 different DNA-binding proteins by gel-shift analysis. Two adjacent protein-binding DNA sequences, designated the BURP and the FROG motifs, act as position and orientation independent silencers when multimerized upstream of the B29 minimal promoter. Neither motif has any obvious homology to known proteinbinding DNA sequences. Both BURP and F R O G regions also negatively regulate several heterologous promoters including mb-1 and c-los, to an extent similar to that of B29. The third protein-binding DNA sequence exactly matches the Bright consensus motif identified in the lg heavy chain intrunic enhancer, but does not bind purified Bright protein. However, this B29 sequence binds nuclear matrices in matrix attachment assays. The BURP and FROG motifs and the B29 Matrix Attachment Region (MAR) are required for maximal negative regulation of the B29 minimal promoter. These features of the B29 silencers resemble other known silencer elements in othcr genes.

$372

Abstracts

J ALLERGYCLIN IMMUNOL JANUARY 1997

1517

1518

Impaired Lymphocyte Development in Mice Treated With Type I Interferon. Q Lin, C Dong, and M D Cooper, Departments of Microbiology and Pediatrics, University of Alabama at Birmingham, and the Howard Hughes Medical Institute, Birmingham, AL 35294. Type I interferons (IFN~/[3) are naturally-produced regulators of cell growth and differentiation. We observed previously that IFNc~/[3 inhibits the IL-7 induced growth and survival of early B lineage cells in vitro. In the present study, we treated newborn mice with an active human IFN~x2/~I hybrid molecule to assess its potential in regulating lymphopoiesis in vivo. IFNc~2/~I injections (4• U) were begun on day 3 and given daily over the next five days. When the mice were sacrificed one or two days later, a pronounced reduction in cellularity was observed in both thymus and bone marrow. The thymus from IFNa2/altreated mice was reduced by approximately 90% in weight and cell number. The CD4+CD8 + thymocyte compartment was greatly decreased, whereas increased proportions of progenitor CD4 CD8 thymocytes and mature CD4 + and CD8 + T cell subsets were observed. Lymphoid cells in bone marrow were found to be much more sensitive to type I interferon treatment than were erythroid and myeloid cells. The percentage of CD43+B220 + early B lineage cells in bone marrow was reduced by approximately 2-fold, whereas the percentage of more mature CD43 B220+ cells were decreased by ->6-fold. The number of B220 + B lineage cells in the neonatal liver was also reduced by 4-fold or greater in mice treated with the IFNcx2/cxl hybrid. These results support the idea that type I interferons may play an important regulatory role at early stages in T and B lymphocyte development. (Supported in part by NIH grant AI39816. MDC is a Howard Hughes Medical Institute Investigator)

The Role of Androgen and Estrogen Receptors in B Lymphopoiesis. G. Smithson, J. F. Couse, K. Korach, and P. W. Kincade. Oklahoma Medical Research Foundation, Oklahoma City, OK. We used estrogen receptor c~ knockout (ERKO) mice and testicular feminization (tfm) mice to determine the role of androgen (AR) and estrogen (ER) receptors in the regulation of B lymphopoiesis. There is a great deal of evidence suggesting sex steroids are an important regulatory component of B cell production in the bone marrow. For example, during pregnancy there is a dramatic reduction in B cell precursors. Estradiol (E2) and dihydrotestosterone treatment also results in a depression of B lymphopoiesis. Alternatively, in animals with reduced sex steroid levels, such as hypogonadal and castrated mice, there is a concomitant increase in pre-B cell production. Tfm mice have a naturally occurring missense mutation in the AR gene. As expected, they had a significant increase in pre-B cells and immature and mature B cells in the spleen. These data clearly show that testosterone utilizes the AR to regulate B cell production. ERKO mice possess an artificial neo gene insertion in the second exon of the ERc~ gene. In contrast to Tfm mice, they had no increase in B lineage cells in the bone marrow or spleen. Moreover, female ERKO mice actually had significantly decreased levels of B cells and B cell precursors. One possible explanation for this finding is that testosterone levels are increased in female ERKO mice and may regulate pre-B cell production through the AR. Alternatively, estrogen may use another receptor. To investigate this possibility, we isolated stromal cells from the bone marrow of male ERKO mice. Previous studies showed that stromal cells are required for E 2 to depress B cell precursor expansion. In co-cultures with ERKO stromal cells and B cell precursors from BALB/C mice, E2 was still able to inhibit precursor expansion. The presence of mRNA for the recently described ERI3 and a mutated form of the ERct, unique to the ERKO mouse, was found in a cloned ERKO stromal cell line. These findings suggest estrogen may utilize an alternate form of the ER. ERKO mice, and stromal cells derived from them, should be valuable for assessing the contribution of the ER to the regulation of lymphopoiesis.

1519

A Regulatory Role for Fc~/Receptor CD16 in the Development of Murine B-Cells. R.G. Lynch, B. de Andres, E. Rakasz, M. Sandor ~, S. Verbeek*, and A. Mueller. Department of Pathology, University of Iowa, Iowa City, Iowa, ^Department of Pathology, University of Wisconsin and *University of Utrecht, The Netherlands. Fc~ receptors (CD16/Fc3,RIII and CD32/Fc-/RII) are expressed early in myeloid and lymphoid cell development. During B-lymphopoiesis CD 16 and CD32 are expressed up until the stage of Ig gene rearrangement, but only CD32 is expressed at later stages. During T-lymphopoiesis CD16 is expressed on prothymocytes but not after TCR gene rearrangement. We presented evidence (PNAS 91:12857, 1994) that alternative (non-Ig) ligands on thymic stromal cells interact with Fc3,R on fetal prothymocytes to influence their rate of differentiation to mature T cells. To test the hypothesis that Fc3,R also plays a role in normal Blymphopoiesis, adult bone marrow was cultured with cytokines (GM-CSF, IL-3. IL-5) plus 2.4G2, a rat mAb that binds to CD16 and CD32, or with normal rat IgG. FACS analysis showed that 2.4G2 stimulated a 2-fold increase in 6B2+/IgM- cells and a 7-fold increase in 6B2+/IgM+ cells by 72 hrs, while control cultures showed a slight reduction in 6B2+/IgM- cells and a 3-fold increase in 6B2+/IgM+ cells. The stimulating effect of 2.4G2 appears to be mediated through CD16, but not CD32, since the effect is not observed in cultures of bone marrow from CD16 k.o. mice. These findings identify a role for CD16 (Fc3,RIII) in the regulation of routine B-cell development.

1520

Regulation of CD72 Gene Expression During B Lymphocyte Development. HYing, JR Parnes. Stanford University, Stanford, CA CD72 is a 45kD glycoprotein that is predominantly expressed on cells of B lineage except plasma cells. Deletion analyses have defined the 255 base pair minimal promoter capable of tissue-specific and developmental stage-specific expression. DNase I footprinting analysis of the CD72 minimal promoter revealed three protected elements FP I, FP II and FP III. FP I or Ill gave increased reporter gene activity specifically in B cells but not in T cells or non-lymphoid cells. FP II, on the other hand, exhibited both tissue-specific and developmental stagespecific activity reflective of the activity of the CD72 gene in vivo. Further analyses revealed that FP I was bound by the transcription factor PU.1/Spi-1 and this interaction was essential for the B cell-specific activity of the CD72 promoter. FP II was specifically recognized by BSAP whose expression correlates with CD72 during B lymphopoiesis. Mutations eliminating the binding site of BSAP in the reporter construct also eliminated the binding site of BSAP in the reporter construct also eliminated the increase of reporter activity in B cells. In addition, cotransfections with reporter constructs plus different amount of the expression plasmids for BSAP showed that CD72 promoter activity was upregulated by BSAP in plasmacytoma cells and T cells in a dose-dependent manner. Moreover, the expression level of CD72 decreased 10 fold on normal plasma cells. And the binding of BSAP was undetectable in those cells. This study strongly suggests that BSAP is critical for the specific activity of the mouse CD72 promoter. The absence of positive factors such as BSAP may be responsible for the loss of mouse CD72 expression in plasma cells which might reflect a common mechanism for down regulation of many molecules at the plasma cell stage.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1521

1522

CDI9 Regulates Peripheral Tolerance in B Lymphocytes. M lnaoki, S said, CC Goodnow, TF Tedder. Dept Immunology, Dukc University Medical Center, Durham, NC, and Howard Hughes Medical Institute, Stanford University, Stanford, CA The CDI9 cell surface molecule establishes signaling thresholds critical for B lymphocyte development and activation. Increasing the cell surface density of CDI9 renders B lymphocytcs hyper-responsive to transmembrane signals. To examine the role of CD19-regulated signaling thresholds in the generation and maintenance of tolerance, mice that overexpress haman CD19 (hCDI9) were crossed with transgcnic mice expressing soluble hen egg lysozyme (sHELl and HEL-specific immunoglobulin (Ig) genes. hCDI9/HEL/lg triple-transgcnic and hCD19/tg doubletransgenie mice werc generated. Overexpression of CDI9 in sHEL/Ig mice did not result in clonal deletion of B cells in the bone marrow, but did result in a decrease in the generation of B cells as occurs in hCDI9 mice. Peripheral B cells from sHEl,/lg mice arc rendered tolerant and thereby produce very little spontaneous HEL-specific antibody. However, hCDIg/sHEL/Ig mice had l(10-fold higher mean serum levels of anti-HEL antibodies. Immunization of hCDI9/sHEL/Ig mice with HEL also induced l(10-fold higher anti-HEL antibody responses than those observed in sHEL/Ig mice. Thcse results suggest that both the development and maintenance of tolerance is regulated by inlraeellular signaling thresholds and that overexpression of CDI9 shifts the balance between tolerance and immunity to immunity by augmenting antigen receptor signaling.

eDNA Cloning And Expression Analysis of CIqR., The Haman CIq/MBL/SPA Receptor That Enhances Phagocytosis. RR Nepomuceno, A Henschen-Edman, WH Burgess, AJ Tenner. U of California, Irvine, CA and American Red Cross ftolland Laboratory, Rockville, MD ClqR v was tirst described as a receptor that enhances phagocytosis triggered by the classical complement component Clq. Subsequent studies have shown that this 126,(100 M, leukocyte surface receptor also modulates phagocytosis when triggered by two other proteins that are structurally related to Clq: mannosc binding lectin (MBL) and pulmonap,, surfactant protein A (SPA). Using an anti-ClqRp monoclonal antibody that blocks the cnhanccd phagocytosis triggered by these three ligands, the receptor protein was purified from U937 cell extracts. Eleven internal pcptide sequences were obtained, as well as 22 amino acids of the amino lerminus. With a combination of RT-PCR and eDNA libra U screening, the eDNA corresponding to the coding region of ClqRp was chmcd and sequenced. All of the sequenced peptides arc encoded within this cDNA. Data base homology searches anti motif analysis show that CIqRP is a novel, ~31 amino acid type I membrane protein, containing a C-typc carbohydrate recognition domain, five EGF-like domains, a single transmembranc domain and a short cytoplasmic tail. Carbohydrate analysis of the receptar indicates dmt CIqR;, contains O-linked glycosylation. The gone for CIqR;, is present in the gcnome as a single copy by Southern Not analysis. Using a combination of FACS analysis, Northern blot, and RT-PCR, ClqRt, was flmnd to be highly expressed in endothelial cells, in addition to cells of myeloid origin as previously reported. These data indicate that this eDNA encodes for the receptor that plays a role in Clq-/MBL-/SPA-mediatcd removal or deslruclion of pathogens and immunc complexes by phagocytosis and suggests added roles for endothelial cells in this process, Supported by file Arthritis Foundation and NIH #AI-41091L

Abstracts

1523

S373

The Clq-binding membrane proteins, gClq-R and cClq-R, associate to form a metal ion-independent complex. Berhane Ghebrehiwet. Phoebe D. Lu, Weibing Zhang, Sue A. Keilbaugh, Leonora E. A. Leigh, Paul Eggh'ton. Kenneth B. M. Reid and Ellmor L B. Peerschke. Departments of Medicine and Pathology, State University of New York, Stony Brook, NY 11794-8161; and MRC lmmunochemistry Unit, University of Oxford, Oxford OXI 3QU, UK. Two types of ccll membrane proteins which display recognition specificity for the two structural domains of Clq have been described: a 60 kDa calreticulin homolog, designated cClq-R, binds to the collagen-like 'stalk', and a 33 kDa glycoprotcin has affinity for the globular 'heads' (gClq-R). Although the two molecules are known to be coexpresscd on a wide range of cell types, and often coclute during purification, there is no direct evidence which shows that the two molecules associate with each other either on the membrane or when examined in a purified system. In this report, we present the first evidence which shows that: (a) purified and biotinylatcd cClq-R binds to recombinant as well as Raji cell membrane derived gClq-R as assessed by solid phase ELISA, (b) a major site for cClq-R is located within the first 22 N-terminal residues of the 'mature' form (residues 74-282) of gClq-R since the binding of cClq-R to gClq-R is inhibited by two monockmal antibodies (mAbs) 60.11 and 46.23 recognizing epitopes within rcsidues 74-96, and (c) biotinylatcd cClq-R binds to microtiter-fixcd intact RNi and K562 cells and this interaction was inhibited by mAbs 6(I.11 and 46.23. Taken together, the evidence presented here suggests, that cClq-R is ablc to form a complex with gClq-R and may be anchored to the ccll surface via the N-terminus of gClq-R. [Supported by ACS-IM771 and NIH-HL5029101]

1524

Identification of Amino Acids Specific for Decay Accelerating Activity (DAA) in Complement Receptor Type 1 (CRI; CD35). M K~. ch, R Hauhart, D Hourcade, B Subramanian andJAtkinson. Washington University School of Medicine, St. Louis, MO USA Previously we showed that complement control protein repeats (CCP) 8-14 (LHR B) and CCP 15-22 (LHR C) which bind C3b and C4b eJficiently had no DAA for either classical (CP) or alternative pathway (AP) convertases. CCP 1-7 (LHR A) accounted for all DAA for the CP convertase. On the other hand, full DAA for the AP convertase was obtained when LHR A was followed by LHR C. We now report that 1) LHR A, with veO, low C\~b binding ability, appears sufficient for DAA for AP C3, but not C5, convertase. The amdysis of deletion mutants showed that CCP 1-3 in LHR A are indispensable for DAA of C3/C5 eonvertases in both pathways. 2) By substitution mutagenesis of CCP 1 and 2, we have identified amino acids and short peptides important h~r DAA. In most cases, mutations have a similar effect on both AP and CP convertases. In many, but not all cases, effect on DAA parallels that on binding. 3) Mutation of two sequcnces, one in CCP 1 (4 amino acids) and the other in CCP 2 (5 amino acids), causes significant reduction of DAA for both pathways without detectable effect on binding of C3b or C4b. Thus, these peptides likely contain residues specific' for DAA. 4) By mutating specific amino acids in CCP 1 and 2 we were able to increase DAA for one or both pathways. This is a potential advantage in the development of an improved complement inhibitor. In conclusion, we identified two peptides which contain residues important ,Vwc!fically for DAA. This, together with previous identification of the amino acids critical for C3b/C4b binding and for cofaetor activity, increases our understanding of structure/function relationships in this protein with more than 2,000 residucs.

$374

1525

Abstracts

Transcriptional Regulation Of Early Complement Components. E Veksler, and KE Sullivan. Children's Hospital of Philadelphia at the University of Pennsylvania School of Medicine, Philadelphia, PA 19104 The early complement component genes (ClqA, C4A/B, C2, and C3) share four conserved promoter motifs. Pustell alignment analysis reveals no other similarities between the four promoters. The promoter motifs are interspersed with multiple initiation sites in each of the four genes. Mutation analysis reveals that the four conserved motifs are important for hepatic expression. Electrophoretic mobility shift assays demonstrate that three of the four motifs bind hepatic and/or monocytic-specific DNA-binding proteins. The fourth motif binds a DNA-binding protein which is ubiquitously expressed. The DNA-binding proteins which bind the four motifs range in molecular weight from 36kD to 69kD. Two of the four motifs bind DNA-binding proteins which require zinc for full activity, suggesting that they belong to the zinc finger family of transcription factors. The DNA-binding proteins which interact with the third and fourth conserved motifs were cloned using a kgtll expression library. These two clones have been sequenced and expressed in a bacterial system. The expressed and partially purified proteins recognize the conserved motifs in a specific manner, confirming their identity. The DNA-binding protein which interacts with the third conserved motif has helicase and proline-rich protein motifs. Both DNA-binding proteins are unique genes. Although the early complement components are not coordinately regulated, the use of conserved transcriptional strategies suggests that their expression may be evolutionarily related.

1526

Membrane Attack Complex (MAC) Triggers Nuclear Factor-kappa B (NF-KB)-Dependent Iuterleukin-8 (IL-8) and Monocyte Chemoattractant Protein-1 (MCP-1) Secretion from Endothelial Cells. JS Warren, KS Kilgore, E Schmid, TP Shanley, CM Flory, V Maheswari, NL Tramontini, H Cohen, PA Ward, HP Friedl. University of Michigan Medical School, Ann Arbor, MI, USA. Assembly of sublytic concentrations of the MAC on cell surfaces can induce a number of proinflammatory activities. Available data indicate that MAC-induced cell activation occurs through a number of signal-transduction pathways, but little is known about the intranuclear mechanisms by which these complement-derived products promote the upregulation of inflammatory mediators. Utilizing purified complement proteins (C5-9) to assemble functional MAC on early passage human umbilical vein endothelial cells (HUVECs), we examined mechanisms of MCP-1 and IL-8 induction, Formation of the MAC promoted an increase in NF-KB DNA binding activity within 60 minutes as determined by serial electrophoretic mobility shift assay, Cytosolic to nuclear translocation of NF-KB was confirmed by Western immunoblot and immunocytochemical analysis. Formation of the C5b-8 complex also promoted NF-KB translocation, but to a lesser degree than observed in HUVECs that bore complete MAC. No cytosolic to nuclear translocation of the NF-KB subunit, p65, was observed in unstimulated HUVECs or in cells incubated with the MAC components devoid of C7. A direct cause and effect linkage between MAC assembly and NF-KB activation was confirmed through examination of the effect of the NF-KB inhibitor, pyrrolidine dithiocarbamate, on IL-8 and MCP-1 expression, This study provides direct in vitro evidence that MAC triggers increases in IL-8 and MCP-1 mRNA concentrations and protein secretion by inducing NF-KB activation.

1527

Novel Gene Regulation by Cell Surface Complement Activation. MC Braun, A E Prada, K Zahedi, JJ Bissler and A E Davis III. Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH Sub-lytic deposition of the terminal components of complement induces a variety of cellular responses. We sought to further characterize complement-mediated gene induction or repression through the use of differential display. Complement was activated on the cell surface of a

J ALLERGY CLIN IMMUNOL JANUARY 1997

monocyte-macrophage cell line using complement fixing antibody and C7 deficient serum, or C7D reconstituted with purified C7. Minimal lysis was determined by trypan blue dye exclusion and LDH release; MAC deposition was confirmed using FACS analysis with monoclonal antibody to the C5b-C9 neoantigen, mRNA was extracted 1, 6, 12, or 24 hours post-incubation, and differential display was performed. Potential differentially expressed bands were identified, and confirmed by Northern blot analysis. Expressed bands were then cloned and sequenced. To date, 20 differentially expressed eDNA species have been examined; two novel gene products have been identified. One is a 500 bp fragment of a MHC class 3 gene with homology with the extra-cellular matrix protein tenascin. No expressed transcripts of this gene have been reported. The second is a 1.5 kb transcript of a novel gene. Both appear to be down regulated by the deposition of the terminal components of complement. Their further characterization will help elucidate the role of the terminal components of complement in the immune response.

1528

Sublytic complement activation affects Schwann cell differentiation. SM Dashiell and CL Koski. Department of Neurology, University of Maryland, Baltimore, MD Although complement (C) effects demyelination in vitro and C5b-9 is transiently deposited on Schwann cells (SchC) in nerves of patients with inflammatory neuropathy, most evidence suggests that fully differentiated, myelin protein expressing-SchC resist C-mediated lysis. We therefore studied the effect of sublytic C on SchC myelin gene expression and progression into S phase. SchC purified from rat sciatic nerve and stimulated in vitro with glial growth factor and forskolin, expressed Po mRNA and protein in Golgi. Treatment of SchC with Ab and 10% NHS as a C source decreased Po and MBP mRNA 49% and 31% respectively by 1 hr, and 71% and 74 % at 3 through 12 hr. Po mRNA in unstimulated SchC remained constant over 6 hr in the presence of Actinomycin D but decreased following incubation with Ab+C, suggesting C activation enhanced Po mRNA degradation. The results in part reflected terminal C complexes (TCC) formation since C7 reconstitution of Ab+C7 depleted serum downregulated Po mRNA over AB+depleted serum alone. Ab +C also increased thymidine uptake of SchC 4.3-+ 0.6 fold over SchC in defined media (N2), N2+Ab, or N2 +Ab +HI-C. The effects of TCC on the Po promoter are currently under investigation. Our data suggest that C activation on SchC contributes to demyelination by promoting dedifferentiation through downregulating myelin gene expression and stimulating cellular proliferation. Supported by NIH P50NS20022.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1529

Use of Exhaled Nitric Oxide (eNO) to Monitor Inflammation in Children with Severe Asthma. MJ Lanz, DYM Leung, SJ Szcqter, CW White. National Jewish Center for Immunology and Respiratory Medicine, Denver, CO Exhaled nitric oxide (eNO) has been shown to be a marker of airway inflammation in asthmatic adults. We measured eNO, serum eosinophilic cationic protein (ECP), serum interleukin 2 receptor (sIL2R), and spiromctry in children with moderate to severe asthma. Peak eNO levels were measured by chemiluminescence during slow expiration. The effort required by a child Io produce a slow expiration for NO measurements is simple and helped with coaching. Six asthmatic children (3 boys, 8-17 yrs, FEVI% <75) on inhaled glucocorticosteroids with no radiographic evidence of acute sinusitis were studied before and after an oral glucocorticosteroid burst of at least 5 days at 1 mg/kg/day. Seven normal children (1 boy, 11-20 yrs, FEV~'>; >80) were also studied. The mean peak eNO level (parts per billion, ppb) for the asthma group before treatment (5[ ppb + 6) was greater than the controls (11 ppb • 2: p < 0.11008). There was no significant difference in baseline wdues with ECP or sIL2R between the asthma and control groups. The difference in peak eNO levels before and after treatment in the asthmatic group was also statistically significant (p < 0.001 ). In all 6 asthmatics, eNO levels decreased after glucocortieosteroid treatment to levels comparable to the control group (p < 0.98).

eNO Control Asthma -pre Asthma -post

Abstracts

ECP

1 lppb + 2 15 +_ 4 51ppb _+ 6*:" 15 _+ 2

slL2R 760 ~ 153 ~55 ~+ 147

10ppb + 6w167 7 + lw 669 :~ 140w

PF%

FEV~%

1(15 + 5 99 + 8 ~5 + 6** 64 + 5** 90 + 4w

89 -'_:7w

Compared to control; **p < 0.flfll PF% - percent predicted peak flow Compared to prc-Rx; w < (I.01, w167< 0.01 In summary, eNO is a sensitive marker for inflammation in asthma compared to controls relative to ECP and slL2R. As a result, eNO appears to be a more useful diagnostic test for persistent inflammation in asthma than ECP and sIL2R. In addition, cNO can be uscd in moderate to severe asthmatic children as a measure of clinical response to anti-inflammatory treatment, The measurement of eNO is an easily reprodueiblc, non-invasive test which benefits the asthmatic child and the physician. 1530

Bronchial hyperreactivity and eosinophilic inflammation in a mouse model of asthma. PD Mehlhol), M van de Ri]n, AB Gohtberg. l'"P Kump, TR Martin and HC Oettgen Childrcn's l lospital, Boston, MA, University of Pennsylvania, Philadelphia, PA, Medical College of Wisconsin, Milwaukee, WI We havc extended a model of allergic airways inflammation induced by the naturally occurring mold allergen, A,v~er~ilh~s .~i~m~gatus tall, to show that repeated airways exposure to Af antigen induccs bronchial hyperrcactivity in mice. 129/SVEV mice were immunized intranasally nine times over three weeks with Af o r normal saline (NS) and studied after the last dose. Bronchoalvcolar lavage (BAL) eosinophil counts were markedly elevated in A{Ltreated mice (mean 9.42 • 105 eos/ml) compared with NS-treated controls (mean 1.02 • l(t ~eos/ml). Histologically, the lungs of mice treated with normal saline appeared normal while those of mice treated with ,41"showed dense cosinophilic infiltrates. Total lgE levels were increased more than tenfold in Af-trcated mice (p < 0.0l). At-specific IgE titers measured by passive cutaneous anaphylaxis (PCA) averaged 140 in A f t r e a t e d micc compared with less than 1() in NS-treated controls. A lLIreatcd mice had increased BHR compared with controls as determined by measurements of pulmonary conductance (GL) and compliance (Cd~,0 h~llowing mcthacholinc (MCh) challenge and whole-body plethysmography, Thc mean MCh doses required to reduce G L and Cdyn to 50% of baseline levels were 30% and 5t)%

S375

lower than in NS controls, respectively. Two-way analysis of w~riance (ANOVA) confirmed this increase was highly significant (p < 0./101). This model provides a valuable tool in the study of asthma. We anticipate using it with available genetically altered mice to assess the impact of thesc alterations on asthma pathophysiology.

1531

Eosinophil percentage and eosinophil cationic protein (ECP) concentration of induced sputum for the assessment of airway inflammation in bronchial asthma (BA). C-S Hong, J W Park. Y B Park, Yonsei University, Seoul, Korea The eosinophil and the level of ECP in induced sputum may be useful in diagnosis and assessment of the variability of the airway inflammation in BA. To ewduate the usefulness of sputum eosinophil and ECP in diagnosis of BA, we measured these parameters in 68 patients with respiratory, complaints. In addition, we followed up 14 BA with variable airflow limitation. BA group (n - 41) showed higher sputum eosinophil % (24.5 + 7.6 vs. 2.2 • 2.9%, p < 0.0001) and higher level of sputum ECP (198.2 vs. 90.6 rag/L, p < 0.05) than none BA group (NBA, n = 27). Sputum ECP and sputum eosinophil % were correlated each other (r = 0.4358, p < 0.till. Sputum eosinophil % correlated with PFR (r (t.4746, p < 0.01) but sputum ECP did not correlate with PFR. Both ECP and eusinophil r~; in sputum did not correhtte with methacholine PQ,~. [n BA group, moderate-severe asthmatics (FEV I <80%, n 14) had higher sputum cosinophil % (43.6 • 14.9 vs. 14.5 _+ 5.8%, p < 0.01) and higher ECP levels (396.0 vs. 138.4 mgjL, p < 0.05) than mild asthmatics (FEVI ->80%, n 27). Between mild asthmatics and none BA group, sputum cosinophil % was higher in mild asthmatics ( 14.5 • 5.8 vs. 2.2 _+ 2.9%, p < (L(tl) but ECP level was not different. In 14 BA who were followed up, the change of PFR did not correlate with the change of sputum ECP level, even though there were certain relationship between changes of PFR and sputum eosinophil % (r = 0.7145, p < 0.01). These results suggest that sputum eosinophil % and sputum ECP level could be helpful for diagnosis of BA but sputum ECP is not satisfactory for assessment of wlriability of airway eosinophilic inflammation during the management of asthma.

$376

1532

1533

1534

Abstracts

Proto-oncogene involvement in apoptosis associated with inflammation in asthma. A.M. Vignola, P Chanez, G Chiappara, A.M. Merendino, L. Siena, F. Guddo, C Reina, G Bonsignore, J Bousquet. CNR-Palermo Italy, INSERM U454 Montpellier,n France Apoptosis may play a crucial role in the regulation of the survival of structural and inflammatory airway cells and in the chronicity of inflammation in asthma. Using the TUNEL technique we evaluated cell apoptosis in bronchial biopsies obtained from 44 asthmatics (A), 39 chronic bronchitics (CB) and 15 control subjects (C). The spontaneous P-53 (pro-apoptotic) and Bcl-2 (anti-apoptotic) expression was studied using immunohistochemistry. Apoptosis in eosinophils and macrophages was evaluated combining APAAP, with mAbs anti-EG2 and anti-CD68, and TUNEL techniques. Finally, the number of apoptotic, P53 and Bcl-2 positive cells was compared with the mucosal expression of GM-CSF. In A and CB apoptosis was restricted to desquamated or metaplastic epithelial cells. On the other hand, in A and CB but not in C basal epithelial cells expressed Bcl-2 and were non-apoptotic. In the submucosa of A the number of apoptotic (p < 0.001) or P53 positive cells was lower than in CB (p < 0.003) or C (p < 0.001) and correlated to Bcl-2 positive cells (p < 0.001). In addition, in A the number of apoptotic cells was inversely correlated to the clinical severity of the disease (p < 0.001). In the submucosa of A, GM-CSF expression was higher than in C and CB and correlated with the clinical severity of asthma and the number of Bcl-2 positive cells (p < 0.001). Finally, a lower number of apoptotic eosinophils and macrophages was found in A than in CB. In conclusion this study indicate that cell apoptosis can play a crucial role in the pathogenesis of the chronicity of airway inflammation in asthma. Enhanced expression of cyclooxygenase isoenzyme 2 (COX-2) in asthmatic airways and its cellular distribution in aspirin-sensitive asthma. AR Sousa, R Pfister* PE Christie* SJ Lane, S Nasser, M Schmitz-Schumann* TH Lee. Dept Allergy & Respiratory Medicine, UMDS, Guy's Hospital, London SEI 9RT. *Hochgebirgsklinik, DavosWolfgang, Switzerland. The expression of Cyclooxygenase (COX)-1 and COX-2 isoenzymes have been studied in the bronchial mucosa of 10 normal and 18 asthmatic subjects, 11 of whom were aspirin-sensitive (ASA) and 7 were aspirin-tolerant (NASA). There was a significant respective 4 and 14-fold increase in the epithelial and submucosal cellular expression of COX-2 (p = 0.002, p = 0.0001), but not COX-l, in asthmatic patients. There was no significant difference in the total number of cells staining for either COX-1 or COX-2 between ASA and NASA subjects, but the number and percentage of mast cells that expressed COX-2 was significantly increased by 6- and 2-fold, respectively, in ASA individuals (p = 0.004, p = 0.008). There was a mean 4-fold increase in the percentage of COX-2-expressing cells that were mast cells in ASA individuals (p = 0.006). The number of eosinophils expressing COX-2 was increased by 2.5-fold in ASA subjects (p = 0.044). COX-2-derived metabolites may play an essential role in the inflammatory processes present in asthmatic airways and development of drugs targeted at this isoenzyme may have therapeutic potential in the therapy of asthma. Longterm predictive value of sequential bee sting challenge in bee venom allergic children. J Forster ~, G Schi2tze 1, P Hauk ~.2, J Kuehr1, tUniversity Children's Hospital, Freiburg, Germany, 2National Jewish Center for Immunology and Respiratory Medicine, Denver, CO Hauk et al. (J Pediatr 1995;126:185) used a sequential sting challenge test to predict further systemic reaction in children allergic to insect venom. In a period of two years (median) they found a reasonable negative predictive value of 97.4%. We reinvestigated the children with bee venom allergy to see whether the prediction holds true for twice that time. Between 1988 and 1992 92 bee venom allergic children (28 girls, 64 boys, median age 8 years) with more than

J ALLERGY CLIN IMMUNOL JANUARY 1997

cutaneous reactions on the index sting were tested by a sequential bee sting challenge. Of these 77 were not eligible for venom immunotherapy by our criteria. We got a structured telefone interview of all children 6.4 years (median) after the test, 36 of them had experienced a field sting 4.1 years (median) after the test. Twenty four of them showed no reaction, 10 only a local one. One child experienced urticaria, one a slight dyspnea; both received only antihistamines. Thus, taking into account both cases the predictive value for no further systemic reaction was 94.4%, but 100% for no severe systemic reaction. Of the 15 patients assigned to receive immunotherapy 12 were restung. At this occasion one experienced severe dyspnea, when he was in his third year of immunotherapy, another - restung half a year after a three year immunotherapy - required adrenalin and i.v. therapy for circulatory problems. We conclude, that sequential bee sting challenge test safely can be used to identify children with no need for immunotherapy. It seems advicable, to monitor closely the etficacy of immunotherapy in those children who by this test are assigned to receive it.

1535

Safety of Three Rush Protocols for Hymenoptera Venom lmmunotherapy. F Purello D'Ambrosio, L Ricciardi, S lsola, S Gangemi, C Levanti, M Cilia, A Musarra, School of Allergy and Clinical Immunology, University of Messina, Italy. The authors report on their experience in 185 patients (mean age 34.5 years), with allergy to honey bee (HB) and yellow jacket (YJ), treated with either a 7 day (group 1:52 pts), 4 day (group 2:58 pts) and 120 rain (group 3:75 pts) rush venom subcutaneous immunotherapy (IT) protocol; a cumulative dose of 231.7 mcg, 195.5 mcg and 100 mcg was reached with each protocol respectively. The patients in the three groups were comparable with regard to clinical characteristics and immunological reactivity determined by skin tests and evaluation of venom specific IgE antibodies. All the rush IT protocols were carried out in an intensive care unit. The patients in the 3 groups were comparable with regard to clinical characteristics and immunological reactivity determined by skin tests and evaluation of specific venom IgE antibodies. Systemic reactions (SR) occurred in 24.7% of patients in group 1, 22.4% in group 2 and 6.6% in group 3; in no case did IT have to be interrupted. l i b venom led to more systemic reactions (14.5%) venom.

The results achieved with this study point out that rush protocols with low cumulative doses have a lesser risk of SR; from a practical point of view they are followed by a better patients compliance reducing the number of working days lost.

ALLERGY CLIN IMMUNOL 'OLUME 99, NUMBER 1, PART 2

1536

1537

Discontinuing Venom Immunotherapy: Long-term Outcome. DBK Golden, A Kagey-Sobotka, L M Lichtenstein, Johns Hopkins University, Baltimore, MD Venom immunotherapy (VIT) rapidly reduces the risk of a systemic sting reaction (SSR) in adults from 30-70% to <2%. When VIT is stopped after 5 years(yrs) or longer the risk of a SSR is 5%-15% during the first few yrs. It is uncertain whether SSR will occur more than 5 yrs after discontinuing VIT, and whether VIT can be safely stopped in some pts after <5 yrs. Among all pts who had VIT at our center, we identified those who stopped VIT: some had dropped out of therapy early (6-24 too), some stopped after 3-4 yrs, and most completed 5 yrs or more of VIT and were advised to stop by the allergist (many as part of our reported studies of discontinuing VIT). Contact was made with 127 pts including telephone interview for sting history and request to visit for skin test and blood sample. Of the 74 pts in our original study, 62 were reached and 311 reported recent stings with 5 SSR (1 of the 5 pts had a previously reported SSR after 3 yrs off VIT). Another 65 pts who had stopped VIT were reached: 40 had ->5 yrs VIT, 10 had 3-4 yrs and 15 had <2 yrs. Of 28 pts stung from this group 15 had >5 yrs VIT (no SSR) and 13 had <5 yrs VIT (1 SSR). It is surprising that 5/6 SSR occurred in our original study group (5-12 yrs after stopping VIT), whereas only 1 SSR occurred in pts who stopped 1-4 yrs earlier. We have now observed a total of 104 pts who had >-5 yrs VIT and were stung after stopping: 16 (15%) had SSR; most were mild but 4 were severe, SSR occurred in 11/96 pts (11.5%) stung in the first 5 yrs off VIT, and 6/42 (14%,) stung >5 yrs off VIT. In 4/6 SSR pts, the ST was negative but venom-lgE positive at the previous cncountcr. All SSR were similar in pattern and severity to pre-VIT reactions in the same pt. We conclude that the risk of SSR when VIT is stopped after 5 yrs or longer remains in the reported range of 5%-15r~f in the 5-10 yrs after stopping VIT. This risk of SSR does not seem to decrease over time, unlike the progressive decline in immunologic markers (skin test and venom-lgE). To prospectively assess the risk of recurrent SSR, there is anccd for sting challenge studies of patients who have been off VIT for 5-10 yrs and patients who have stopped VIT after just 3-4 yrs treatment. Development of sensitization and desensitization to mosquito bites and concurrent IgE and IgG responses during long-term exposure. Z Peng and FER Simons. University of Manitoba, Winnipeg, Canada. Prospective studies of skin rcactivity to mosquito bites during long-term exposure are incomplete, and concurrent antibody responses during exposure have yet to be investigated. In a pilot study, we monitored the skin and antibody responses of a human and a rabbit during king-term exposure to mosquito bites from a species which had not bitten them previously. A healthy male received 100 Culex quinquefaciatus (Cr. q) bites every 2 weeks (wk) and a rabbit received lOOAedes aegypti (Ae. a) bites weekly over 10 months. The skin immediate reactkm was measured at 20 rain and the delayed reaction at 24 h, hi-weekly for the human and weekly for the rabbit. Using ELISA, serum Cx. q salivary gland-specific lgE and lgG were evaluated in the human every 4 wk, and Ae. a saliva-specific lgG was evaluated in the rabbit every 2 wk. In the human, both skin immediate and delayed reactions began at wk 2, peaked at wk 5-13 and wk 5-19 for the delayed and the immediate reactions, respectively. The delayed reaction disappeared at wk 28, while the immediate reaction decreased to a very small size at wk 28 and remained small till wk 43. Both Cx. q-lgE and -IgG peaked at wk 5. The lgE decreased to baseline at wk 26, and the lgG decreased to 1/3 of the peak at wk 19 and stayed at this level until wk 39. In the rabbit, skin reactions developed by wk 3 (delayed) and by wk 5 (immediate), peaked at wk 7, and then decreased to a very small size by wk 14 and stayed small to wk 49. The Ae. a-IgG level started to rise at wk 5 when skin reactions had developed, peaked at wk 16 when they had decreased, and then gradually declined after they had disappeared. The results indicate that: 1) natural desensitization to mosquito bites eventually occurs during exposure; 2) in

Abstracts

S377

humans, both IgE and lgG are involved in the development of sensitization; 3) in rabbits, IgG acts as a blocking antibody which increases and then decreases during desensitization.

1538

Antigen 5 and phospholipase (PL) from Polistes dominnlus differ significantly in amino acid sequence from those of North American Polistes venoms. DR Hoffman, East Carolina University School of Medicine, Greenville, NC The old world paper wasp, P. dominulus, has become established in much of the northeastern United States and is common in many areas. In 1990 we demonstrated using sera from allergic patients from the U. S. and Spain that P. dominulus venom was antigenically different from the venoms of North American Polistes. In 1995 Sanchez et al. and Campi et al. confirmed that there were significant differences by RAST inhibition. Ag 5 and PL were isolated from P. dominulus venom by gel filtration and cation exchange chromatography. The purified proteins were reduced and alkylated, digested with enzymes, and the peptides isolated for amino acid sequencing by reversed phase HPLC. The complete amino acid sequence of P. dominulus ag 5 was determined. It showed 15% difference from North American species, which differ by only 1 to 5% among the 4 species studied. There are no complete sequences known for Polistes PL, so a number of peptides were sequenced from P. exclamans PL. Peptides corresponding to 108 of about 304 residues could be compared for P. dominulus and P. exclamans. The peptide sequences were 24% different, with 38% of the substitutions being non-conservative. Several involved changes in charge. These structural studies support the conclusions of the studies of immunologic cross-reactivity, Old world Polistes have significant differences in their venom allergens from North American species. Venom extract manufacturers should provide an appropriate extract for use in Europe, Asia and Africa, and P. dominulus extract should be made available in the United States.

1539

The role of CD28/B7 interactions in the development of an in vivo type 2 immune response. R. Greenwald ~, M. Hah'orson l, P. Lu 3, X.-D. Zhotd, S.-J. Chen l, K.B. Madden I, S.C. Morris 2, P.S. Linsley ~, F.D. Finkelrnan 2, and J.F. Urban 4, and W.C. Gause( tUSUHS, Bethesda, MD, 2Univ. of Cinn., Cinn., OH, 3Bristol-Myers Squibb, Seattle, WA, 4USDA, Beltsville, MD. Although CTLA4Ig, which blocks B7 interactions with CD28/CTLA4, inhibits the development of IL-4 producing T cells in vivo, few in vivo studies have examined the role of B7-1 vs. B7-2 and CD28 vs. CTLA-4. We have previously shown that CTLA4Ig administration blocks T cell IL-4 production and the associated type 2 immune response to injection of an immunogenic anti-lgD antibody and to oral inoculation with the nematode parasite, H. poly~'rus. We have now examined differentiation to T cell effector function during both immune responses in C57BL/6.CD28 KO mice. These mice fail to make IL-4, germinal center, IgG1, or lgE responses to anti-lgD antibody, but make strong IL-4, IgG1, and IgE responses when inoculated with H. poly~rus. CTLA-4Ig administration inhibits the IL-4 response to 14. p o l y ~ m s in CD28KO mice, indicating that a B7 ligand other than CD28 provides the costimulatory signal. We have also examined the effect of blocking B7-1 and/or B7-2 and found that T ceil IL-4 production and the associated type 2 immune response to H. polygyrus is unaffected by administration of either anti-BT-1 or antiB7-2 mAbs but that administration of the combination of anti-B7-1 and anti-B7-2 mAbs blocked the response. These observations indicated that: 1) a CD28/B7 interaction is required to induce some, but not all, type 2 cytokine responses: and 2) an interaction between B7-1 and a B7 ligand other than CD28 is able to costimulate a type 2 cytokine response to some antigens.

S378

1540

Abstracts

Analysis of T helper cell responses in wild-type and 1L-4 deficient BALB/c mutant mice and their responsiveness to IL-12. Pascale Kropf, Robert Etges 1, Lisa Schopf, Charles Chung e, Joe Sypek 2, and lngrid Miiller 1. 1University of Notre Dame, Department of Biological Sciences, Notre Dame, IN 46556, ZGenetics Institute, Cambridge, MA 02140 Interleukin-4 is considered to be the key factor driving polarized Th responses: early production of IL-4 is thought to be essential both to drive Th2 development resulting in further IL-4 production, and in the suppression of IFN-'ysecreting Thl cells. Based on the observation that anti-IL-4 mAb-treated BALB/c mice are resistant to Leishmania major infection, we were surprised that IL-4-/- gene-disrupted BALB/c mice remained susceptible to leishmaniasis. These data indicate that there is an IL-4 independent pathway of Th2 cell differentiation and establishment of susceptibility. We tested the hypothesis that susceptibility of BALB/c mice to L. major infection is due to the inability of helper T ceils to maintain responsiveness to IL-12. We tested this hypothesis on a population level in L. major infected BALB/c wild-type and 1L-4 - / - BALB/c mutant mice as well as in immunomanipulated mice. We determined first in an accessory cell-free system in vitro whether naive purified CD4 + T cells from unimmunized wild-type and IL-4 - / - mutant BALB/c mice can be induced to differentiate into cells with a polarized Thl or Th2 phenotype. We find that naive CD4 + T cells from both wild-type and IL-4-/- BALB/c mice can respond to IL-12 and IL-4 in vitro. The nonhealing response of both types of mice to L. major infection can be redirected in vivo by immunintervention so that they can heal their lesions and clear the parasite load. We show that CD4 + T ceils activated, differentiated and committed in vivo in response to L. major infection in both wild-type and IL-4-/- BALB/c mice do not lose their ability to respond to IL-12 on a population level,

J ALLERGY CLIN IMMUNOL JANUARY 1997

1542

Zhou, Myra C. Sieve, Ram P. Tewari*, and Robert A. Seder. NIAID, Bethesda, MD, *Southern Illinois Medical School, Springfield, IL Histoplasma Capsulatum is a dimorphic fungus found in the soil of different geographic regions. Inhalation of H. capsulatum results in a sub-clinical infection in immunocompetent hosts due to an effective cellular immune response. In a previous study it was shown that neutralization of endogenous IL-12, IFNg or TNFa resulted in accelerated mortality, while exogenous 1L-12 treatment protected the animals from a fatal outcome. In this study we were interested in the factors involved in the maintenance of immunity in response to a secondary infection with H. capsulatum. To address this, normal C57B1/6 mice were first infected with a sublethal dose of H. capsulatum (2• Three weeks later, animals reinfected with a lethal dose of H. capsulatum (6• survived reinfection and developed sterilizing immunity. In addition, animals treated with neutralizing antibodies against IL-12, TNFa, IFNg, IFNgR or a combination of all three survived the infection and had little to no detectable 14. capsulatum from spleen cells up to 90 days post infection. Animals treating with antibodies to deplete neutrophils, CD4 or CD8+ T cells also survived the infection. However, animals depleted of both CD4 and CD8+ T cells had a fatal outcome 20 days post reinfection with increased H. capsulatum tissue burden from spleen cells. These results suggest that CD4 and CD8+ T cells are required to maintain an effective memory immune response that is independent of IFNg.

1543

1541

Leishmania major infected C3H mice treated with antiIL-12 do not maintain a Th2 response. B. Hondowicz, T. Scharton-Kersten, and P. Scott. University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA Leishmania major infected BALB/c mice are susceptible and develop a Th2 response, while normally self-healing C3H mice given anti-IL-12 monoclonal antibodies during the first three weeks post-infection develop a similar Th2 response with enhanced susceptibility. We now demonstrate, however, that while anti-IL-12 treated C3H mice develop a Th2 response with large lesions at six weeks, after eight weeks the C3H mice begin to resolve their infections. At two weeks after infection lymph node cells from BALB/c and anti-IL-12 treated C3H mice produced comparable levels of IL-4, IL-10, and IFN-~. However, by 24 weeks anti-IL-12 treated C3H mice had switched to a Thl response similar to control C3H mice. Although the levels of IFN-~/, IL-4, and IL-10 were the same in anti-IL-12 treated C3H and BALB/c mice, the levels of IL-12 were significantly higher in anti-lL-12 treated C3H mice compared to BALB/c mice. When we treated C3H mice continuously with an anti-IL-12 monoclonal antibody they remained susceptible, demonstrating the importance of this IL-12 production for healing and the development of a Thl response in the presence of a predominate Th2 cell population. The ability of anti-IL-12 to augment Th2 development and susceptibility was also observed in the normally resistant C57BL/6 and DBA/2 mouse strains. However, a reversal to a Thl phenotype was only seen in C3H and C57BL/6 mice. Moreover, anti-IFN-3, monoclonal antibodies induced similar Th2 responses in C3H and C57BL/6, which later reverted to a Thl phenotype and healing. These data suggest that maintaining a stable Th2 response requires reduced levels of IL-12, and that the ability to modulate IL-12 levels may be genetically determined.

Protection against Histoplasma Capsulatum Infection does not Require IL-12 or IFNg in a Secondary Response. Ping

Innate resistance to Mycobacterium bovis (BCG) is not compromised by ILl2 ablation. L Thompson-Snipes, E Skamene, M Boule, D Radzioch. Montreal General Hospital Research Institute, Montreal, QC Canada. During mycobacterial infection, ILl2 induces protective T-cells and IFN~, production. However, it is not known whether ILl2 is essential for an effective Thl cytokine response to intracellular pathogens such as BCG and whether ILl2 participates in the innate immunity associated with the murine Bcg gene. To examine this, we compared BCG resistant mice, B10.A (bcgr), to the susceptible congenic strain B10.A (bcg~) following administration of a monoclonal antibody to ILl2 (10F6), which inhibits LPS-induced IFN3, production. Three groups of mice from each strain were compared over 21 days of BCG infection for pulmonary and splenic CFUs, as well as splenic cytokine mRNA expression profiles, by semiquantitative PCR, indicative of either a Thl or Th2 response. The three groups did not differ significantly in their production of IFN',/ However, the bcg~ animals had a small increase in IFN~/mRNA as early as day 3 of infection irrespective of anti-ILl2 treatment. By day 21, bcg' animals showed a significant increase in IFN'y mRNA that was mildly attenuated by anti-ILl2. In addition, in anti-ILl2 treated susceptible animals, there was a 2-fold increase in spleen CFUs by day 21 and only these animals showed lung CFUs. In contrast, anti-ILl2 treatment had little or no effect on the bcgr animals' response to infection. In summary, during BCG infection, the bcgr animals mount an early Thl response whereas the bcg~ animals show a significant mycobacterium load by day 21 and produce increased IFN~/. Thus, it appears that the inherent resistance of bcff animals to mycobacterium does not depend on lL12 to maintain an effective immune response. Supported by MRC of Canada.

J ALLERGY CLIN [MMUNOL VOLUME 99, NUMBER 1, PART 2

1544

1545

1546

Inducible nitric oxide is not required for IL-12/IFN-3, dependent innate resistance to the intracellnlar pathogen, Toxoplasma gondii. TM Scharton-Kers'ten, G Yap, A Sher National Institutes of Health, N1A1D, LPD, Bethesda, MD The induction by IFN-y of reactive nitrogen intermediates has been postulated as a major mechanism of host resistance to intracellular pathogens. To formally test this hypnthcsis in vivo, the course of Toxoplasma gondii infection was assessed in nitric oxide synthasc (iNOS) knockout mice. As expected, macrophages from these animals displayed defective microbicidal activity against the parasite in vitro. Nevertheless, iNOS deficient, in contrast to IFN-y or IL-12 knockout animals, survived acute infection and controlled parasite growth at the site of inoculation. This innate resistance was ablated by neutralization of l FN-3, or IL-12 in vivo and markedly diminished by depiction of ncutrophils demonstrating the existence of previously unappreciated NO independent mechanisms operating against the parasite during early infection. Importantly, iNOS knockout mice eventually succumbed to 7/ go~ulii. At that stage (3 wks post-infection) parasite expansion was evident in the central nervous system but not the periphery suggesting that the protective role of nitric oxide against this intracellular infection may be tissue specific rather than systemic. CD40L stimulates human macrophages to express proThl factors (IL-12, IL-18, B7) and HIV-suppressive [3-chemokines. RS Kornbhlth, K Kee, DD Richman. Univ. Calif. San Diego & VA Medical (?enter, La Jolla, CA Resting macrophages are poor antigen-presenting cells (APCs) and must be stimulated to express essential costimulatory fi~ctors and modulatory eytokines. CD4t) ligand (CD40L) upregulates the APC functions of B cells and dendritic ccHs (DC), and CD40L is known to stimulate macrophages to produce IL-12. To study this further, macrophages were prepared by culturing monocytes from healthy donors for 7-14 days. 293 cells stably transfected with a human CD40L construct (or the control empty vector) were then added to the cultures, and supernatants and cells were collccted. By ELISA, CD40L-293 cells (but not control 293 cells) induced the production of 1L-12 heterodimcr, By RT-PCR, IL-18 (also called 'IGIF') and B7.1 were also induced by CD40L. These factors promote the release of IFN-y by T cells and NK cells, and favor the development of type I T cell responses. Also, CD40L induced significant production of the [3-chemokines MIPlc< MIP-1[3, and RANTES at levels capable of preventing HIV infection of CD4+ T cells. In contrast, IFN-~ or GM-CSF alone did not induce these chemokines. The association of CD40L with chcmokine production may explain why abrogation of CD40L suppresses the infiltration of lymphocytes into immunological lesions. LPS, a "danger" signal to the immune system, had similar effects to CD40L in all assays. Since macrophages greatly outnumber DC in ahnost all tissues of the body, the APC and cffector functions of CI)4t)L-stimulated macrophages may play an important role in CD4t)L-initiated immunological responses.

Leishmania major in mice lacking TNF receptors. M. Nashh'anas, M. Moore* and P. Scott. University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA and *Genetech, Inc., South San Francisco, CA Resistance to the intraccllular protozoan, Leishmania marl)r, is dependent upon IFN-gamma mediated macrophage activation. In vitro studies have demonstrated that thc leishmanicidal activity of maerophagcs is dependent upon the production of TNF and the production of nitric oxide. Recently, we demonstrated that C57BL/6 mice ~acking the TNFRp55 were able to control and eliminate L. marl;r, although interestingly, they failed to heal their lesions (J. Immured., 1t~96, 157:827). In order to determine if TNF signalling through either the p55 or p75 receptor was required to control the parasites, we infected genetically resistant mice that lacked the p75 receptor (TNFRp75-/-), or mice lacking both receptors (TNFRp55p75-/ ), and followed the course of infection

Abstracts

S379

that was similar to wild-type mice, both with regard to parasite number and lesion resolution. In contrast, T N F R p 5 5 p 7 5 - / - mice exhibited a course of infection similar to that previously reported with T N F R p 5 5 - / mice. Thus, T N F R p 5 5 p 7 5 - / - mice maintained lesions for greater than 12 weeks, but similar to the T N F R p 5 5 - / mice were able to clear the parasites in the lesions. Furthermore, we found that both TNFRp75-/ and TNFRp55p75 / - mice developed a Thl response, accompanied by increases in iNOS mRNA gene expression within the lesions. These data suggest that a TNF-independent pathway exists for nitric oxide production and clearance of ixishmania.

1547

Multiple pathways for lymphotoxin(LT)to affect lymph node genesis and lymphocyte organization. PD Rennert, JL Browning and PS Hochrnan Biogen Inc, Cambridge, MA Treatment of pregnant mice with a LT[3-RIg protein specific for the LT membrane form, LTcq3, prevents progeny from developing Peyer's patches and a subset of lymph nodes(LN), disrupts splenic lymphocyte organization and eliminates MAdCAM-I expression on marginal zone cells. This suggests the absence of LN in LT~3-R Igtreated mice is due to inhibition of MAdCAM-1 which is expressed early in LN development and recruits the first cell populations into the LN anlage. Thus we performed an analysis of the genesis and organization of LN and MAdCAM-1 expression in mice treated during gestation with LT[3R-Ig. We now report mesenteric, sacral, cervical and lumbar LN were present in these mice. In normal adult mice these LN expressed either a mucosal or a peripheral addressin phenotypc. LT{3-RIg also affected lymphocyte positkming and expression of addressins and sialoadhesin in the LN; LN regained normal phenotype once LTI3-RIg dosing ceased, Treating with soluble LT/TNF specific antagonist TNF-R551g did not affect LN genesis but disrupted lymphocyte positioning in spleen and LN and eliminated splenic MAdCAM-1 expression, Thus we have shown a) there exist multiple LT dependent pathways for LN genesis-one requiring LTcq3 expression and other LTe~ pathway(s) independent of known receptors, b) membrane and soluble LT influence addressin expression and lymphocyte positioning to affect immune function, c) MAdCAM-I expression is not absolutely required for genesis of all LN and d) the pathway of genesis of a LN, its role in immunity and its addressin phcnotype do not absolutely corrdate.

1548

13-Chemokine Gene Expression in a MIP-I~ Knockout Mouse During an Influenza Viral Infection. J.F. Sheridan, .I. Jung, D.A. Padgett, HdP. Lafuse, P.M. Mantcha. The Ohio State University, Health Sciences Center, Columbus, Ohio MIP-Ic~ is an important signal for the recruitment of leukocytes to sites of viral-induced inflammation. Disruption of the MIP-Ic~ genc results in diminished inflammatory infiltration and delayed clearance of the virus in the lungs of influenza A/PR8 virus-infected mice (Science 269:1583, 1995). As a [3-chemokine, MIP-le~ shares activities with other members of the family including an ability to bind to the same receptors. To determine if deletion of the M1P-lc~ genc results in compensatory expression of other members of the [3-chemokinc family, a systematic examination of [A-chemokine gene expression during infection was undertaken in the lungs of A/PR8 infected mice. In this study, MIMICs fl)r MIP-Ic~ and MIP-I[3 were constructed and used for competitive RT-PCR to quantitate mRNA levels in the lungs of wildtypc {+/+) and MIP-la mutant ( - / - ) mice during infection. At 5 and 7 days post infection, the + / + mice expressed mRNA for both MIP-I~ and MIP-I[A, while the / - mice expressed only mRNA for MIP-I[L When MIP-IJ3 mRNA expression was compared in + / + and - / mice, similar levels of expression were found during infection. Thus, deletion of the MIP-I~ gene did not result in compensatory expression of the MIP-1[3 gene in the lungs of A/PR8 virus-infected mice.

$380

1549

1550

1551

Abstracts

A Possible Role for Interferon-gamma as a Regulator of B Cell Proliferation During an Immune Response. DyanaK. Dalton* Laura Haynes* Paula JardieuN, Siamon Gordon, Susan L. Swain* Satish Keshav. *The Trudeau Institute, Saranac Lake, NY/qGenenteeh Inc., South San Francisco, CA The University of Oxford, Oxford, UK The focus of this study is to define the roles of interferon-gamma in the regulation of the immune response. As a model we are using interferon-gamma knockout (gko) mice that are infected with the intracellular pathogen Mycobacterium bovis (BCG). By comparing the immune response of gko mice to wild-type (wt) mice we will be able to identify in vivo functions of interferon-gamma. Using the technique of immunohistochemistry, we have observed numerous large clusters of B cells in the liver of BCG-infected gko mice. We have measured much higher levels of circulating total IgG1 (4237 tLg/ml versus 514 ixg/ml) and total lgE (91.5 ~g/ml versus 11.7 I~g/ml) antibodies in the serum of BCG-infected gko mice compared to wt. In vitro, splenocytes from BCG-infected gko mice proliferate to BCG and Con A to a much greater extent from days 1 to 5 of culture than BCG-infeeted wt splenocytes. At the end of the culture period, there are more live cells in the gko cultures than wt cultures. The majority of cells remaining in both gko and wt splenocyte cultures are B cells. Using BrDUlabeling of proliferating cells, we are investigating a possible role for interferon-gamma in vivo as a regulator of B cell proliferation during the immune response. Lack of type 2 T-cell independent B-cell responses and defect in isotype switching in TNF-LTa deficient mice. Bernhard Ryffel, Franco di Padova, Max H. Schreier, Michel Le Hir, Hans-Pietro Eugster and Valerie F. J. Quesniaux Institute of Pathology, University of Basel, Sch6nbeinstrasse 40, CH-4031 Basel, Switzerland TNF is implicated in in vitro immunoglobulin (Ig) production but its role in vivo is not clearly defined. Our previous studies had shown that TNF-LTa double deficient mice have defective IgM and IgG primary antibody response to the T-cell dependent (TD) antigen SRBC. We now extend these studies to secondary responses and to T-cell independent (TI) B-cell responses. Injections of the TD antigen SRBC did not induce germinal centre formation in the spleen of TNF-LTa deficient mice. Associated with the morphological defect, there was a defective IgG antibody response and a secondary hyper-IgM response to the TD antigen in TNF-LTa deficient mice. The response to the TI antigen type 2 DNP-Alanyl-Gycyl-Glycyl-Ficoll (DAGG-FicoII) was essentially absent in TNF-LTa deficient mice, while that to the TI antigen type 1 TNP-LPS was significantly reduced only for IgG3 isotype. Transplantation of bone marrow cells from wild-type mice into irradiated TNF-LTa deficient mice restored the formation of splenic germinal centres and corrected the IgM and IgG responses to both TD and TI antigens. These data suggest that TNF and/or LTa signalling are critically required for germinal centre formation and for the IgM and IgG responses to both TD and TI type 2 antigens. Expression of Human C-Reactive Protein (CLIP) by CRPtrausgenic (CRPtg) IL-6-Deficient (IL-6 - / - ) Mice. AJ Szalai, F W Van Ginkel, JR McGhee, R Murray, JE Volanakis. University of Alabama at Birmingham, Birmingham, AL. To examine the CRP regulatory role of IL-6 in vivo, we produced CRPtg, IL-6 /- mice by selective breeding. Mice were screened for presence of the CRP tg by PCR, and basal and LPS-induced (18 h) serum CRP was quantitated by ELISA. Serum IL-6 was quantitated 2 h after LPS injection using ELISA. IL-6 -/ mice produced no detectable serum IL-6, whereas IL-6 +/- had 84___24 ng IL-6/ml serum. Like the parental CRPtg/IL-6 +/+ stock, male CRPtg/IL-6 +/- mice expressed CRP constitutively (94_+15 ixg/ml), whereas females did not. In both males and females CRP increased in response to LPS (424_+80 and 199_+38 ttg/ml, respectively). In male CRPtg/IL-6 - / - mice, CRP was expressed constitutively (67_+20 txg/ml), and was induced modestly by LPS (108_+38 txg/ml) and by mouse rlL-6 (219_+55 ixg/ml). In stark contrast female CRPtg/IL-

J ALLERGY CLIN IMMUNOL JANUARY 1997

6 -/ , expressed no CRP constitutively or after treatment with LPS, rlL-6, or both. By comparison, mouse serum amyloid P was expressed constitutively equally by both sexes (123_+19 Ixg/ml), and responded to LPS and rlL-6 equally in IL-6 -/ mice of either sex. We conclude that in vivo, IL-6 induction of the human CRP-transgene only occurs if the transgene is already "on" as it is in males. Supported by NIH grant # AI 15607. 1552

Tumor necrosis factor receptor-deficient islets are protected from diabetogenic T cell attack. S V Pakala, ML Chivetta and JD Katz Department of Pathology, Washington University School of Medicine, St. Louis, Mo Insulin-dependent diabetes mellitus is caused by the autoimmune destruction of the insulin-producing pancreatic beta cells. T lymphocytes have been implicated in this destructive process but the exact mechanism(s) of beta cell death have not been identified. In this study we address this issue using cytokine receptor deficient islet transplants. We transplanted interferon gamma receptor- (IFN~/-R-/-), tumor necrosis factor alpha receptor (TNFa-R1/R2 -/ )- deficient or control islets under the kidney capsule of diabetic streptozotocin-treated NOD.scid mice. After 7-10 days T cells from diabetic NOD mice were transferred into these mice. We found that mice transplanted with TNFc~-R1/R2 -/ islets remain normoglycemic while those transplanted with either normal islets or IFN3,-R - / - islets become rapidly diabetic. Moreover diabetes was blocked at the stage of initial T cell infiltration. We evaluated the role of the individual receptor chains,(p55 (TNF~x-R1 - / - ) and p75 (TNFc~-R2 /-)) and found that TNFa-R1 /- islets were protected while the TNF-R2 -/ islets were destroyed as rapidly as wild type islets. TNF has been reported to induce the expression of Fas on cells by signaling through the p55 receptor. We transplanted lpr islets which are Fas deficient and found that these islets were destroyed too indicating that TNFc~ does not function by inducing Fas. In conclusion, we report in this study that TNF-R1 /islets are protected from autoimmune destruction implying a direct role for TNFa in the formation of insulitis and the subsequent destruction of islets.

1553

Induction of Anergy in Naive T Cell Populations. PJ Lucas 1, FP Wange, D Y Loh 3, and RE Gress( 1EIB, NCI, NIH, Bethesda, MD 2Washington University, St. Louis, MO 3Nippon Roche Research Center, Kamakura, Kanagawa, Japan T cell activation is thought to require at least two signals, antigen receptor engagement and costimulation. Receptor engagement without costimulation has been shown to result in a state of non-responsiveness, termed anergy. To investigate mechanisms of anergy induction in naive T cell populations, we utilized a TCR transgenic (Tg) mouse, DO11.10, which contains a transgene encoding a TCR with specificity for cOVA when presented in the context of MHC I-Ad. T cells from these mice are phenotypically naive by all criteria tested, including low expression levels of CD44, CD25, CD69 and high expression levels of CD62L. Anergy induction was performed by incubating purified T splenocytes with antigen coated fixed-antigen presenting cells. These conditions were sufficient to cause anergy in T cell clones isolated from the Tg mice, but were ineffective against naive T cells. Instead, naive T cells gave an enhanced response to conditions able to cause anergy induction in the clones. To better understand this resistance to anergy induction, we incubated naive T cells with a high concentration of calcium ionophore, which has been shown to cause the induction of anergy in T cell clones in a receptor independent manner. High calcium ionophore concentrations did induce anergy in the Tg T cell clones, but did not affect naive T cell activation. To better understand this phenomenon, the minimal requirements for the induction of anergy of naive T cells was determined. These results demonstrated that only one antigen stimulation was sufficient to allow anergy induction of many T cells, however a second antigen stimulation was required to cause anergy induction in the majority of T cells.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1554

1555

Characterization of Anergy Induction in the Presence of Functional Antigen Presenting Cells in a Human T Cell Clone. RD Garman, CM Counsell, M Luqman, lmmuLogic Pharmaceutical Corporation, Waltham, MA Understanding the requirements for human T cell activation and inactivation could enable us to specifically enhance the beneficial T cell responses and to inhibit the undesired T cell responses. In vitro experiments with human T cell clones specific for a particular antigen can be used as a model to examine in vivo T cell activities. T cell clones can be rendered unresponsive by either presenting their cognate ligand in the absence of costimulation or by forcing MHC class II + T cells to function as antigen presenting cells. It is generally believed that T cell activation under these conditions is incomplete, resulting in failure to secrete cytokines associated with T cell activation. A CD4 ~ T cell clone was derived specific for a peptide from Fel d 1, the major allergen of the domestic cat. Using this model system, it was demonstrated that treatment of the T cell clone with a high dose of its cognate peptide ligand rendered it nonresponsive to subsequent stimulation as measured by proliferation and cytokine secretion. T cells that were treated with a high dose of peptide, and subsequently became nonresponsive, secreted greater amounts of cytokines during the treatment than T cells treated with a low dose of peptide, which remained responsive. By limiting the presentation of the peptide figand to antigen presenting cells, it was demonstrated that the induction of anergy was dependent on the dose of the peptide and not on the presentation of peptide by T cells to each other. These results demonstrate that the stimulation o f t cells by a high concentration of their cognate peptide ligand presented on functional antigen presenting cells initially induces high levels of cytokine secretion and T cell proliferation, but renders them unresponsive to subsequent challenge. The ability to control the responsiveness of antigen-specific T cells by treating them with different amounts of peptide ligand has tremendous therapeutic potential.

Galectin-I and TCR Synergize to Signal Protein Tyrosine Phosphorylation, ERK Activation, and Apoptosis. GNR Vespa, R Archer, J Ngt~ven, L Baum, MC Miceli. Departments of Microbiology and Immunology and Pathology, UCLA School of Medicine, Los Angeles, CA TCR ligation can drive a number of distinct responses including proliferation, cytokine production, anergy, and apoptosis. T cells may discriminate between stimuli in a variety of ways, including the type of Ag (pcptide, superantigen, or allorecognition), the magnitude of the signal through the TCR, the coordinate engagement of accessory molecules, or the presence of cytokines. Recent reports indicate that galectin-I can induce apoptosis in activated T cells through a mechanism which requires expression of CD45. Galectin-1 is a = 13-galactoside binding protein expressed by thymic and lymph node stromal cells at sites of cell death during normal T cell development. In order to determine whether TCR and galectin-I signals cooperate in signal transduction and the mechanism by which galectin-1 signals cell death, we have analyzed the effects of independent or coordinate ligation of galectin-I and TCR on cell death and early signal transduction events. Our data indicate that galcctin-I and TCR synergize to induce cell death in a T cell hybridoma and in freshly isolated thymocytes. Here we demonstrate that galectin-I induces the tyrosine phosphorylation of several proteins, indicating that galectin-1 signals through a tyrosine kinase dependent pathway. Crnsslinking the TCR has prcviously been demonstrated to result in the activation of MAP/ERK kinase cascade, which has been implicated in contributing to signals for mitogenesis and apoptosis in a number of signal transduction systems. We demonstrate that galectin-1 and soluble anti-TCR mAb synergize to activate ERK, suggesting a potential mechanism by which galectin-1 can modulate TCR activity.

Abstracts

S381

1556

CD9 delivers an apoptotic costimulatory signal into T cell receptor-stimulated naive T cells. X-G Tai 1, K Toyooka 1, Y Yashiro 1, R Abe 2, C-S Park I, T Hamaoka 1, M Kobayashi "~, S Neben s, H Fujiwara 1. tBiomedical Research Center, Osaka University Medical School, Osaka, Japan; 2Research Institute for Biological Sciences, Science University of Tokyo, Chiba, Japan; ~Genetics Institute, lnc., Cambridge, MA. The induction of full activation or death in TCR-triggered T cells depends largely on whether appropriate costimulatory signals are provided. Here, we show that the costimulation of CD9 on virgin T cells during TCR stimulation results in transient albeit potent activation followed by apoptosis rather than full activation. Anti-CD9 mAb synergized with suboptimal doses of anti-CD3 mAb in inducing T cell activation. 3H-TdR incorporation determined 2 days after CD9 costimulation was as potent as that induced by CD28 costimulation. However, in contrast to progressive T cell proliferation induced by CD28 costimulation, CD9 costimulation led to the induction of apoptosis of once activated T cells. CD9 costimulation only transiently produced a small amount of IL-2 and induced apparently low levels of bcl-xL and bcl-2 compared to those observed in CD28 costimulation. Addition of rIL-2 to cultures of CD9 costimulation induced strikingly enhanced expression of bcl proteins especially of bcl-Xl, and protected TCR-stimulated T cells from apoptosis. These data indicate that CD9 on T cells serves as a molecule that delivers an apoptotic costimulatory signal as the result of failure to generate a positive signal fnr sufficient levels of IL-2 production.

1557

CD30-Mediated Apoptosis in Murine CD8 T Cells After Cessation of TCR Signals. W.G. TelJbrd and R.A. Miller, University of Michigan Medical School, Ann Arbor, MI 48109. A variety of culture systems have been developed to study mechanisms of activation-induced cell death in peripheral T lymphocytes either during the initial period after exposure to an activating stimulus, or following repeated stimulation of activated T cells. In this study wc developed a new culture model for the analysis of apoptosis after withdrawal of TCR signals from activated T cells. T cells activated by anti-CD3 antibodies for 48 hr and subsequently in the presence of IL-2 but absence of further CD3/TCR stimulation underwent dramatic cell death approximately four days following removal of the TCR stimulus. Apoptotic cells generated in this protocol, unlike those produced by hyperstimulation, retained substantial levels of non-degraded DNA, consistent with death in the G0/G1 phase of the cell cycle. This "agonist withdrawal'" cell death occurred largely within the CD8 T cell subset, with CD4 cells showing greater resistance to apoptotic death. Apoptosis induced by agonist withdrawal was not affected either by Fas antigen fusion protein or by antiTNFa neutralizing antibodies, but was inhibited by approximately 50% by a CD30 fusion protein. CD30 was found to be transiently expressed on CD8 T cells immediately prior to death, with minimal expression on CD4 cells, while CD30 ligand was found to be expressed coincidentally with CD30 on both CD8 and CD4 T cells. These results suggest a role for CD30 in the induction of apoptosis in CD8 T cells after interruption of CD3/TCR signals.

$382

Abstracts

J ALLERGYCLIN IMMUNOL JANUARY 1997

1558

Fas Ligand (CD95L) and B7 Expression on Dendritic Cells Provide Counter-Regulatory Signals for T Cell Survival and Proliferation. A W Thomson, L Lu, S Qian, WA Rudert, PA Hershberger* DH Lynch. Thomas E Starzl Transplantation Institute, Pittsburgh, PA and *Immunex Corporation, Seattle, WA Highly purified DC (DEC-205 +, MHC class II hl, B7-2 + [CD86+], CD40 +, C D l l c +) grown from normal mouse bone marrow in response to GM-CSF + IL-4 were found to express FasL (CD95L) mRNA by reverse transcriptase PCR and to uniformly express FasL by both flow cytometric and immunocytochemical analyses. These cells but not DC propagated from FasL-deficient (B6.gld) mice induced dose-dependent increases in DNA fragmentation in Fas + Jurkat T cells over 18 h co-culture. The addition of mouse Fas-Fc fusion protein at the start of the cultures diminished this effect. Even at high relative concentrations however, B7-2h~ DC could induce only low levels of DNA fragmentation in Con-A or allo-activated splenic T cells, as determined by radio- or spectrofluorometric assays and by in situ nick end labeling. However, in the presence of CTLA4-Ig, a molecule that blocks the B7-CD28 costimulatory pathway, DC that failed to stimulate in primary MLR induced markedly augmented levels of apoptosis in alloactivated T cells. CTLA4-1g treatment also increased the level of DNA fragmentation induced by FasL-deficient DC, indicating the existence of additional (Fas-independent) pathways of DC-induced T cell death. These findings suggest that the costimulatory (B7-CD28) and T cell death-inducing pathways play important counter-regulatory roles in dictating the outcome of (allogeneic) DC-T cell interactions. The molecular interactions involved may determine the balance between tolerance and immunity, with implications for the development of DC-based therapeutic strategies of immune-mediated disorders.

1559

Neutrophil Activation by B. burgdorferi Lipoproteins. Tom B. Momson and Janis J. Weis. University of Utah School of Medicine, Department of Pathology, Salt Lake City, UT 84132 Lyme disease and Lyme arthritis has been correlated with the direct invasion and persistence of B. burgdorferi in afflicted tissues. Spirochete infiltration causes recruitment of circulating leukocytes into the extravascular space, which if not cleared, leads to disease pathology. Borrelial outer surface membrane contain a lipid modified protein with stimulatory properties which may contribute to the host evasion and disease pathology. Neutrophils could be a target cell for the lipoproteins since they are likely critical for clearance of the spirochete, and the arthritis tissue pathology. We examined the ability of OspA, a model B. burgdorferi lipoprotein, to induce neutrophil responses consistent with activation. Neutrophils responded to OspA treatment by: up regulating surface expression of Mac-I and CD10; down regulating L-selectin; inducing IL-8 production; and enhanced binding to extracellular matrix. In addition, OspA primed PMNs for fMLP-induced production of superoxide and azurophil degranulation. Only the lipid modified OspA demonstrated the above activities and these activities were not due to lipopolysacaride (LPS) contamination. By comparing the dose-response of OspA with two other bacterial stimulants LPS and fMLP, we determined that OspA was a very strong neutrophil agonist, with half-maximal responses in the 100 pM range. The magnitude and rapidity of the OspA response was also comparable to LPS and fMLP. However, unlike LPS, OspA did not require serum components for neutrophil stimulation. These results indicate that OspA and other B. burgdorferi lipoproteins could play a direct role in the activation of neutrophils that leads to localized disease pathology.

1560

Class 1 MHC And TAP-1 Down-Expression In Human papillomavirus (HPV) Infected Papillomas; Mechanism For Evasion Of Cytotoxic T-Cell (CTC) Recognition. A Vambutis, V Bonagura, BM Steinberg. Long Island Jewish Medical Center, New Hyde Park, NY. Previously we observed that class I MHC expression by HPV infected papillomas from patients with recurrent

respiratory papillomatosis (RRP) was down-expressed. To determine the mechanism that causes this, we studied papillomas for their expression of transporter associated with antigen presentation (TAP-I) protein by immunohistology using indirect staining o sequential sections of frozen papillomas, or cultured papilloma cells, stained with monoclonal antibodies specific for class I MHC ~x chains (anti-HLA-A, -B, -C with [32 microglobulin W6/32), and human TAP-1. Class 1 MHC and TAP-1 proteins in RRP derived papillomas are co-down-expressed. There is a correlation between TAP-1 levels and time to RRP recurrence. Patients who frequently relapse show lowest TAP-1 expression. Papilloma cells in culture also show a down-regulation of TAP-l, suggesting that they can be used to study the mechanism of TAP-1 regulation. We conclude that RRP-derived papillomas are like HPV infected cervical carcinomas that also show decreased or absent class I MHC expression linked with down-expression of TAP-1. These observations imply that at the level of the virus, an important mechanism employed by HPV in evading CTC recognition and function is interference with TAP gene regulation/function that causes class 1 MHC down-expression.

1561

Signalling of Macrophage Functions by Glycoinositol Phospholipids (GIPLs) from Trypanosoma cruzi: Distinct Responses elicited by Oligosaccharide and Ceramide GIPL Domains. GA DosReis, CG Freire de Lima, JO Previato, L Mendonfa-Previato, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil We have previously demonstrated down-regulation of host T lymphocyte activation induced by G1PLs purified from Tcruzi, and mapped the T-cell suppressive activity to the GIPL ceramide domain (Gomes NA et al (1996) J lmmunol 156, 628-635). Since host macrophages interact with T.cruzi during infection, we started to investigate biological effects of T.cruzi GIPLs on macrophage function. GIPLs, or their isolated oligosaccharide domain, induced increased spreading and proliferation in the J774 macrophage cell line, as well as metabolic activation in peritonial resident macrophages, as judged by the MTT assay. On the other hand, the isolated GIPL ceramide induced morphological changes in both J774 cells and resident macrophages, including intense vacuole formation, but did not affect the cell cycle. Moreover, in the presence of the cytokine IFN-% GIPL ceramide induced an intense cell death response in J774 cells, similar to apoptosis. This reaction could not be induced by LPS plus IFN-% and IFN-3, could not be substituted by TNF-~. Agarose gel analysis showed DNA fragmentation in supernatants from resident macrophages and J774 cells treated with GIPL ceramide plus 1FN-% The results indicate that T.cruzi GIPLs have distinct signalling effects on macrophages, depending on engagement of the oligosaccharide or the ceramide domains. The results also suggest that parasite GIPLs could play an important role in subverting host macrophage function.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1562

Early IL-4 Induction by Toxoplasma gondii Contributes to Host Susceptibility During Acute Infection. EY Denkers and Y Zhang, Dept Microbiol and lmmunol, College Vet Med, Cornell Univ, Ithaca NY To assess early IL-4 induction after murine infection with high (RH) and low (NED) virulence T. gondii strains, a competitive PCR protocol was employed. RH induced splenic IL-4 within 2 hr of infection, a response that diminished to background levels by 72 h, with a concurrent modest rise in IFN-gamma mRNA. Infection with NED induced a weaker IL-4 response (6-fold lower at 2 hr of infection), but by day 6, IL-12(p40) and 1FN-gamma transcripts were 5-6 fold greater. Histological examination revealed a large infiltrate of granulocytes in spleens of day 6 RH, but not NED, infected animals. Peritoneal cavities of RH-infected mice contained numerous tachyzoites, but in NED-infected mice, a massive lymphocyte-like infiltrate with few tachyzoites was instead found. To directly evaluate the role of IL-4 in RH virulence, IL-4 knockout (KO) mice were infected with the latter parasite strain, then survival and gross pathology monitored. Although IL-4 KO continued to display mortality during acute infection, the animals succumbed with a delayed kinetics (approximately 24 hr) relative to wild-type (WT). In day 6 infected mice, while WT peritoneal cavities possessed numerous tachyzoites, few could be found in that of the IL-4 KO, which instead was infiltrated with large numbers of polymorphonuclear granulocytes. Nevertheless, day 8 infected IL-4 KO (a time immediately precceding death) contained high numbers of peritoneal tachyzoites, and spleens, as well as livers, displayed extensive necrosis. We conclude that 1L-4 induction plays a role in pathology of RH infection, but that additional, as yet unknown, factors also contribute to the virulent properties of the latter parasite strain.

1563

Induction of lnterleukin-10 by HSV and Sendal Virus in Human PBMC. F Payvandi, J Lee and P FitzgeraldBocarsly, UMDNJ-New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, NJ Human IL-10 (hIL-10) is a potent suppressor of cytokine production. We have previously shown that exogenous hIL-l(/ inhibits production of IFN-r by PBMC induced with herpes simplex virus (HSV), Sendal virus, Newcastle disease virus (NDV) and vesicular stomatitis virus. We further demonstrated that endogenous IL-10 inhibits IFN-c~ production by PBMC induced with HSV. To investigate the source of endogenous IL-10, we examined the secretion of IL-10 by PBMC stimulated with different viruses. PBMC were incubated with HSV or Sendal virus and tested for IL-10 after 2, 4, 8, 24 and 48 hr by ELISA assay. In a representative experiment, secreted IL-10 was detectable at 4 hr with concentrations of 12 and 123 pg/ml and maximized at 24 hr with concentrations of 42 and 462 p~ml in response to Sendal virus and HSV, respectively. Both live and UV-inactivated HSV induced IL-10 to the same levels but UV-Sendai virus did not induce IL-10. IL-10 was also induced by PBMC in response to NDV, human influenza A virus, and HIV. To determine the cell populations producing IL-I(), purified B, T, monocytc, NK and dendritic cells were stimulated with either HSV or Sendai virus for 24 hr. The monocyte population secreted 47 and 394 pg/ml 1L-10 in response to Sendal virus and HSV, respectively whereas none of the other cell populations secreted IL-10 in response to either virus. Furthermore, RT-PCR indicated that both viruses induced IL-10 mRNA as early as 2 hr, with a peak by 8 hr, with HSV Sendal virus. We conclude that hIL-10 is rapidly produced by PBMC in response to different viruses. This IL- 10 can be expected to play immunomodulatory roles such as we have described for inhibition of IFN-c~ production.

1564

Evidence for persistent infection or spontaneous reactivation of HSV-I in the trigeminal ganglia (TG) of HSV-1 latent mice. DJJ Cart and WP Halford. Department of Microbiology and Immunology, LSU Medical Center, New Orleans, LA. We have previously detected Thl and Th2 cytokine transcripts and evidence for CD4 + and CD8 + cells present

Abstracts

S383

in the TG of latent HSV-I infected mice 120 days post infection (PI). In addition, the continuous elevation in the serum levels of anti-HSV-1 antibody is detected out to 120 days PI. To test the hypothesis that either persistent, low level HSV-1 replication or spontaneous HSV-1 reactivation is occurring in the TG of mice after primary infection, mice were treated for 12{) days with a concentration of acyclovir (3 mg/ml in drinking water) that blocks HSV-I replication in the TG during an acute infection. Whereas there were no differences in the anti-HSV-1 titers in the serum 20, 35, 60, 90, and 120 days PI comparing mice treated with or without acyclovir as determined by ELISA, there was a reduction in the detection or levels of cytokine transcripts (including 1FN-y, TNF-u, and RANTES) at 120 but not 35 or 60 days PI in mice treated with acyclovir as determined by RT-PCR. Similarly, there was a reduction in the level of CD4 + and CDg ~ transcripts at 120 days but not 35 or 60 days PI in mice treated with acyclovir compared to untreated controls. However, latency associated transcript (LAT) detection remained consistent in all mice treated with or without acyclovir suggesting that there is no loss in the latent infected neurons. Collectively, the results suggest that the persistent expression of cytokines in the TG of latent HSV-I inlected mice is due to the local reactivation of virus or a low level replication of HSV-1 in the TG.

This work was supported l~v RO1 NS35470 from the N1NDS, NIH.

1565

Pathogenic THI and Protective TH2 Clones Differ in Their Recognition of the Autoantigenic Peptide of Myelin Proteolipid Protein. Mercy Prabhu Das, Lindsay B Nicholson, Alessandro Sette and Vijay K Kuchroo. Center for Neurologic Diseases, Harvard Medical School, Boston, MA and Cytel Corporation, San Diego, CA. We previously generated a panel of Thl clones specific for an encephalitogcnic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF). The majority of the clones induced EAE upon adoptive transfer. To identify the structure of PLP 139-151 recognized by the clones, a panel of alanine and lysine substituted peptides of the PLP 139-151 peptide were used to identify the TcR and MHC binding residues, In spite of the differences in TcR gene usage, all the Thl clones required W144 and H147 as the TcR contact residues and L145 and P148 as the MHC binding residues. W144 however, was the primary and most critical residue required for the activation of these clones. In this study we determined the T cell receptor (TCR) contact residues in two panels of Th2 clones, one that was specific (or the PLP peptide 139-151 and a second one that was generated against the altered peptide, Q144, (HSLGKQLGHPDKF). The Q144 specific Th2 clones were degenerate in their recognition and besides Q144 they also recognized the native peptide. Using the panel of alanine substituted peptide analogues of the native PLP and Q144 pcptides we show that in contrast to the Thl clones the Th2 clones have shifted their primary contact residue and recognize LI41/GI42 and/or K143 as their primary TcR contact residue. Preimmunization with the A144 altered peptide which preferentially activates Th2 clones inhibits EAE induced with the native peptide. These data suggest that T cell differentiation associated with TCR recognition of distinct regions of an antigen may define the resulting T cell phenotype and alter autoimmune disease course. Furthermore, these data may explain the crossreactivity of the Th2 clones generated against the altered peptide.

S384

1566

Abstracts

Treatment of MBP plus MOG-Induced Experimental Auo toimmune Encephalomyelitis with a Peptide of MBP. MP Happ, E Leadbetter, C Bourque, B Devaux, G Sunshine, D Smilek, S Hirani and B Wallner, ImmuLogic Pharmaceutical Corp, Waltham, MA Numerous reports have previously described reductions in severity of myelin basic protein (MBP) or proteolipid protein (PLP)-induced EAE upon administration of soluble peptides encoding major T cell epitopes of the inducing myelin antigen. The predicted applicability of this approach to the treatment of human multiple sclerosis is unclear, as this autoimmune disease is generally hypothesized to involve reactivity to more than one myelin antigen, including MBP, PLP and myelin oligodendrocyte glycoprotein (MOG). We have been able to induce EAE in both SJL and (PL/JxSJL)F1 mice by immunization with the extracellular domain of the MOG protein. This disease was ameliorated by administration of a T cell epitope-containing peptide of MOG. In addition, we have developed a new EAE model in which immunization with MBP and MOG together results in a more aggressive disease course than that produced with either antigen alone. This comparatively severe disease was treated with a combination of one MOG-derived epitope peptide and one MBP-derived epitope peptide. The same treatment effect was also obtained with an equivalent total dose of the MBP peptide alone, although this peptide did not treat the disease induced only with MOG. The amelioration of disease symptoms that was observed included a reduction in daily mean clinical scores, a decrease in incidence of disease and in disease-associated mortality, and a reduction in severity of leukocyte infiltration in the brain and spinal cord. These results suggest that peptide immunotherapy is an effective means of disease control even when the treatment is targeted to only one component epitope or one component protein antigen of a diverse autoimmune response.

1567

Unexpectedly high frequencies of activated autoreactive T cells in peripheral blood of patients with multiple sclerosis. KD Bieganowska, LJAusubel, and DA Hailer. Brigham and Women's Hospital and Harvard Medical School, Boston, MA The frequency of clonally expanded and persistent T cells recognizing the immunodominant myelin basic protein (MBP)p85-99 epitope was directly measured ex vivo in subjects with typical relapsing remitting multiple sclerosis. Single T cells expressing mRNA transcripts encoding T-cell receptor (TCR) a and b chains found in T-cell clones previously isolated from these subjects recognizing the MBPp85-99 epitope were examined. In contrast to frequencies of one in 105 to 106 as measured by limiting dilution analysis (LDA) estimates of the T-cell frequencies expressing MBPp85-99 associated TCR chain transcripts were as high as one in 300. MBPp85-99 TCR transcripts were present in IL-2 receptor a (IL-2Ra) positive T cells which were induced to undergo Fas mediated cell death upon antigen stimulation. These data demonstrate that at least a subpopulation of patients with MS can have a very high frequency of activated autoreactive T cells.

1568

Spontaneous lg Peptide-mediated T cell Help, a Mechanism for Autoantibody Production in Aging Mice. RR Singh, FM Ebfing, BH Hahn, UCLA, Los Angeles, CA The mechanism(s) of autoantibody production are unclear. We have previously shown that SLE-prone NZBAV F1 [BW], but not MHC class II-matched normal mice, spontaneously develop T cells that react with peptides from the VH region of syngeneic anti-DNA Ab (Singh et al, J Clin Invest 1995). These peptides accelerate in vivo autoantibody production and disease in BW mice (Singh et al, J Exp Med 1995). The number of activating T helper peptides across the Ig molecule increases as autoimmunity progresses with age in BW mice. Thus, Ig peptide-mediated T cell help is an important physiological mechanism of maintenance of autoimmunity in SLE-prone individuals. Here, we asked if a similar mechanism is operative in autoantibody production seen in older individuals. First, we

J ALLERGY CLIN rMMUNOL JANUARY 1997

demonstrated that two MHC class II (H-2dU)-matched strains of BW, namely BALB/c • NZW F1 [CW] and NZB • B10.PI [BP] mice do not develop autoantibodies until 35 weeks of age, when they have low levels of antinuclear and IgG anti-DNA antibodies. Both strains of mice do not develop fatal nephritis, and 100% survive one year of follow up. To determine the role of Ig peptidemediated T cell help in autoantibody production in older mice, splenic T and B cells from CW or BP mice of different age groups were cultured with previously defined Ig-derived T helper determinants (p34, p58 and p84) and 23 overlapping 15-mer peptides representing the entire VH region of mAb anti-DNA, A6.1, and IgG anti-dsDNA antibody forming cells (AFC) were enumerated in an in vitro ELISPOT assay. Two and four VH peptides induced 2 to 6 fold increase in IgG anti-DNA AFC in 35-week-old CW and BP mice, respectively, but none of the peptides elicited any increase in anti-DNA AFC in younger mice (20-25-week-old). Thus, appearance of autoantibodies in older normal mice coincides with the development of Ig-derived T helper determinants. In conclusion, we describe this mechanism of autoimmunity induction, where a reciprocal cross-reactive T-B cell stimulation may be responsible for autoantibody production in individuals with SLE and old age.

1569

Transgenic T Cells that Recognize CD1 on B Cells Induce or Prevent Murine Lupus; Role of Thl and Th2 Subsets. D. Zeng, M. Dick, M. Amano, L. Cheng, S. Jones and S. Strober. Stanford University School of Medicine, Stanford, CA 94305 Murine Lupus with immune complex glomerulonephritis is characterized by anti-dsDNA antibodies, proteinuria and ascites. Several laboratories have reported that T cells play an important role in the production of the pathogenic anti-dsDNA antibodies, but a homogenous population of T cells has not been previously shown to induce lupus in non-autoimmuoe mice. In the current study, we show that CD4 + and CD8 + T cells or CD4 + T cells alone, obtained from BALB/c mice expressing TCR ~ and !3 transgenes encoding receptors that recognize CD1 on transfected cell lines and on syngeneic B cells, induce lupus after intravenous injection into irradiated wild-type BALB/c nu/nu adoptive recipients. The recipients develop serum antidsDNA antibodies and proteinuria about 50 days after the cell injection, and ascites at about 75 days. Those with ascites have profound anemia, hypoalbuminemia, immune complex glomerulonephritis and die by 100 days. The T cells that induce the disease show a Thl-like cytokine secretion pattern in vitro with large amounts of IL-2 and IFN-',/and small amounts of IL-4. Purified double negative (CD4 CD8-) T cells which express the identical TCR and [3 transgenes, but which show a Th-2-1ike cytokine secretion pattern in vitro (large amounts of IL-4 but little amounts of IL-2 and IFN-~) prevented the disease after co-injection with the T cells that induced the disease. Thus, Thl- and Th2-1ike subsets of T cells which recognize autoantigens on B cells are capable of inducing or preventing routine lupus, presumably by regulating polyclonal activation of B cells and secretion of pathogenic autoantibodies.

J ALLERGYCLIN IMMUNOL

Abstracts

S385

VOLUME 99, NUMBER 1, PART 2

1570

1571

Self-Reactive T-cells in the Generation of Autoimmune Responses Against Ro60. US Deshmukh, CJ Kannapell, JE Lewis, F Gaskin, YH Lou, S T Waters, KS Tung, SM Fu. University of Virginia, Charlottesville, VA. Autoantibodies to Ro60, a cytoplasmic/nuclear ribonucleoprotein, arc detected in patients with autoimmune diseases such as systemic lupus erythematosus. Although self reactive T-cell arc presumed to be inw~lved, the precise mechanisms responsible for the development of these autoantibodies are not known. T-cell epitopes on Ro60 were mapped in mice (SJL/J) immunized with recombinant human Ro6t). Using a panel of synthetic overlapping pcptides spanning the entire sequence of Ru611, tin immunodominant T-cell response was localized to a region encompassing amino acids 316-335. Mice immunized with peptide 316-335 generated a very strong T-cell response against the peptide. The minimal T-cpitope was mapped to a 15mer, 316-330. These mice also developed autoantibodies rcacting with the native mouse Ru60, La and other intraccllular constituents, showing evidence of epitope spreading. To determine whether this phenomenon occurs with autologous mouse Ro peptide, it was necessary to clone and sequence mouse Ro60. Mouse Ro60 cDNA was cloned and sequenced from WEHI 7.1 ccll line. The mouse and human Ru60 sharc an homology of 85.2r tit the cDNA level and 89.8% at the protein level. The T-cell rcsponse generated against the human peptidc cross-reacted with thc murinc peptide, which differs at 3 amino acids. Moreover, animals immunized with the self peptidc generated a strong T-cell rcsponsc and autoantibody responses similar to those describcd above wcrc seen. Our results indicate a lack of tolerance against Ro60 at the T-cell level and establish the role of these autorcactivc T-cells in thc development of autoantibody responsc against Ro60. Whelher these T-cells are activated by cruss-reactivc determinants or molecular mimics needs to be determined.

Disruption of positive selection of T cells on self-peptides in the thymus induces autoimmunity. A Kretz-Rommel and RL Rubm, The Scripps Research Institute, La Jolla, CA We previously demonstrated in a mouse model of druginduced lupus that intrathymic injection of procainamidehydroxylamine (PAHA, a reactive metabolitc of the lupusinducing drug pmeainamide) induced chromatin-reactive T cells and anti-chromatin autoantibodies in vivo. Wc tested the capacity of PAHA to affect central T cell tulcrance using thymus organ culture followed by in vitro expansion of T cells in the absence of PAHA. Exposure of 0-2 day-old thymi from C57BL/6xDBA/2 mice to as low as 5 IxM PAHA allowed a 10-fold expansion of chromatin-reactivc T cells in vitro. No gross changes in the quantity of CD4 and CD8 double or single positive T cells were detected in the thymus after PAHA Ireatment, and PAHA had no affect on negative sdcction of V,~8-spccifie T cells by Staplo,Iococcus enterotoxin B. Several groups suggest that thymic T cell selection occurs thruugh cognate low affinity interactiuns with pcptides derived from self-mulecules, resulting in positively selected T cells anergic (unresponsive) to the selecting peptide. Using an in vitr~ model of T cell anergy, concentrations as low as 2 I.~M PAHA completely prevented energy induction by anti-CD3 treatment of the T helper cell chine 12.11 as measured by ~H-thymidinc incorporation and interferon-y production. We suggest that PAItA may also prevent induction of anergy during positive selection of chromatin-specific T cells and that peptides involved in positive T cell selection are commonly derived from chromatin because of thc high abundance of this material in the thymus. To test this we subjected thymi to PAHA from mice expressing the transgene neu-autoantigen pigeon cytochrome c (PCC). Thymucytes derived from these PAHA-exposcd thymi showed a 12-fold increase in proliferative response to PC(7 and a 8-fold inercasc in response to chromatin. These studies support the view that T cclls arc positively selected on self-peptide, and suggest that PAHA induces autoimmunitv by disrup-

tion of self-tolerance during positive selection of T cells in the thymus.

1572

Allergen-induced migration of the human cells in the lungs, spleen and thymus of allergic hu-SCID mice. (7.. Duez PhD, J. Pestel PhD, P. Lassalh" MD, A. Tsicopoulos MD and A.B. Tonnel MD. INSERM U416, [nstitut Pasteur, Lille, France. Severe combined immunodeficient (SCID) mice reconstituted with mononuclear cells of allergic patients sensitive to Dermatophagoides pteronyssinus (Dpt) produce human IgE and develop a pulmonary inflammatory-type reaction after allergen exposure (Eur. J. Immunol., 1996, 26:1088). To understand the mechanisms of the human cell migration in SCID mice, their phenotypic profile was analysed in the lungs, spleen, and thymus 2 months after Dpt inhalation, The CD45+ human cell recruitment was only observed in hu-SCID mice after Dpt exposure. Immunohistochemical analysis of sections of these organs gave the following results (expressed as the median and the interquartile range of the number of cells per field): respectively in the lungs, spleen and thymus of hu-SCID mice exposed to allergen 72 (104), 28 (t02), and 7 (108), but in hu-SCID mice not exposed to allergen 0 (0), 0 (1), and 1 (1). Moreover human cells recruited into the 3 organs were preferentially memory (CD45 RO) and activated (HLADR) cells. In the lungs and spleen, a greater influx of CD4+ versus CD8+ T cells (with a ration 2:1) was observed. CD20+ B cells were predominately found in the spleen and thymus (respectively 27.7 _+ 10.9% and 23 _+ 12%, of CD45+ cells) and some IgE expressing cells were also evidenced. CD23+ cells were more frequently detected in the thymus (14.6 + 1(1%) than in the spleen (2 _+ 1.2%). Whereas in the spleen and thymus no difference in the pattern of adhesion molecule expression (CDI la, VLA-4, ICAM-I) was observed, in the lungs, the percentage of human leukocytes expressing the c~ chain of the LFA-1 (CDlla) was higher than those of VLA-4+ or ICAM-I+ cells (respectively 53.2 _+ 10%, 10 + 5%, 2.7 _+ 1%,). Thus human LFA-I and its murine counterreceptor ICAM-I may play a role in the human cell migration towards SCID mice lungs. In conclusion, as the pulmonary infiltrate of allergic hu-SCID mice shows a cell phenotypic pattern similar to that observed in asthmatic patients, this model could be useful to study the factors implicated in the cellular migration during an allergic reaction.

$386

Abstracts

J ALLERGYCLIN IMMUNOL JANUARY 1997

1573

VLA-4 expression on memory/activated CD4+ T cells and their adhesion are upregulated by antigen stimulation. M. Tarkowski, K. Pacheco, LJ. Rosenwasser, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO VLA-4 is expressed on T lymphocytes, and after stimulation it acutely increases its binding avidity. We have shown that allergen stimulation increases VLA-4 receptor density on human CD3+ T cells over 24 to 48 hrs. We hypothesized that the rise was specific for CD4+ cells and correlated with increased cell binding to the counter ligand. Human T cell lines were established after 2 cycles of stimulation with Lol p I allergen (10 mg/ml) or Tetanus toxoid (.2 lfu/ml). Cells were analyzed by flow cytometry at 0, 24, and 48 hrs for staining with antibodies against CD49d, CD4, CD45RO and CD45RA. Parallel T cell samples were labelled with Cr 5~ and incubated for 1 hr in wells coated with the CS-1 fragment of fibronectin or whole plasma fibronectin. Messengar RNA for expression of a-4, b-1 and b-7 chains of VLA-4 was analyzed by RT-PCR. VLA-4 receptor density increased by 85% (p < 0.05) 24 hrs after antigen stimulation, exclusively on CD45RO +/CD4 + cells. Binding to CS-1 was coordinately upregulated from 4.5% at baseline to 21% 24 hrs after stimulation. The increased surface expression of VLA-4 correlated with increased a-4 and b-1 chain mRNA expression, but not with b-7 mRNA. Increases were seen with both Lol p I and Tetanus stimulation. These findings indicate that allergen and antigen stimulation induces increased VLA-4 expression on CD45RO+/CD4+ T cells and functionally correlates with enhanced binding to CS-1. We postulate this may be one of the mechanisms to localize allergen specific CD45RO+/CD4+ cells to sites of allergic inflammation.

1574

Regulation of 1CAM-1 and VCAM-1 Expression in the Human Bronchial Epithelial Cell Line BEAS-2B and Involvement in Eosinophil Adhesion. JAtsuta, SA Sterbinsky, L M Schwiebert, BS Bochner, RP Schleimer, Johns Hopkins Asthma and Allergy Center, Baltimore, MD We have demonstrated previously (JACI 292:97) that cytokines induce surface expression of ICAM-1 and VCAM-1 on a human bronchial epithelial cell line (BEAS2B) in vitro. We have now studied 1) mRNA expression of ICAM-1 and VCAM-1 induced by cytokines, 2) relevance of ICAM-1 and VCAM-1 expression on BEAS-2B to eosinophil (EOS) adhesion, and 3) the effect of glucocorticoid. Using Northern blot analysis, ICAM-1 and VCAM-1 mRNA expression was detected in BEAS-2B cells stimulated with TNFc~ (1 ng/ml, 2 hr). Treatment of BEAS-2B monolayers with TNFc~ (10 ng/ml, 24 hr) significantly increased adhesion of EOS (from 5.7+_0.3% to 15.7%_+3.0 adhesion, p<0.01). Blocking antibody to ICAM-1 had no significant effect on levels of EOS adhesion. In contrast, antibody to VCAM-1 completely decreased net EOS adhesion (104.3• inhibition, p<0.01). Glucocorticoid (10 -7 M) had no significant effect on TNFe~-induced expression of either 1CAM-1 protein or mRNA but significantly inhibited both TNFc~-induced VCAM-1 protein and mRNA expression. These results suggest that VCAM-1 on airway epithelium may functionally interact with EOS and that suppression of epithelial VCAM-1 expression by glucocorticoids may contribute to their antiinflammatory effects.

1575

Intercellular Adhesion Molecule-l(CD54) on Eosinophils Is Involved in Cytokine-Stimulated Eosinophil Degranulation. S Horie, Y Okubo, M Hossain, T Momose, M Sekiguchi, Shinshu University, Matsumoto city, Japan Recent evidence suggests that adhesion molecules play important roles in eosinophil functions such as degranulation and superoxide anion production. CD11b/CD18 (Mac-l) and CD49d/CD29 (VLA-4) are involved in eosinophil-endothelial adhesion through their counter ligands, intercellular adhesion molecule-1 (CD54) and vascular cell adhesion molecule-l, respectively. CD54 is also induced on eosinophils by cytokine stimulation. We hypothesized that CD54 on human eosinophils may participate in eosinophil degranulation. Eosinophils were isolated from venous

blood of normal volunteers by using magnetic cell separation system. CD54 was induced on purified eosinophils by a combination of 10 ng/ml GM-CSF and 10 ng/ml TNF-c~ within 2 hours of incubation as determined by flow cytometric analysis. GM-CSF alone also induced slight but significant CD54 expression on eosinophils. Eosinophil degranulation was induced by 10 ng/ml GM-CSF on 96well tissue culture plate coated with human serum albumin and this effect was synergistically enhanced by adding 10 ng/ml TNF-a. To determine the role of newly expressed CD54 in eosinophil degranulation, a blocking assay was performed using monoclonal Abs (mAb) against CD54 and CDI8. Anti-CD18 mAb and anti-CD54 mAb markedly inhibited eosinophil degranulation induced by GM-CSF or a combination of GM-CSF and TNF-cx (GM-CSF/TNF-cx). On the other hand, anti-CD54 mAb had little effect on eosinophil adhesion induced by GM-CSF or GM-CSF/ TNF-c~, whereas anti-CDl8 mAb significantly inhibited eosinophil adhesion. These results indicate that CD54 on eosinophils plays an important role in the eosinophil degranulation by interacting with 132 integrins expressed on eosinophils.

1576

Expression of a novel 1~2 integrin (~dl~2) on human leukocytes and mast cells. MH Grayson, M Van der Vieren, * WM Gallatin, * PA Ho~fman, * BS Bochner, Baltimore, MD and *Bothell, WA 132 integrins are involved in leukocyte adhesion and migration. Recently, a fourth member of the 132 integrin subfamily, c~d, was identified. We studied the relative distribution of c~d on human leukocyte subtypes and skin mast cells. Partially purified leukocytes or dispersed skin mast cells were analyzed by dual color cytometry for expression of 132 integrin subunits using the following murine mAbs: C D l l a (MHM24), C D l l b (H5A4), C D l l c (BU-15), and c~d (169A) (a non-binding IgG1 mAb was used as a control), cxd was expressed on all peripheral blood leukocytes but not on skin mast cells. Overall, monocytes expressed the highest density of ~d (10.7 • 1.8 fold IgG control; -x • SEM, n = 9) followed by a 30% subpopulation of CD8+ lymphocytes (9.5 • 3.4, n = 8), basophils (8.2 _+ 1.8, n = 7), CD16+ lymphocytes (6.5 -+ 2.8, n = 8), neutrophils (6.1 _+ 0.8, n - 7), CD19+ lymphocytes (6.1 _+ 3.2, n = 8), CD4+ lymphocytes (3.4 _+ 1.3, n = 8), and eosinophils (3.0 _+ 0.6, n = ll). For most cells, levels of C D l l a and C D l l b were at least 4 times the levels of ~xd. Levels of CD 11c and c~d were similar except for monocytes and neutrophils where C D l l c was present at twice the density. Eosinophils appear to have preformed stores of both CD1 lb and cxd, because incubation with phorbol ester (10 ng/ml, 15 min, 37~ caused a 3 fold increase in expression of c~d and a 2 fold increase in C D l l b . We conclude that ~d is expressed, albeit at different levels, on most circulating leukocytes and, in eosinophils, can be acutely upregulated with phorbol ester. This differs from c~d distribution in tissues, where its expression occurs in a more restricted pattern on subsets of leukocytes. The role of ~d132 integrins in leukocyte adhesion and migration remains to be determined.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2 1577

Abstracts

levels in 90 children selected from a geographically based birth cohort, with cord blood IgE ->0.56 lU/ml. Concentrations of IgE in serum and cotiniue in urine were measured every 6 and 2 months, respectively. Airborne and dust concentrations of Fel d L Derf l, and Der p I were measured in bedroom samples every month for 2 years thcn every other month. The method of generalized estimating equations was used to find the combination of variables most predictive of IgE concentrations over the four years of observation. Analyses were based on 489 IgE, 2084 air and dust allergen, and 471 urine cotinine measurements, an average of 5.4, 23.2, and 5.2 per child, respectively. Allergic disease was reported for 31.1% of mothers and 29.3% of fathers while 15.6% mothers and 22.5% of fathers were smokers. The children were predominantly white, middle-class; with 45.6% of mothers and 51.1%, of fathers having college degrees. When considered together, the variables most predictive of total serum IgE concentrations (p values) were: age (0.(101), cord blood IgE (0.005), D e r f l in dust (0.015), D e r f l in air (0.00t), and season of birth (0.//01). The model was highly significant R2=(l.27, p<0.0001. The season of birth associated with the highest subsequent [gE levels was fall (Sept, Oct, Nov) while summer (Jun, Jul, Aug) birth was associated with the lowest IgE levels (p=0.004). Other variables considered, but insignificant in the model, were: parental allergic disease (allergic rhinitis or asthma), child's sex, parental education, Fet d t and Der p I in either air or dust, urine cotinine concentrations, and reported parental smoking. In a prospective, population based, study, we found that, in addition to increasing age; cord blood lgE, season of birth and Der f 1 exposure are the best predictors of developing total serum IgE concentrations.

Mast cells express connexins and may communicate in vitro with fibroblasts through gap junctions. H Vliagofiis, CK Oh, and DD Metcalfe. NIH, Bethesda, MD and HarborUCLA, Torrance, CA. Interactions of mast cells with their microenvironment is fundamental for their differentiation, proliferatkm and survival. Gap junctions, channels that link the interiors of adjacent cells, mediate the cell-to-cell exchange of growth regulatory factors. We thus hypothesized that mast cells might express connexins and communicate with cells in their microenvironment through gap junctions. C57 mast cells and bone marrow cultured mast cells (BMCMC) express mRNA for connexin 43 (Cx43) and Cx32 as determined by RT-PCR and Northern analysis. The expression of Cx43 and Cx32 was verified by Western blotting and flow cytometry. To examine the assembly of functional gap junctions, mast cells were loaded with the membrane impermeable dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and dye transfer from mast cells to fibroblasts was quantitated by flow cytometry. Co-culture of BCECF-labeled C57 cells or BMCMC with NIH-3T3 fibroblasts demonstrated significant dye transfer m the fibroblasts. Dye transfer was evident at 15 min and 40% of the NIH-3T3 cells were labeled by 1 b. The dye transfer was bi directional. The addition of increasing numbers of BCECF-labeled mast cells to the NIH-3T3 monolayers resulted in increased numbers of labeled NIH-3T3 cells. The specific gap ]unction inhibitors l-heptanol (3.5 mM) and 1-octanol (500 p.M), significantly inhibited the dye transfer. PMA, which has also been shown to inhibit the communication of cells through gap junctions, inhibited transfer by 60%. Preincubation of BMCMC with an antic-kit antibody that inhibits the adhesion of mast cells to fibroblasts, inhibited the dye transfer by 50%. Thus, mast cells express mRNA and protein for Cx43 and Cx32 and during co-culture they transfer dye to fibroblasts in a manner characteristic of that described for gap junctions, Mast cells may thus be able to communicate with cells in their microenvironment through gap junctions. 1580

1578

Mast CelI-Fibroblast Interaction Induces Histamine Release and C-C Chemokine Production. N.W. Lukacs, R.M. Strieter*, M. Glass* D.D. Taub** and S.L. Ktmkel. University of Michigan Medical School, Dept. of Pathology, Dept. of [nternal Medicine* and NCI**. Mast cell activation can be induced by mu[tipte mechanisms, including IgE-, complemcnt-, and stem cell factormediated pathways. In addition, the interaction of mast cells with particular cell populations, such as fibroblasts, have also demonstrated increased mast cell. In these studies we have investigated the role of fibroblast-mast cell interaction for induction of histamine release and cytokine production. Primary pulmonary fibroblast cell lines were grown in culture and used throughout these studies. Mast cell lines were grown in parallel with the fibroblast lines, derived from the same mouse, by incubation of upper airways with SCF and IL-3. Cell lines were used after 3 to 4 weeks in culture and contained few or no contaminating cell populations, initial studies demonstrated that during mast cell-fibroblast interaction degranulation of the mast cells was observed as determined by histamine release into culture, as compared to mast cells cultured alone. In addition, a significant increase in C-C chemokine production was also observed. In particular, the production of MIP-I[3, C10, and MCP-1 were significantly increased during the fibroblast-mast cell interaction, as compared to mast cells of fibroblasts alone. These studies suggest that fibroblasts-mast cell interactions may play a role in exacerbation of chemokine production and disease progression during local responses in tissue. This work was supported by NIH grants, A136302 and HL35276.

1579

Factors Related to Total Serum lgE from Birth to Four Years of Age. DR Ownby, MD, CC Johnson, PhD, and EL Peter~on, PhD Henry Ford Hospital, Detroit, MI. Total serum IgE concentrations are related to the risk of allergic disease and asthma. We prospectively evaluated factors thought to be related to the development of IgE

$387

Preventive effect of the mite-blocking bedding encasing on mite sensitization and the onset of atopic asthma. K

Nishioka, H Yasueda, M Ebisawa, K Akiyama, Y Iikura, H Saito, National Sagamihara Hospital, Kanagawa and National Children's Medical Research Center, Tokyo, Japan. Background: The method for preventing mite sensitization and atopic asthma is not well established, although the levels of mite allergens are known to be related to those. We investigated whether bedding encasing made from mierofine fibers, Microguard (Allerguard) can prevent infants from becoming mite sensitization and atopic asthma. Methods: Microguard was made from densely woven microfine fibers and was found to block mite allergens passing through. Fifty-seven infants (< I year old) who had IgE antibodies to egg white or milk or soy bean but not to house dust mites were randomly divided into the following two groups. Thirty families of the atopic infants were instructed to decrease mite allergens by throughout cleaning (group A), whereas 27 families received the same instruction and were chosen to use Microguard for encasing all quilts and mattresses of the family members (group B). We examined the levels of group 1 mite allergen (Der 1) in the mattresses, the levels of mite sensitization, and clinical symptoms of asthma over a 1 year study period. After 1 year, we statistically analyzed these values obtained from the two groups. Resuhs: The use of Microguard markedly reduced the levels of Der 1 (126 ng/m e for group A vs. 8 ng/m 2 for B, p < 0.001) and prevented the increase in the levels of Dermatophagoides farinae-specific IgE antibodies (2.5 RAST unit for A vs. l).7 RAST unit for B,p < 0.05) and the onset of asthmatic symptoms (37% for A vs. 11% for B,p < 0.05) during the study period. Conch~sion: The use of mite-blocking bedding encasings such as Microguard for atopic infants seems to be an efficient and safe method for preventing them from becoming mite sensitization and atopic asthma.

S388

1581

Abstracts

Epidemiology of asthma phenotype in extended families.

J ALLERGY CLIN IMMUNOL JANUARY 1997

1583

Environmental Control (EC) can Effectively Reduce Cat Allergen (Fel d I) in House Dust Samples Without Removal of the Cat. S Jufiusson, S Jakobinudottir, V Runarsdottir, T Blondal, D Gislason, US Bjornsdottir, University Hospitals, Reykjavik, Iceland Indoor allergens, especially cat and house dust mites are a leading cause of allergies. Treatment of allergies consists of removal of the offending allergen, pharmacotherapy and immunotherapy. Pet owners are often unwilling to give up their animals despite clinically relevant allergies. We examined whether Fel d I levels can be decreased in homes of cat allergic patients with stringent environmental control measures without removal of the cat. We also assessed the patients ability to comply with these measures. Fel d I levels were measured monthly in house dust samples by ELISA during an l I month study period before and after institution of EC in 12 homes, and in 12 homes with an unchanged environment (UE). Following EC, a gradual decrease in Fel d I levels was found. After 11 months, levels had decreased by 91.4% (p = 0.007). In the U E group, Fel d I levels remained unchanged throughout the study period. Throughout the 11 months, all patients (12/12) in the EC group used Acb mattress covers and 10/12 used Acb pillow covers (Allergy Control T M Products). 12/12 complied with instruction to wash bedding weekly at 60 ~ C and 11/12 washed the cat every 2 weeks. 8/12 did not have, or removed carpets from the bedroom. However, only 5/12 kept the cat out of the bedroom and 5/12 used tannic acid to upholstery and carpets as instructed. These data demonstrate that with stringent EC, Fel d I levels can be decreased significantly without removal of the cat. Compliance issues presented a difficulty in the group instructed in EC, especially with regards to keeping pets out of bedrooms. This is of great concern as this group was specifically targeted with education on the specific allergen, consequences of its persistence, and the possible benefit of its removal.

1584

The Effect Of A HEPA Room Air Cleaner On Cat-Induced Asthma And Rhinitis. Robert A. Wood, Elizabeth Flana-

CE McEvoy, SS Rich, D Drury, LR Daniel,, MN Blumenthal, University of Minnesota, Minneapolis, MN and Bowman Gray School of Medicine, Winston-Salem, NC. One difficulty in identifying specific asthma causing genes has been its phenotypic heterogeneity speculated to be a result of gene-environmental interactions. We studied 20 large extended Caucasian families, ascertained by asthmatic probands over the past 20 years. The mean family size was n = 28 (range 9 - 65) and included an average of 3 generations and 557 individuals. Twenty-six percent (n = 146) of family members were identified as being affected with asthma (defined as: 2 or more of 3 symptoms of cough, wheeze, shortness of breath; a doctor's diagnosis of asthma; use of asthma medications; <3 pack-year history of tobacco use; and, either a 20% drop in FEV~ at any dose of methacholine or 15% increase in FEV~ after bronchodilator). The mean age of onset in those affected was 11.11 +_ ! 1.42 years. There were significant differences in the log total serum IgE levels between the affected and unaffected (mean log (IgE) affected = 2.1 _ 0.6 IU, unaffected = 1.7 +_ 0.7 IU, p < 0.0001) but not in the presence of atopy (one or more positive antigen skin tests) 84.2% of affected and 65.0% of unaffected family members, (p = 0.08). There were more affected females than males (OR = 1.24, 95% CI, 1.09 to 1.41; p = 0.1)3); this relationship persisted after adjusting for tobacco use (OR = 1.34, 95% CI, 1.11 to 1.61; p = 0.006). Report of passive tobacco exposure was evaluated as an environmental factor. Family members within this strictly defined phenotype were less likely to report a history of tobacco exposure either during fetal development (OR = 0.58; 95% CI, 0.39 to 0.87; p = 0.03) or early childhood (OR = 0.60%; 95% CI, 0.40 to 0.89; p = 0.04). This suggests that the relative impact of certain suspected environmental factors may vary with the underlying genetic risk. Further studies evaluating the effects of other potentially important environmental stimuli on the asthma phenotype are needed. 1582

Effect of Allergen Avoidance on Infant Lung Function and Bronchial Reactivity. SD Pipis, EK McArdle, A Custovic, A Smith, K Hawkins and A Woodcock, North West Lung Centre, Manchester, UK It has been suggested that primary sensitisation to mite allergens may occur in utero (Allergy 1996; 51: 447-51). Allergic sensitisation is a major risk factor for bronchial hyperreactivity (BHR) in childhood. The aim of this study was to establish whether mite allergen avoidance during pregnancy affects lung function and BHR in high risk infants (both parents atopic) at the age 1 month. 24 full-term infants (15 boys) underwent lung function tests under sedation to determine maximal flow at FRC (Vm~,xrRc) and to assess BHR to histamine (PC~()) by the squeeze technique. Differences in Vm, x Fr~c and PC3o between the groups were analysed using Mann-Whitney U test. By the 16th week of pregnancy mattress, pillow and quilt covers for maternal bed and high filtration vacuum cleaner were supplied to 12 cases (active group), whilst no environmental intervention was implemented in remaining 12 (control group). Dust samples were collected from maternal mattress (M) and bedroom floor (BF) before, and then 6 months after the introduction of avoidance measures, and Der p 1 levels measured by mAb based ELISA. There was no difference in Der p 1 at the start of study (M-I.1 and 1.7 p.g/g; BF-0.7 and 0.7 ~g/g in the active and control group, respectively). Der p 1 levels fell significantly in the active group (M-0.22 Ixg/g, BF-0.27 txg/g), with no change in the controls. Baseline Vm,,~F~c was obtained in all 24 infants. 6 infants (3 in each group) were flow-limited at baseline, and thus not challenged. There was no difference in V,,,~xwc between the groups (%pred; median 68.8, 95% CI 37.1-88.4; median 67.1, 95% CI 42.7-109.3, in active and controls, respectively). Similarly, PC3~) was not different in two groups (median 3.5, 95% CI 0.8-27.2; median 5.6, 95% CI 0.3-32.0, in active and controls, respectively). These results indicate that infant pulmonary function and BHR in high-risk infants are not likely to be affected by mite allergen avoidance during pregnancy and the first month of life.

gun, Mark Van Natta, Pei Hua Chert, Peyton A. Eggleston, Baltimore, MD. To evaluate the effect of a H E P A room air cleaner on cat induced asthma and rhinitis, 35 cat allergic subjects who were living with one or more cats were studied in a double-blind, placebo-controlled trial. After a 1 month baseline period, subjects' bedrooms were equipped with an active or placebo air cleaner and mattress/pillow covers. Cats were kept out of the bedroom and bedding was washed weekly. The interventions were continued for 3 months. Evaluations included monthly cat allergen levels in bedroom air and settled dust, AM, PM, and nighttime nasal and chest symptom scores, twice daily peak flow rates, daily medication requirements, monthly spirometry, and methacholine challenge pre- and post-study. 35/35 subjects had rhinitis and 28/35 had asthma, all with regular symptoms and medication use at baseline. Filter compliance (measured by timer, compliance = >80% of expected use) was documented in 31/35 subjects. Airborne allergen levels were reduced in the active filter group compared to the placebo group (p = .045; active group: log mean airborne Fel d 1 reduced from 3.0 ng/m3 at baseline to 1.7 ng/m3 at month 3: placebo group: 2.6 ng/m3 at baseline to 2.8 ng/m3 at month 3). However, no differences were seen in settled dust FeI d i levels (p = .41), AM, PM, or nighttime nasal symptom scores (p = .77, .53, and .14, respectively), chest symptom scores (p = .39, .18, and .22), sleep disturbance (p = .10), AM or PM peak flow rates (p = .42 and .68), or medication requirements (nasal, p = .16; chest, p = .65). We conclude that while a combination of a H E P A room air cleaner, mattress/pillow covers, and cat exclusion from the bedroom did reduce airborne cat allergen levels in bedrooms in cat containing homes, these reductions did not significantly reduce disease activity by any parameter studied.

J ALLERGYCLIN IMMUNOL

Abstracts

S389

VOLUME 99, NUMBER 1, PART 2

1585

Environmental Control (EC) with Cat in situ, Reduces Cat Allergen (Fel d I) in House Dust Samples--but does it alter clinical symptoms? US Bjornsd6ttir, S Jakobinudottir, V Runar~dottir, Th Blondal, S Juliusson, University Hospitals, Reykjavik, Iceland Treatment of animal dander sensitivity relies on reducing allergen exposure, pharmacotherapy and immnnotherapy. We examined whether environmental control (EC) without removal of cat has an impact on clinical symptoms. Fel d I levels were measured monthly during an 11 month study period before and after institution of environmental control in 12 homes, and in 12 homes with an unchanged environment (UE). The EC and UE groups were further randomised into subgroups to receive either Nasal Budeson)de (BUD) (Rhinocort| 400 ~xg/day) or placebo. Before entry into the study and at 3 month intervals, allergen challenge with Fel d I was performed. Following EC, a gradual decrease in Fel d I levels was found. After 11 months, levels had decreased by 91.4% (p = 0.007). In the UE, Fel d I levels remained unchanged. In the subgroups randomised to receive BUD, EC significantly (p 0.043) improved nasal congestion (score 2.0, SEM 0.97), as compared to the UE group (score 2.7, SEM {).34). However, EC alone did not significantly alter symptom scores (3.55, SEM 1.14) compared to UE (6.12, SEM 1.18). BUD alone (2.86, SEM 0.34) improved symptom scores significantly more (p = 0.048) than EC alone (3.78, SEM 0.76). These data demonstrate that with stringent environmental control methods, Fel d I levels can be decreased significantly without removal of the cat. It is likely that longer periods of EC (>11 months) and lower levels of Fel d I are necessary to decrease allergic inflammation in the airway mucosa for marked clinical improvement to be evident. Our data show small, but significant improvements in the groups treated with EC and BUD as compared with BUD alone. We suggest that initial therapy for cat allergic pet owners should include both extensive EC and pharmacotherapy.

1586

A 15 kD protein containing sequence for an AIt al epitope. F Hu, D. Rosenthal, C Barnes, J Landuyt, F Pacheco, and J Portnov, The Children's Mercy hospital, Kansas City, MO. DNA and protein sequencing studies have provided significant progress toward understanding the nature of allergenic materials. In order to further define the allergens of Alternaria (ALT) the following experiments were conducted. A 247 base pair DNA sequence coding for a portion of a previously identified Air al epitope was produced from ALT mRNA by RT-PCR utilizing primers encoding the epitope sequence, mRNA encoding this sequence was identified in 8 individual strains of ALT by northern blotting experiments. The 247 BP sequence was labeled with ~2p and utilized as a probe to screen a cDNA library produced from mycelia of ALT strain CBS 1/)333. Six individual clones containing this sequence were identified. The clone containing the longest insert was sequenced. The sequence contained an open reading frame with both start codon and stop codons. The entire sequence encompassed 507 nucleotides and the open reading frame translated into a protein with a molecular weight of 14.9 kD. The sequence included a region of 60 nucleotides with very high homology for sequence from a previously reported Alt al epitope of 20 amino acids. The calculated pl for this protein is 9.9 and there are several possible carbohydrate attachment sites. Initially, Alt al was identified only by molecular size on SDS-PAGE. The reported size of the protein varied from 28 to 31 kD in the mmreduced state and the molecule reduced into fragments of 14 and 16 kD. The 20 amino acids of the N-terminal were previously reported and two different fusion proteins containing this sequence were shown to have lgE binding potential. This protein is known to be in 8 strains of ALT. It contains sequence coding for a known Alt epitope and was sequenced directly form clones of a ALT eDNA library. The contribution of this protein to the overall allergen)city of ALT is yet to be determined.

1587

Long-term (>5 years) follow-up of occupational asthma after removal from exposure. A Cartier, L Perfetti, H Ghezzo, JL Malo, H6pital du Sacr&Coeur, Montr6al, Canada. Background: Asthma symptoms and bronchial hyperresponsiveness persist in the majority of subjects with occupational asthma (OA) after removal from exposure. Existing data are based on relatively short-term ( < 5 yrs) follow-ups. Aim: To investigate whether some improvement is found in a follow-up after more than 5 yrs. Methods: 30 subjects with OA to high (n = 14)- or low (n = 16)-molecular-weight agents were studied 8.8 _+ 2.1 yrs (> 5 yrs in every subject) after the diagnosis coinciding with cessation of exposure. Fourteen subjects had received inhaled steroids at one time or another between the two visits. On the follow-up visit, questionnaire, spirometry and methacholine (Mch) tests were administered and results compared with those obtained at the time of diagnosis. Results: On the follow-up visit, 7 subjects had become completely asymptomatic, 14 required bronchodilators occasionally and the remaining 9 were on inhaled steroids. No significant change in baseline FEV 1 (in % pred) was observed, while forced vital capacity significantly decreased (p < 0./)5). Bronchial responsiveness to Mch significantly improved (p < 0.05) (all subjects but one had increased responsiveness at the time of diagnosis); however, 14/30 subjects (47%,) still had significantly increased responsiveness (PC20 -< 8 mg/ml): among those, there were more subjects with OA to high-molecular-weight agents (HMW) (10/14, 71%; p < 0.05). PC20 at the last visit inversely correlated with duration of exposure (r - -0.37; p < 0.05) and directly with both FEVI (r = 0.48 p < 0.01) and PC20 (r = 0.70; p < 0.001) at the time of diagnosis. PC20 at the last visit did not correlate with the duration of the followup. Conclusion: This long-term ( > 5 yrs) follow-up of patients with OA after removal from exposure shows better clinical and functional figures than those reported in previously published reports of shorter follow-ups in our center. This can be explained either by the use of inhaled steroids in some subjects or by the greater time lapse after cessation of exposure. Supported by the Reseau qu6b6cois d'excellenee en sant6 respiratoire and the International Council for Canadian Studies (support to L.P.)

$390

1588

Abstracts

J ALLERGY CLIN IMMUNOL JANUARY 1997

Particle size characteristics of airborne cultural fungi. S Mishra, T Randolph, D Pierson, H Burge*, NASA, Houston TX, *Harvard School of Public Health, Boston, MA. Exposure to airborne fungal spores is a recognized cause of hypersensitivity diseases. Particle size is the principal factor controlling site of deposition in the respiratory tract, but remains poorly known for most fungal exposure situations. We used a 2-stage Andersen cultural impactor to collect more than 300 samples outdoors and in a large Texas office building over three years. We used DG18 and malt extract agars, counted and identified all colonies after one week incubation at room temperature, and converted counts for multiple impactions. Results by stage are:

Taxon

In: % 1st

In: % 2nd

Out: % 1st

Out: % 2nd

Penicillium Cladosporium Alternaria Curvularia

22.5 23.3 23.3 68.6

77.5 76.7 76.7 31.4

32.3 35.2 30.6 41.1

41.0 64.8 69.4 40.0

Most fungi were recovered on the second stage which recovers all spores between - 1 and 8~m in aerodynamic diameter. Curvularia, however, was most abundant on the first stage, indicating (as expected) an aerodynamic diameter >8~m. Average particle sizes for Penicillium, Cladosporium, and Alternaria were larger in outdoor than in indoor air; those for Cun,ularia were largest indoors. Alternaria spores are 9-18p, m in smallest diameter and should have been captured on the 1st stage. Either the spores travel in a collapsed form, or small, immature spores dominated our collections. These data indicate that most fungal spores (including those of Alternaria, important in inducing asthma) are "respirable', and can penetrate beyond nasopharyngeal region. Spores of Curvularia (which is a recognized cause of allergic fungal sinusitis) probably impact in the nasal passages. These data also provide some support for the hypothesis that indoor fungal exposure rather than outdoor is more important as a stimulant for acute asthma attacks. 1589

Airborne and Dustborne Fungi on Airplanes, Trains, Buses, and Subways. M Muilenberg, T Dumyahn, D Forster, J Spengler, H Burge. Harvard School of Public Health, Boston, MA. Air quality in airplane cabins and other modes of public transportation has recently come under scrutiny because of apparent increases in incidence of contagious and hypersensitivity diseases. Airborne fungal concentrations on a new model airplane (N = 4), trains (6), interstate buses (6) and subways (8) were compared using l-rain, samples onto DG18 agar in a Portable Burkard Culture Plate Sampler (44 Lpm). 6 air samples were exposed during each travel segment. Using standardized areas and collection times, dust samples were collected from fabric-covered seats into 10 • 18 cm cloth bags using a portable vacuum cleaner. After sieving, a measured amount of dust (25 mg) was suspended in 2 ml 0.02% Tween 20 in distilled water, 10X serially diluted, and 0.1 ml of the suspension spread-plated onto duplicate DG18-filled Petri dishes. The Mann-Whitney U statistic was used on log-transformed data for between-vehicle comparisons. Total fungal concentrations in dust (GM -+ 1 s.d.) were 44,800 (20,700-96,800) CFU/gm in airplanes and between 64,500 and 71,000 CFU/gm on buses, subways and trains (no significant difference between any two vehicle types; p > 0.1). Yeasts, non-sporulating fungi, Aureobasidium, and Cladosporium predominated in all vehicles. Similar concentrations have been recovered from carpeted living rooms in the Boston area; GM = 85,800 (25,100-103,000) CFU/gm dust. Total fungal recoveries from dust and air were not well correlated except in trains (Spearman coefficient = 0.657). Average total airborne fungal concentrations were lowest on airplanes; GM = 32 (12-90) CFU/m3 of air, and highest on trains; 135 (69-262). Cladosporium, yeasts, non-sporu-

lating fungi, and Aspergillus were the four most frequently recovered fungal groups for all vehicle types. These data suggest that exposure to potentially allergenic fungi on some aircraft is relatively low while exposures on ground based public transportation is similar to that in a residential environment.

1590

Indoor Survey of Molds and Prevalence of Mold Atopy. Y.

Katz, ~ H. Verleger,1 J. Ban', I M. Rachrniel, 1 S. Kiviti,: E. S. Kuttin s. Zerifin, ~ Tel-Aviv,2 Ness-Ziona, 3 Israel. Aim of the study: To determine the significance and the contribution of molds to allergic manifestations. Methods: Prevalence of allergic rhinitis and asthma in 395 members of a rural community were examined by questionnaire and inquire of medical files. The atopic status in general and allergy to molds was determined by SPT to a panel of aeroallergens including Aspergillus, Penicillium, Alternaria and Cladosporium. An indoor mold survey was performed by placing SDA plates at houses, identification and counting mold of colonies from each species. Results: 42 subjects, composing 10.9% of the study group had positive SPT to molds. 61.9% of the participants that were allergic to molds, were classified as symptomatic. Among individuals with a borderline positive SPT to molds (wheal = 2-3 ram) additional 23 had positive SPT to molds. Among the whole 65 mold positive subjects, 13 were allergic to molds solely, only 2 of these had allergic symptoms. All 59 houses contained viable molds. The most common mold was Aspergillus, followed by Penicillium, Alternaria and Cladosporium. Aspergillus was also the most abundant mold in houses. There was no significant correlation between the abundance of molds, positive SPT to that mold and symptomatology. However, for Penicillium and Alternaria there was an inclination toward higher rate of symptomatology in patients with positive SPT to molds when high quantities of that mold was recovered. Conclusions: Viable molds are common in houses in temperate climate. Allergy to molds itself has low predictive value to development of allergic symptoms, but allergy to molds in otherwise atopic subjects increases the risk of symptomatic disease.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1591

1592

Fungal Growth on Different Fiberglass Duct Materials. CY Rao, HA Burw, Harvard School of Public Health, Boston, MA Fungi in indoor air can cause allergic reactions and other air quality problems. Though infiltration from outdoors is the most common indoor source of fungal spores, indoor amplification is bclievcd to bc the major source of diseasecausing fungal exposure in indoor environments. Of particular concern is amplification of fungi on fiberglass materials in air distribution ducts. To evaluate the environmental conditions that promote fungal growth on fiberglass materials and thc effects of growth on these materials, we studied Clado,sporium sphaero,spemzum and A.spert,,illus vetwicolor growth on wet fiberglass duct liners and duct boards from four manufacturers. Each duct material, which wits either clean (but not sterilized), pre-coated with malt extract broth, or precoated with ventilation system dust, was inoculated on the airstream surface side with a fungus and incubated at room temperature and l(l(lC,/ relative humidity. Growth was examined after various incubation times by stereo and high-powered light microscopy and scanning electron microscopy. Growth occurred on some clean, wetted fiberglass samples when inoculated with spores. However, amount of growth varied with soiling (either by malt extract broth or dust), manufacturer, and the presence of a resin coat. Growth of brown Cladosporimn sphaero,v)emzum was evident to the naked eye while light-colored Ast)etgillus versicolor was only apparent using microscopy. Analyses by light microscopy did not demonstrate that the fungi were degrading the fiberglass. Scanning electron microscopy studies are in progress to verify these findings. Fungal growth can occur on wet fiberglass materials if spores are present and soiling enhances growth. Examination of field samples by the naked eye may not be adequate for evaluation of contamination. The potential for release of spores and other materials from contaminated fiberglass into the occupied space remains to be studied.

Incidence of Rodent Dander Sensitivity in an Urban Population with Allergic Respiratory Disease. DH Lin MD, MF Dimaio MD, W Mlzit; DJ Valaeer MD. New York, New York. IgE mediated immediate hypersensitivity to protein antigens originating from mice or rats is described as an occupational disease occurring in laboratory researchers and animal handlers. Both allergic rhinitis (AR) and allergic asthma (AA} duc to rodent dander hypersensitivity (RDH) are reported in thcse adult populations. Urban dwellers may bc perennially exposed to rodent derived proteins including rodent dander to wlrying degrees in public and home environments. We tested 531 pediatric and adult patients with AR and/or AA at three different New York City ambulatory ccnters serving urban patients from a wide socioeconomic spectrum over a three year period. Ahmg with commercial rat dander and mouse dander extracts (klollister-Stier, Spokane WA) we employed a standard immediate hypersensitivity skin testing panel including dust mite, cockroach, dog and cat dander, and standard controls. The incidence of RDH detected R)r all patients was 18~ for rat and 20% for mouse; for AA patients with or without AR it was 20gf for rat and 22% for mouse; tot AR patients without AA it was 2l)('~ for rat and 21% for mouse. There was no diffcrencc in thc incidence of RDH observed bctwcen the three centers, reflecting sensitization across sociocconomic groups. Concurrent skin tests were negativc to dog and cat in 16% of patients with RDH making cross reactive antibodies to these mammalian danders an unlikely causc for the results observed. We conclude that RDH exists at a significant Icvel in urban adults and children with AA and/or AR and may contribute to perennial symptoms.

Abstracts

S391

1593

Timed Elution of the Allergens of Epicoccum nigrum. T D'Rosario, S Fudembe~, A Dixit, Washington Univ Mud School, St Louis, MO WE have previously demonstrated presence of 44 allergenic proteins in the spores and mycelia of Epieoccum nigrum (EN) (J.A.C.I 90:11-20, 1992). Timed clution studies were performed to optimizc the elution of these allergens. Pure spores (90%) and mycclial components (95c/r,) from a single strain of EN were frozen in liquid N> powdered and homogenized in 0.125M ammonium bicarbonate buffer with 5raM EDTA. Extracted material was immediately centrifuged. Supernate was collected (0 hr extract). The pellet was resuspended in an equal w)lume of buffer and supernates were similarly collected thereafter at 3 hr, 16 hr, 20 hr, and 24 hr intervals. Crude extracts were analyzed by SDS-PAGE and proteins were visualized by silver staining, hnmunoblotting was also performed using 6 sera pools from 30 patients who were skin test positive (2+ to 4+) to EN. A greater number of IgE binding bands were detected in spore extracts its compared to mycelial extracts in all 6 pools. The largest number of silver stained proteins was detected in the [) hr extracts, with gradual reduction until no proteins were found in the 24 hr extracts. Similarly, on immunoblotting, (} hr and 3 hr extracts showed the maximum number of IgE binding bands, suggesting that most allergens were eluted early. A limited number of proteins within molecular weight range of 45 to 66.2kd bound IgE in extracts from 0 hr to 20 hr. These same bands bound IgE from all sera pools. Thus, these major idlergens are eluted for extended periods. These proteins were common to both spore and mycelium extracts. Other continually etuted proteins, in the 32 to 33kd range, bound lgE only in spore extracts. Our results confirm that elution of allergens is time dependent. These data demonstrate that timed elution may be important in preparing mold extract, particularly for the purification of important major allergens.

1594

IgE-Binding Epitopes of the Recombinant AIternaria Allergen, AIt a 2. H Sanchez. D Geisler, R Bush, Wm S Middleton VA Hospital and University of Wisconsin, Madison, WI We have previously cloned and expressed an important Alternaria alternata allergen, r Alt a 2. In this study we sought to identify human IgE-binding epitopes on the molecules. Based on the eDNA sequence of the clone encoding for r Alt a 2, the 189 amino acid sequence was deduced. Using Chou-Fosman algorithm predictions, an antigenic epitope map was constructed. The analysis showed that r Alt a 2 contains 34 potential antibody binding sites located at six different regions on the protein. Region 3 located between amino acids 57-84 was predicted to have the largest number of potential antibody binding sites. Two polypeptides, one of 18-mer (amino acid residues 57-74) and of 22-met (57-78) were synthesized. The synthesized peptides were applied to nitrocellulose membranes and probed from IgE binding by dot-immunoblotting. Using pooled sera from Alternaria sensitive asthmatics, we detected IgE binding to both peptides. The detection of IgE binding epitopes on r Air a 2 will be useful in understanding the immunobiology of Alternaria sensitivity and its treatment,

S392

1595

Abstracts

Detection of serum IgE antibodies to the mite allergen Der p 2 using phage display and enzyme-labelled anti-phage antibodies. RC Aalberse, E Vermeulen, I Bulder, Gill Hakkaart, J Schuurman; CLB, Amsterdam, The Netherlands. Introduction: For the measurement of IgE specific for a major allergen the phage display technology has great potential, because the allergen-phage complex is easily produced in bulk and the phage particle can be detected with high sensitivity. The catching procedure described below avoids interference by competing IgG antibodies. Methods: Der p 2 (cDNA provided by Dr. W.R. Thomas) was expressed on the surface of the filamentous phage M13 as fusion protein with the phage tail protein III. Serum samples were incubated in microtiter plates coated with monoclonal anti-IgE. After washing, allergen-phage particles were added. Bound phage was detected using enzyme-labelled anti-phage antibodies. Results: The assay was optimized using chimeric mouse/ human monoclonal IgE anti-Der p 2 antibodies for spiking RAST-negative human sera. Allergen-specific IgE can be detected in the nanogram range without phage amplification. A further increase in sensitivity can be achieved by amplification of the bound phage, but this procedure has not been optimized yet. Der p 2-specific IgE was detectable in 9/11 sera with a positive RAST to total mite extract. The specificity of the phage-binding was established by inhibition with mite extract. Conclusion: The phage display procedure is suitable for the detection of IgE antibodies to recombinant allergens.

1596

A Worldwide External Quality Control Program for the House Dust Mite Allergen, Der p 1. R. Siebers, N. Rains, P. Fitzharris, J. Crane. Wellington Asthma Research Group, Wellington School of Medicine, Wellington, New Zealand Research laboratories worldwide are estimating the house dust mite allergen Der p 1 as a risk factor for asthma. In to compare allergen concentrations from different geographic locations it is imperative that results obtained by different laboratories are similar in. We report here preliminary results of a worldwide external Der p I quality control program. Two sifted dust samples (A and B) containing no live mites were sent to 20 volunteer laboratories worldwide known to measure Der p 1. A questionnaire regarding laboratory methodology accompanied the samples. Der p 1 concentrations were measured using monoclonal antibody ELISA by all bar two (RIA) participating laboratories. Mean Der p 1 results for sample A was 19.4 ~g/g (s.d: 2.6; range: 6.8-49.7), and for sample B was 18.1 Ixg/g (s.d: 10.1; range: 6.0-39.9). Laboratories extracting Der p 1 at 4oC (n =9) returned lower Der p 1 results (sample A mean: 14.0 ~g/g; 95% CI: 9.7-18.3. Sample B mean: 12.8 ~g/g; 95% CI: 8.6-17.1), than laboratories extracting at room temperature (sample A mean: 23.8 I~g/g; 95% CI: 14.832.8. Sample B mean: 22.4 Ixg/g; 95% CI: 14.8-29.9) independent of buffer type, time of extraction, source of primary standard, or differences in monoclonal antibodies (p=0.056 and 0.032 respectively for samples A and B). This preliminary study has demonstrated a 6 to 7 fold variability in Der p l results worldwide which in part appears to be due to extraction temperature differences.

1597

Identification of T cell epitopes within the rat urinary protein. MG Jones 1, A Felton 2, JR Lamb and AJ Newman Taylor. Imperial College School of Medicine at the National Heart and Lung Institute I, London and Perkin Elmer, Cheshire 2, U.K. The IgE binding allergens of rat urinary protein have been well characterised in individuals with laboratory animal allergy (LAA), however little is currently known regarding the T cell epitopes. We have preliminary data on the T cell epitopes recognised within rat urinary protein. T cell proliferation from 15 individuals with LAA was determined with rat urinary protein, the 17 kDa protein of rat urinary protein which is a major IgE binding allergen in

J ALLERGY CLIN IMMUNOL JANUARY 1997

89% of LAA cases (1), and 3 peptides synthesised from the 17 kDa protein, namely 35-50, 80-108 and 140-159. In all individuals there was a significant T cell response to both rat urinary protein and the 17 kDa protein (stimulation index > 3). The T cell response to the 17 kDa protein compared with the whole rat urinary protein was variable, ranging from 16%, indicating a minor component, to 100% suggesting it represents the immunodominant region of rat urinary protein. Peptide 80-108 of the 17 kDa was found to be positive in 6 of 15 individuals with a stimulation index ranging from 3 to 29, and peptide 140159 of the 17 kDa was positive in 4 of 15 individuals with a stimulation index ranging from 3 to 12. Reactivity to both peptides was present in 2 of 15 individuals. Peptide 35-50 was not found to be positive with any of the 15 LAA individuals (stimulation index < 3). Our data has identified the 17 kDa protein as being recognised by all 15 LAA individuals. In some individuals 17 kDa is immunodominant whereas in other individuals it represents a small component of the cellular response to rat urinary protein. Two regions within the 17 kDa have been identified as T cell epitopes, namely peptides 80-108 and 140-159. (1) Gordon S, Tee RD and Newman Taylor AJ. JACI 1993,92,298-305.

1598

Purification and sequencing of the main soybean hull allergen Gly m 2, responsible for the Barcelona asthma outbreaks. Codina R, Lockey RF and Rama R. * James A Haley VA Hospital and University of South Florida, Tampa, FL and *University of Barcelona, Barcelona, Spain. Two soybean hull isoallergens, Gly m 1A and Gly m 1B, were associated with asthma outbreaks that occurred in Cartagena, Spain. Using sera of asthmatic epidemic patients (AEP) from Barcelona, 3 main allergens, 2 of them with MWs and pls identical to those reported for Gly m 1A and Gly m 1B, were identified. The purposes of this study were to purify and to study the N-terminal aminoacid sequence (NTS) of the third allergen, which has a MW of 8 KDa. The purification combined double dialysis and preparative IEF. Specific IgE to the fractions obtained demonstrated 3 peaks, one of them corresponding to the 8 KDa allergen. The pooled fractions containing this allergen were studied by SDS-PAGE, SDS-PAGE/Western blot and IEF/ Western blot. Only a band with a MW of 8 KDa and a pl of 6 was obtained. Allergenicity of soybean hull correlated with the presence of the 8 KDa allergen. The NTS of the first 20 a.a., which was registered as the NTS of Gly m 2, was determined by the Edman degradation method. This NTS lacks homology with that reported for Gly m 1 but has a homology of 71% with a storage protein of Vigna radiata (cow pea) and 64% with a "disease response protein" from Pisum sativum (green pea). These results suggest that Gly m 2 in soybeans could protect against diseases which affect soybean plants. In addition, Gly m 1 could be another soybean allergen responsible for the Barcelona asthma outbreaks.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1599

Complete DiGeorge Syndrome: Persistence of Profound lmmunodeficiency. ML Markert, TJ Watson, TM McLaughlin. DS Hummel. HM Rosenblatt, SE Schiffl TO Han'ille, L Williams. RI Sch~ff RH Bucklo', Duke University Medical Center, Durham, Vanderbilt University Medical Center, Nashville, TN, Baylor College of Medicine and Texas Children's Hospital, Houston, TX. DiGeorgc Syndrome is characterized by developmental defects of the heart, parathyroids, and thymus with variable T cell function, ranging from essentially normal to a complete absence. Approximately 5% of thcsc patients have complete DiGeorge Syndrome defined by having profoundly depressed T cell function as measured by proliferative responses to mitogens. The purpose of this study was to determine whether the T cell function of patients with complete DiGeorge Syndrome might spontaneously improve with time. DiGeorge Syndrome patients were identified by chart review. All patients had been evaluated by immunophcnotyping with flow cytometry and proliferative responses to mitogens. Patients with low T cell numbers but significant T cell function were excluded. Eight patients were identified who had presented with profoundly depressed proliferative responses to mitogcns. These responses did not spontaneously improve, and clinical immunodeficicncy persisted with follow up as long as 2 years. Two of the patients died while under observation to determine if T cell function would spontaneously improve. One patient remains alive at 4 months with m~ therapy, one is alive and well after imnmnorceonstitution from thymic transplantation, the othcrs either died early from complications of their disease: as gastroesophageal reflux with aspiration (1), infection (1), or after attempts at immunorestorative therapy such as IL-2 therapy, thymus transplantation, or bone marrow transphmtation (3). Since T cell function did not improve in any patient whose initial proliferative responses to mitogens were profoundly depressed, we recommend thymus or bonc marrow transphmtation for complete DiGcorge Syndrome patients as soon as the diagnosis is confirmed.

1600

Maturation of Cellular Immunity in Chromosome 22q 11.2 Deletion Syndrome (DiGeorge Syndrome). KE. Sullivan, D Driscoll, B Ernamlal, D McDonald MeGinn, and E Zackai. Children's Hospital of Philadelphia, Philadelphia, PA We examined the maturation of cellular immunity in patients with chromosome 22ql 1.2 deletion syndrome (DiGeorge syndrome) to detcrminc whether limitation of thymic epithelium would alter the normal maturation patterns of T cells. The spectrum of immunodeficiency in this syndrome is broad. Typically, T cell production is impaired early in life with relative preservation of T cell function. We followed 21 patients with chromosome 22ql 1.2 deletion syndrome prospectively over the first two years of life to characterize the maturation of their T cell production and function over time. 70% of the study population had low numbers ofCD3 T cells in the first two months of life compared to 60% of the population by 24 months of life. 90% of the study population had lower than normal numbers of CD4 cells in the first two months of life comparcd to 30% with lowcr than normal CD4 counts by 24 months of age. CD8 counts were uniformly depressed throughout the first two years of life. CD45RA conversion to CD45RO was delayed in the study population suggesting a delay in antigen induced maturational changes. In contrast, B cell numbers wcrc low in the first two months of life and then expanded such that most patients had higher than normal numbers of B cells at 24 months of age. Natural killer cells also underwent expansion in the first few months of life. Responses to eoncanavalin A were always normal, but responses to phytohemagglutinin and pokeweed mitogen were decreased initially and normalized over 24 months. This study examincs the dynamic nature of the cellular immunodeficicncy seen in chromosome 22q11.2 deletion syndrome and demonstrates that lymphocyte homeostasis is abnormal for prolonged periods of time.

Abstracts

1601

S393

Protein Losing Enteropathy Presenting as Recurrent Sinusitis, Hypogammaglobulinemia and Severe Lymphopenia. V Bommanna, M Ballow, KM O'Neil. The Children's Hospital of Buffalo, Buffalo, NY Protein losing enteropathy (PLE), or protein loss into the gut causing hypoproteinemia, and edema, has been reported following the modified Fortran Procedure (FP) in children with congenital heart disease. We present a patient who developed recurrent sinusitis and otitis, hypogammaglobulinemia and severe lymphocytopenia after FP. This 5V~, girl was born with complex congenital heart disease (patent ductus arteriosus, confutation of aorta with hypoplastic left heart, mmsposition of great vessels, and single ventricle). She underwent palliative surgery in the neonatal period and a FP at 3 years of age. She required continuous digoxin and diuretics for low cardiac output and persistently elevated right atrial pressure ( 19 mm Hg). One year after the FP, she developed rccurrent infections of the ears and sinuses. There was no family history of immunodeficiency. Physical exam showed her to have postnasal drip, a cardiac systolic murmur, and minimal ascites. Laboratory evaluation revealed a low IgG of 263 mg/dl, lgM of 79 mg/dl, and IgA of 48 mg/dl. She had a low but protective tetanus titer at 0.16.1U/ml, but no significant antibody to 9 common respiratory pathogens. Following Pneumovax administration, she made antihodie to only 1 of 4 types tested. She had lymphopenia affecting CD3 (328/ uL), and CD4 cells (28/uL), with normal CD8 (237/uL) and CD[ cells (118/uL). In vitro antigen and mitogen (PHA, Con A) responses were impaired. Her serum protein and albumin were low at 4.2 and 2. gm/dl, respectively. She was treated with monthly IV gammaglobulin (IVIG) infuskms at 400 mg/kg. Sinusitis persisted despite IVIG and prophylactic antibiotics. Trough IgG concentrations never exceeded 265 mg/dl despite dose and frequency escalation to 725 mg/kg every 3 weeks. A stool alpha-1 antitrypsin of 12 mg/gm stool (nl <2 mg/gm) confirmed protein loss. The parents refused further work up of PLE. PLE should be considered in the differential diagnosis of hypogammaglobulinemia following FP.

$394

1602

Abstracts

In Vitro Formation of the Human Thymic Microenviron-

ment: Similarity to SCID Thymus. DD Patel, LP Whichard, GD Miralles, JS Sundy, BF Haynes. Duke University Medical Center, Durham, NC Thymus transplantation has been successful in patients with congenital immunodeficiency, but disadvantages to current methods include the low availability of fresh, HLA-matched fetal thymic tissue and contamination with donor T cells. With the potential need for thymus transplantation in diseases where the self-regenerating pool of T cells is destroyed such as in AIDS, we have begun studies to develop thymic stromal microenvironments in vitro from cultured thymic fibroblasts (TF) and thymic epithelial (TE) cells. Using an artificial capillary system, human TE cells and TF aggregated into nodules containing TE cells in an intertwined pattern encapsulated by fibroblasts (N=7). Unlike TE cells cultured in monolayers, TE cells in micronodules did not differentiate as determined by reactivity with mAbs STE1, STE2 and 11.24 (CD44v9), nor did they form Hassall's bodies. Thus, recapitulating the non-lymphoid thymic stroma of patients with severe combined immunodeficiency (SCID) which lack Hassall's bodies in vivo and in which undifferentiated TE cells and TF are intermixed. SCID-derived TE cells can, however, differentiate when cultured in monolayers in vitro (N=3). Thus, suggesting a role for T cells in TE differentiation in vivo. By RT-PCR, the thymic stromal nodules expressed mRNA for stroinal cell-derived cytokines ILlc~, ILl[3, IL6, ILS, TGF[31, TGFI32, TGFc~, G-CSF, GM-CSF and M-CSF, but not for EGF or the T cell-derived cytokines TNFa, TNF[3, IFN3', IL2, IL3, IL4, IL5, 1L7, IL9 or ILl3 (N=2). When co-cultured with thymic micronodules, lin- progenitor cells from umbilical cord blood (UCB) proliferated and migrated to the thymic micronodules (N=2). Mieronodules seeded with lin- UCB cells contained numerous CDla hi, CD3-, CD7-, CD33 r~ cells with a dendritic morphology (N=2). Thus, thymic nodules formed m vitro from cultured TF and TE cells are morphologically similar to human SCID thymus, and are able to support the development of CDla + dendritic-like cells in vitro.

1603

2'-Deoxycoformycin Blocks T Cell Differentiation in Murine Fetal Thymic Organ Culture: A Model for Adenosine Deaminase Deficiency. R Resta, H Jiang, S W Hooker, A B Laurent, TB Knudsen. and LF Thompson, Okla. Med. Res. Fndn. and Univ. of Okla., Oklahoma City, OK and Jefferson Med. College, Philadelphia, PA Compromised immune function in adenosine deaminase (ADA) deficiency involves lymphospecific toxicity from accumulated adenosine (Ado) and/or deoxyadenosine, but the relative contributions of these ADA substrates are unknown. Current models of ADA deficiency have significant shortcomings, such as the necessity of adding exogenous ADA substrates to in vitro systems and the hepatic toxicity in ADA knock out mice. We propose that murine fetal thymic organ culture with the ADA inhibitor 2'dcoxycoformycin (dCF) is an ideal system to delineate the biochemical mechanism responsible for the block in T cell differentiation in ADA deficiency. Day 15 fetal thymic lobes cultured with 5 IxM dCF for 5 days exhibited a 91% inhibition of thymocyte production without addition of exogenous ADA substrates. The phenotype of the residual cells was: 51% CD4-CDS-, 14% CD4+CD8 +, 34% CD8+CD4 -, 1.4% CD8-CD4 § 38% CD3 +, and 34% ~,8 + as compared to 8% CD4-CD8-, 74% CD4+CD8 +, 7% CD8+CD4 -, 11% CD8-CD4 +, 10% CD3-, and 4% ~,8 + in control cultures. These data are consistent with a block in T cell differentiation prior to the rearrangement of T cell receptor c~ genes, and are similar to those obtained with cAMP elevating agents. Since Ado can elevate cAMP either directly or via Ado receptors, these data suggest that Ado may play a role in blocking thymocyte differentiation in dCF-treated cultures. Indeed, dCF-treated mice exhibited a 30-fold elevation in thymic Ado to 50 p,M, and Ado A2a, A2b, and A3 receptors were found in day 15 murine fetal thymus. The role of Ado and/or Ado receptors in the pathogenesis of ADA deficiency should be re-evaluated.

J ALLERGY CLIN IMMUNOL JANUARY 1997

1604

Gene transfer of CD40 iigand into T cell lines using an adeno-associated virus based vector. RL Fuleihan, EA Cooper, and P Zhang, Yale University School of Medicine, New Haven, CT. X-linked hyper IgM (HIGMX-1) is a rare inherited immune deficiency disease resulting from defects in the gene for CD40 ligand (CD40L). Patients suffer from recurrent bacterial and opportunistic infections, autoimmune disease, and lymphoproliferative disease. Therapy of this severe immunodeficiency is limited to intravenous immunoglobulin or bone marrow transplantation (BMT) when possible. Gene transfer might provide a better therapeutic approach especially for patients who do not have suitable BMT donors. CD40L expression is highly regulated in a tissue specific and activation-dependent manner. Therefore, we have chosen to examine gene transfer of CD40L into T lymphocytes under the regulatory control of the IL-2 promoter (IL-2p) using adeno-associated virus (AAV). AAV is a nonpathogenic human virus that requires only the inverted terminal repeats (ITR) for integration. We have generated a construct expressing human (h) CD40L under the regulatory control of the IL-2p in the vector pBluescript II and then subcloned the construct into an AAV based vector. Transient transfection of either the pBII or pAAV vector resulted in inducible expression of hCD40L in the murine thymoma cell line EL-4. The pAAV/IL-2p/CD40L vector was used to generate recombinant AAV virus (at the Gene Therapy Center of the University of North Carolina). Neither EL-4 cells nor the human Jurkat T cells expressed hCD40L after infection with rAAV/IL-2p/CD40L at 37~ C. It has been shown that second strand synthesis of the single stranded rAAV genome is rate limiting and can be enhanced by adcnovirus or heat shock at 42.5 ~ C. Infected EL-4 and Jurkat cells were incubated at 42.5~ C for 6 hrs. EL-4 cells did not survive heat shock but Jurkat cells survived and expressed hCD40L. Heat shocked but uninfected Jurkat cells did not express hCD40L. These results indicate that the IL-2p provides activation-dependent expression of CD40L and that rAAV can provide activation-dependent gene transfer of CD40L.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1605

1606

Granulocyte Colony Stimulating Factor (G-CSF) Corrects Neutropenia in X linked Hyper IgM Syndrome (HIGMX1). JE Stahhnan, LB Bacharier, RS Geha, and LC Sehneider, Children's Hospital, Boston, MA. HIGMX-I is characterized by absent IgG and IgA levels with normal or increased levels of lgM and is due to mutations in the CD4I) ligand gene. Patients are susceptible to recurrent bacterial and opportunistic infections, lymphoid hyperplasia, autoimmunity, and oral ulcers associated with neutropenia. We report the case of a patient with HIGMX-I and recurrent, profound stomatitis associated with neutropenia responsive to G-CSF. During the first year of life, the patient developed recurrent otitis media and interstitial pneumonia which resolved with IV TMP-SMX and IM gammaglobulin (GG). HIGMX-I was diagnosed based on near absent lgG and IgA levels with a normal IgM and was confirmed by the absence of CD4I) ligand on stimulated T cells. He did well until agc 9 years on IMGG. Over the next several years, he had episodic stomatitis associated with febrile neutropenia poorly responsive to high dose IVIG and plasmapheresis on 1 occasion. At 17 years of age, he presented with severe stomatitis, intermittent fevers and 14kg weight loss. His absolute neutrophil count (ANC) was 29(1 mm ~. HSV was not detected. No clinical response to acyclovir or antibiotics was noted. Bone marrow biopsy revealed hypocellularity with decreased myeloid precursors. Recombinant G-CSF was instituted at 5 mcg/kg/day SC without response and was increased to l0 mcg/kg/day resulting in gradually increasing ANC and resolving of the stomatitis. Over the next year, he was maintained with 6.5 mc~k~d. He did well with significant improvement of his stomatitis symptoms compared to previous years. Only 2 neutropenic episodes required therapy; one associated with post-traumatic cellulitis and the second in the setting of a URI. Both resolved with increased doses of G-CSF (10 mcg/kg/d). The etiology of neutropenia in HIGMX-I is unclear. Wc belicve that G-CSF is effective in treating neutropcnia associated with this disorder. Dusing may be adjusted based on the severity of neutropcnia or clinical situation. Deficiency of Zap 70 kinase and reduction in levels of other T-cell second messenger proteins in child with immune deficiency and inflammatory bowel disease. C. McCusker, W.. Sommervi[le, .d. l/ei[lette. B. Mazet, R. Schreiber. Division of Allergy and Immunology, and Gastroenterology, Montreal Children's Hospital, Montreal, Quebec, Canada. Zap 70 is a protein tyrosine kinase involved in T-cell receptor (TCR) mediated signaling events. It is found in association with thc phosphorylated ~ and CD3 chains following antigen-MCH complex interaction with the TCR and its phosphorylation ultimately results in T-cell activation. Zap 70 kinase deficiency, a rare form of SCID, is characterized by the absence of CD8 ~ T cells, and thc presence of CD4 ~ T-cells which are not responsive to TCR-mediated stimulation. We have identified a child of consanguineous parents with selective T-cell deficiency, hypogammaglobulinemia, and granulomatous colitis. This boy, now age 14, has low peripheral CD8 + and normal C D 4 T cells. He has diminished responses to standard mitogens. However, activation of his CD4 + cells using phorbol cstcr/ionomycin (+ IL2), the combination of which bypasses the TCR, results in near normal proliferations. Additiomdly, there was limited ability to increase cytosolic Ca > levcls when T cells were stimulated with PHA. Recently we have defined the nature of this T-cell defect. Northern blot analysis of peripheral blood lymphocytes of the patient compared to control PBL's and the Jurkat T-cell line show a marked decrease in expression of Zap-70 mRNA in the patient, mRNA of a related early signaling protein, lck, was normal. Additionally, Western blot analysis of PBL lysates shows reduction in expression of the Zap 70 protein when compared to normal controls. Expression of another tyrosine kinase, FYN, was also reduced compared to controls in two experiments. Unlike previous descriptions of children with Zap 70 deficiency who present with SCID, this child's primary clinical manifestation was IBD. In the mouse, IBD is associated with

Abstracts

S395

such defects as IL2 or 1L10 knockouts, but to our knowledge this is the first indication in humans that an isolated T cell signaling defect may be associated with immunodeficiency and IBD.

1607

Familial Occurrence of Job's Syndrome of Hyper-lgE and Recurrent Infections. HR Hill, NH Augustine, G Alexander, JC Carey, HD Ochs, RJ Wedgwood, RJ Faville, PG Quie and MF Leppert. University of Utah, Salt Lake City, UT, University of Washington, Seattle, WA. and University of Minnesota, Minneapolis, MN. Job's syndrome of hyperimmunoglobulinemia E and recurrent infections is a mild to very severe immunodeficiency disorder characterized by eczema, peculiar facies, bone fractures, and recurrent infections of the skin and respiratory tract. The most intriguing hypothesis of the pathophysiology of the disorder involves an imbalance in the production of IFN~/, a major activator of phagocytes, and IL4 which increases the production of IgE. Although isolated instances have been reported of familial occurrence of Job's syndrome, a thorough analysis of the inheritance of this disorder has not been undertaken. We have recently examined 5 kindreds in which more than one immediate family member has classic features of Job's syndrome. Transmission has occurred from affected father to two daughters, as well as from mother to affected son. In one kindred, three generations including a great grandfather, grandfather, mother and child were affected. Occurrence of the syndrome in the five kindreds we have studied strongly suggests autosomal dominant inheritance with incomplete penetrance. A search continues for the specific gene defect involved.

1608

Transient Wiskott-Aldrich (WAS) Like Immunodeficiency in a 4 Year Old Boy With Recurrent Infections and Thrombocytopenia. E Ru&-Huidobro, HM Rosenblatt, Z Dreyer, ME Paul, 1C Hanson, SL Nbramson, WT Shearer. Baylor College of Medicine, Houston, TX. WAS is an X-linked immunodeficiency characterized by thrombocytopenia with low platelet volume (MPV), eczema, and defective T cell function and abnormal antibody production to polysaccharide antigens. Isohemagglutinins are absent and serum IgM levels are usually low. In addition, CD43 is decreased and lymphocyte microvillous morphology is abnormal on electron microscopy (EM). The patient reported here is a 13 mo boy who presented with a history of >20 episodes of otitis, recurrent sinopulmonary infections and platelet count of 35,000 /ul and MPV of 5-6 (hi > 7). Serum IgG, A, and M were normal and isohemagglutinins were present. Lymphocyte phenotyping revealed normal percentages and numbers of T and B cell subsets with normal to decreased proliferative responses to PHA, ConA and PWM. Maternal X chromosome inactivation studies showed random inactivation in T, B, and myeloid lines. Although CD43 was quantitatively normal, lymphocytes EM was abnormal in 2 labs with scores as follows: (Pt 2.79 _+ .51; nl 3.62 _+ .22; WAS 2.89 _+ .27). On follow-up at 4 yo, platelet counts had risen to 110,000 /ul with MPV 5.7; serum immunoglobulins were normal and immunization resulted in normal responses to diphtheria, tetanus and HiB conjugate vaccines; baseline antibody levels to pneumococcal serotypes 3, 7, 9, and 14 were absent. In vitro phenotyping and proliferative responses to mitogens were normal with variable responses to specific antigens. He had been clinically well with only I episode of pneumonia since 3 yo. Cases such as this with transient or mild characteristics of WAS or X-linked thrombocytopenia present problems for genetic counseling and therapeutic and diagnostic intervention which may be resolved by mutation analysis of the newly discovered WASP gene locus.

$396

1609

1610

Abstracts

Immune Response to Oral Poliovirus Vaccine in lgA Deficient Patients. Magda MS Carneiro-Sampaio 1, Silvana B Castrignano1"2,Barhro Carlsson~ Lars A Hanson 2 1 Dept. of Immunology, 1 Univ. of S Paulo, Brazil, 2 Dept. of Clinical Immunology, Univ. of GOteborg, Sweden Although vaccine-associated paralytic poliomyelitis (VAPP) is rare, immune deficient individuals in general are at high risk of developing it. There is not a clear-cut recommendation about the use of oral poliovirus vaccine (OPV) in individuals with IgA deficiency (IgAD), the most common primary immunodeficiency. We evaluated 34 IgA deficient children and adolescents who had received OPV (at least 4 doses as basic immunization schedule) before they had their immunodeficiency diagnosed. We searched for history as well as clinical signs of paralysis, analysed serum antibody titers to the 3 poliovirus serotypes (neutralization technique), and measured serum and salivary antibody levels to poliovirus type 1 (ELISA). No patient presented paralysis or other kind of side reactions after vaccination, not even the patient with associated IgG2 deficiency. Neutralizing antibody titers to the 3 poliovirus serotypes were considered to be protective (->8) in 31/32 patients. Serum IgG and IgM anti-poliovirus I antibody levels from patients were not significantly different from age-matched healthy control group (n = 34). In saliva samples, IgM anti-poliovirus type 1 antibody levels were significantly higher in the patient group compared to the control one, whereas no significant differences were observed in IgG antibody levels. Our results suggest that IgAD individuals are not at increased risk of acquiring VAPP compared to general population. This finding is corroborated by the fact that no case of VAPP in IgAD individuals has been described in the literature. Increased production of specific IgM antibodies in mucosa may compensate IgA deficiency. Financial Support: Brazilian Research Council (CNPq), Swedish Institute, Swedish Medical Council and Ellen, Walter and Lennart Hesselman Foundation.

Interferon-gamma (IFN-y) production in IgG2 deficient patients. CM Kokron, MD; N Ramesh, PhD; DJ Ahern; RS Geha, MD. Children's Hospital, Harvard Medical School, Boston, MA. IgG2 deficiency is the most frequent immunoglobulin subclass deficiency in children and the defect that causes this immunodeficiency is unknown. Deletions of the heavy chain constant region have rarely been shown in symptomatic individuals with IgG2 deficiency. On the other hand, the role of IFN-~/ as a switch factor in inducing isotype switching to IgG2a has been well established in the murine system. IFN-~ has been shown to induce C~/2 germ-line transcripts in human peripheral blood ceils, however the role of IFN-~/as a switch factor for IgG2 synthesis is not conclusive in humans. To examine the potential role of IFN-~ in human IgG2 deficiency we studied 13 patients whose ages varied between 2 and 16 years. The patients were divided in 3 groups according to their age because of the gradual maturation of IFN-~/ production with age. PBMCs from patients and age matched controls were stimulated with OKT3, PHA or TSST1 for 48 hours. Our results show no statistical difference in IFN-y production with all 3 stimuli in IgG2 deficient patients and age matched controls. We also evaluated the IFN-y receptor function by stimulating PBMCs with IFN--~ and measuring HLA-DR expression. Normal receptor function was found in IgG2 deficient patients. Finally, we evaluated the possible role of IFN-y as switch factor by examining IgG2 synthesis in CD40 stimulated PBMCs from patients with X-linked hyperIgM syndrome. No IgG2 production was observed whereas lgE synthesis occurred in response to anti-CD40 plus IL-4. This suggests that IFN-y is not a switch factor. Our data suggests that IFN-y is not a switch factor for IgG2 synthesis in humans.

J ALLERGY CLIN IMMUNOL JANUARY 1997

1611

Molecular Analysis of Chronic Granulomatous Disease Caused by Defect of gp91-phox. PJ Pa&io, JE Perez, JA Lopez, JH Botero, A Condino-Neto, D Gate& de O, JT Curnutte. l.aboratory of Immunology, School of Medicine, University of Antioquia, Medellin, Colombia, Department of Immunology, Genentech Inc, So. San Francisco, CA, USA. Chronic granulomatous disease (CGD) is an inherited disorder of phagocytes in which a defective respiratory burst leads to severe recurrent bacterial and fungal infections. CGD is a consequence of a mutation in one of the four molecules that conform the NADPH oxidase system. The critical component of this electron transporting system is a very unusual flavocytochrome b localized in the plasma membrane. Mutations in the gene encoding the [3 subunit of this flavocytochrome (gp9l-phox), localized in the short arm of the X chromosome are responsible for 60% to 65% of all cases of CGD. Here we report the molecular characterization of seven unrelated kindreds with gp91phox deficiency from Colombia and Brazil. In four cases we found nonsense mutations (C271 --~ T, C469 ~ T, C868 --->T and G318 --->A) that predict the conversion of an Arg (157, 244 and 290) or a Trpm 6 to a premature stop codons which predict truncated proteins with variable lenght. Other two patients showed missense mutations (G674 --->A, G731 ---->A; predict Glu225 --> Val and Cys244 ~ Tyr, respectively) which alter the expression or function of gp91-phox. The last patient revealed a substitution of g to a in the penultimate nucleotide of intron 12 which is a 3' splice consensus site. This mutation predicts a defect of pre-mRNA processing that splices out the exon 13 of the mRNA. In six of these kindreds the mothers were carriers. One mother did not present any change in her gp91-phox gen which indicates a de novo mutation. Thus, these family-specific mutations in gp91-phox produce different structural defects that alter the expression or function of an esential component of phagocyte oxidase.

1612

Clq Deficiency Presenting with Escherichia coli Sepsis and Brain Abscess in the Neonatal Period. PC Avila, A Dorenbaum, D Wffliams-Herman, ME Elder, RS Shames, and DW Warn. University of California San Francisco, San Francisco, CA. About 30 cases of Clq deficiency have been described. Patients usually present with SLE during childhood or early adulthood. We describe an 8 day-old Caucasian boy who presented to the ER with sepsis, manifested as tonic-clonic seizures, respiratory distress, marked metabolic acidosis, hypoglycemia, pancytopenia, and pulscless electrical activity needing cardiopulmonary resuscitation. Blood and urine cultures yielded E. coli, and a head MRI disclosed a left thalamic abscess. He received a total of 7 weeks of IV antibiotics and was discharged at 39 days of age. His umbilical cord stump separated at 10 weeks. Follow-up imaging studies showed progressive internal hydrocephalus, and a VP shunt was placed at 5 months of age. He developed cow's milk-induced allergy, but at 13 months of age he was thriving. Family history was unremarkable. Maternal HIV serology was negative. Immune work up demonstrated: eosinophilia (1,800/1~1); normal lymphocyte phenotyping (CD3, CD4, CD8, CD16/56), except for elevated B-cell count (CDI9: 2,439/1~1, normal (nl): 113-775); normal T-cell function (PHA 104,105 cpm, nl >32,000, MLC 58,992 cpm, nl >18,000); normal immunoglobulins at 9 months (lgG 379 mg/dl, IgM 46 mg/dl, IgA 10.5 mg/dl); present isoagglutinin titers (anti-B 1:8 and anti-A 1:32); a protective anti-tetanus toxoid antibody titer (>3.0 U/ml); and normal integrin markers (CD18, CDlla, CDllb). In contrast, the CH50 showed no complement hemolytic activity. Nephelometric studies revealed normal C2, C3, C4 levels; but Clq was low (<3.5 mg/dl, hi: 5 - 8.6). Both parents had normal CH50 and Clq. Conclusion: Early complement component deficiency should be considered in the differential diagnosis of severe neonatal sepsis even if non-encapsulated organisms are involved.

J ALLERGY CLIN IMMUNOL VOLUME 99, NUMBER 1, PART 2

1613

1614

1615

Innate Immunity: Mannose Binding Protein Deficiency Is More Frequent Than lgA Deficiency. S Kagen, R Muthiah, J Slupianek. Kagen Allergy Clinic, Appleton, WI Mannose binding protcin (MBP) is an acute phase reactant produced in the liver which is an important component of human innate immunity. When it is bound to bacterial cell wall carbohydrates, MBP acts as a nonspecific lectin which can activate complcment and facilitate phagocytosis without antibody participation. We studied 106 chronic sinusitis patients for MBP deficiency. Other studies included assays of lgA, IgG, IgM and IgG subclasses. Deficiency in serum MBP was present in 10/106 patients (9.4%). Below normal levels of lgA were found in 4/100 patients (4%); low total IgG in 20/100 paticnts (20%); and low IgM in 1/1()0 patients (1%). Bclow normal lgGl levels were present in 2/71 patients (2.8%)" low IgG2 in 2/71 (2.N%): low lgG3 in g/71 (11.3%); and low IgG4 in 7/71 (1(}%). MBP deficiency is morc common than IgA deficiency. Mannosc binding protein determinations perlk)rmed in patients with chronic sinusitis may assist clinicians in making the diagnosis of MBP deficiency, and in determining which patients may benefit from prophylactic use of antibiotic therapy. Cell surface heparan sulfate mediates anti-HIV-I activity of CC cemokines. T. Oravecz, M. Pal~, J. Wang and M. A. Norcross; FDA/CBER, Bcthesda, MD The chemokine receptor CCR-5 functions as a coreceptor for entry of monocytotropic HIV-1 strains and serves as the target for the antiviral activity of CC chemokines. In addition to specific receptors, chemokines are also known to interact with heparan sulfate (HS) proteoglycans. This study wits designed to investigate the role of HS in the antiviral effects of chemokines and in regulating chemokinc binding to T cells and macrophages. Wc observed that digestion of cell surface HS from PM-1 cDz"u cells with heparitinasc renders the cells resistant to the antiviral activity of RANTES and MIP-113, especially at low chemokine Icvcls. Flow cytometry experiments rcvcaled a two phase binding interaction of RANTES with the cell surface in T-cell lines. Binding sites with high (10-1tl00 ng/ml) and low (1-10 ~g/ml) affinity were detected and heparitinase treatment prevented only high affinity RANTES binding. In contrast, resting or activated monocytes bound RANTES only at high chcmokine levels which was not sensitive to enzyme treatment. Consistent with the binding data, int;zction of macrophages was only partially blocked by high concentrations of RANTES and this inhibition was not prevented by heparitinasc treatment. Our results support a role of HS proteoglycans in facilitating high alfinity intcractions of RANTES will) the cell surface to mediate the anti-HIV-I activity of C(' chemokines. The absence of ItS dependent chemokine binding may explain the resistance of macrophagcs to file antiviral effects of chcmokincs. Cytokine Profile in HIV Long Term Survivor with Hyper IgE and Normal CD4+ T Cell Number. C Serot~9 C White, A Dorenhattm, M Elder; D Warn, UCSF, San Francisco, CA A prominent tinding in HIV infection is immunoglobulin dysregulation. It is widely accepted that progression of HIV infection is correlated with a T helper 2 (Th2) cytokine profile. Many studies have demonstrated increased immunoglobulin lcvels in HIV infection and increasing IgE levels correlate with falling CD4+ T cell numbers. We have been following a long term surviw)r of vertically acquired HIV infection for twelve years with an lgE level of greater than l(t,000 IU/ml. The patient has had recurrent StaphylococCllS ElllFCIIS skin and multi-organ abscesscs. She has not taken antirctroviral therapy for several years and has age appropriate CD4+ T ccll numbers (absolute CD4 1600) and a relatively low H1V viral burden of 30,000 copies/ml. We studied the cytokincs produced by stimulated unseparated PBMC and highly purified T cell subsets in vitro. Similar levels of IFN~, TNFc~, IL2, and ILl0 were observed in PMA + ionomycin stimulated cells from the patient and

Abstracts

S397

HIV infected and uninfected controls. In contrast, production of IL4 and IL5 was increased in the patient's CD4+ T cells compared to control cells (2011 pg/ml vs. 100 pg/ml; 1600 pg/ml vs. 200 pg/ml, respectively). AntiHIV lgE antibodies were not detected using an immunoblot technique. We conclude that despite increased levels of Th2 cytokines and extremely high IgE levels, this patient has maintained normal CD4+ T cell numbers and relatively low viral burden for years. We hypothesize that the Th2 phenotypc may have limited disease progression in this patient. Anti HIV IgE antibodies do not appear to be involved in the control of HIV infection in this patient and work is in progress to further elucidate the presence of protcctivc antibodies.

1616

Encephalitogenic Role of Citrullinated Myelin Basic Protein in EAE. L Cao, Q Hart, JN Whitaker, UAB Medical School, Birmingham, AL The citrullinated myelin basic protein (MBP-Cg) is overexpressed in multiple sclerosis (MS) brain, and T cells recognizing MBP-C8 exist in the blood of MS and normal individuals. To investigate the encephalitogenic role of MBP-C8, Lewis rats wcrc immunized with guinea pig brain MBP-C8 and a non-citrullinated isomer, MBP-C1, in CFA for EAE induction. Comparable to the results with MBPC1, MBP-C8 could induce EAE at a dose as low as 25 p,g/rat. T cell lines derived from the lymph node cells of rats immunized with MBP-Cg, were preferential in their response to MBP-C8 over MBP-CI, recognized the dominant cpitope on MBP pcptide 68-88 and were encephalitogenic in adoptive transfer. There was a minor T cell response to human MBP, in contrast to a high serological response to the human MBP. Unlike MBP-C1, MBP-C8 could reinduct active EAE in 70% of rats which had recovered from MBP-Cl-induced EAE. With reinduction there were mild clinical manifestation and moderate CNS infiltration with some demyelination. These findings imply that the structural features of MBP-C8 may permit additional or altered cpitopes to be expressed that are different from MBP-C1 for encephalitogenicity or immunoregulation. The abnormally abundant MBP-C8 in MS brain may play a role in the chronicity of MS through cpitopc spreading.

1617

Cerebral Vasculitis Associated With Chronic Mucocutaneous Candidiasis. M Grouhi, I Dalal, CM Roifman, Univcrsity of Toronto, Hospital for sick Children, Toronto, Ontario, Canada Chronic Mucocutaneous Candidiasis (CMCC) is a complex disorder that includes chronic or recurrent infections of the skin, nails and mucous membranes with candida albicans. A variety of autoimmune disorders such as endocrinopathies, hematological abnormalities, alopccia, vitiligo, malabsorption syndromes, thymoma and interstitial keratitis may also occur in these patients. Most patients with CMCC lack the ability to develop and express effective cell-mediated immune response against candida, but a subset of them has a more profound cell-mediated and/or humoral immunodeficicncy. We report here, for the first time, two patients with CMCC who experienced ccrehrovascular accident (CVA) at the second decade of life that caused permanent hemiplegia. Angiography in patient 1 demonstrated a diffuse vasculopathy. All four major arteries were involved with multiple fusiform aneurysmal dilatation of numerous branches of different arteries. Brain biopsy confirmed the diagnosis of vasculitis in both small and large vessels. Angiography in patient 2 revealed a severe narrowing of the internal carotid artery with a near total occlusion of the intracercbral branches. These findings are consistent with Moyamoya disease. We conclude that: A) Patients with CMCC are at higher risk to experience CVA. B) In cases of "unexplained" CVA, mainly in the first 3 decades of life a diagnosis of CMCC should be entertained.

$398

Abstracts

J ALLERGYCLIN IMMUNOL JANUARY 1997

1618

"Of mice and men": CD40 ligand is not required for T cell priming in humans. FM Lobo, EA Cooper and RL Fuleihan, Yale University School of Medicine, New Haven, CT. X-linked hyper IgM (HIGMX-1) results from defects in the gene for CD40 ligand (CD40L). Patients suffer from recurrent bacterial and opportunistic infections, autoimmune disease, and lymphoproliferative disease Recent evidence from gene targeted mice demonstrates that CD40L is necessary for T cell priming. We have investigated a 23 year old patient with H1GMX-1 who has been followed at Yale since early childhood. His T cells failed to express CD40L after stimulation. Sequence analysis of his CD40L cDNA showed the deletion of exon 3 which was subsequently found to be due to a point mutation (a ---,g) at the - 2 position in the acceptor site of exon 3. In contrast to CD40L-deficient mice, this patient demonstrated reactivity against recall antigens by T cell proliferation in-vitro and by positive delayed-type hypersensitivity skin testing in-vivo. Furthermore, the patient's circulating T cells contained a primed population of CD4+ T cells as determined by CD45RO expression. Next, we examined T cells from the patient's mother, an obligate carrier of HIGMX-]. Because the defect in CD40L is not lethal to T cells, T cells from HIGMX-1 carriers express either normal or defective CD40L creating a situation in which CD40L-defective T cells compete with normal T cells. Approximately 50% of the CD4+ T cells from the patient's mother expressed normal CD40L upon stimulation. Normal CD40L did not provide an advantage for CD4+ T cell priming, as 47% of the mother's CD4+/CD45RO+ T cells expressed CD40L compared to 80% of control CD4+/CD45RO+ T cells. Taken together, these results suggest that in contrast to the results found in mice, CD40L is not required and provides no advantage for T cell priming in humans.

characterized the effect of a novel cytokine IL-15, which stimulates T and NK cells and binds the IL-2R~/, on toxicity of neonatal mononuclear cells (MNC). MNC were separated from blood of normal adults and newborns (cord blood) and cultured in IL-15 for 1 week. Cytotoxicity assays were performed using K562 and CEM.NKR cells coated with HIV gpl20 antigen. The table illustrates percent lysis determined by 5JCr release at an effector:target ratio of 25:1 using a microplate assay. K562 Cytokines

Cord

None IL-15(t0ng/mL) IL-12(lng/mL) IL-12+IL-15 IL-2(U/mE)

24-+4(20) 74-+2(20)* 70-+3(12)* 42 -+ 6 (16)? 75-+2(21)*

1620

Defective Cell Mediated Immunity in the X-linked Hyper lmmunoglobulin M Syndrome. R Ameratunga 1"2, HM Lederman 1, KE Sullivan 3, HD Ochs4, S Seyama 4, JK French 2, R Prestidge 5, J Marbrook e, WC Fanslow 6 JA Winkelstein 2. Johns Hopkins University, Baltimore, MD t, University of Auckland, New Zealand 2, Childrens Hospital of Philadelphia, PA 3, University of Washington WA 4, Genesis Corporation, Auckland, New Zealand 5, Immunex Corporation WA 6. The X-linked form of the hyper Immunoglobulin M syndrome (XHIM) is a primary immune deficiency disorder characterised by an increased susceptibility to recurrent bacterial infections as well as to Pneumocystis carinii and Cryptosporidium. While the defect in immunoglobulin isotype switching and the susceptibility to bacterial infections can be explained by the defective CD40 ligand function, the basis for their susceptibility to pathogens associated with defects in cellular immunity is unclear. We have examined T cell proliferation in five patients with well characterised XHIM using a panel of soluble antigens (Candida, Tetanus toxoid and Diptheria toxoid) and lectins (PWM, PHA and Con A). All patients had impaired proliferative responses to to one or more antigens. In contrast, their lee?in responses were normal. Three of the five XHIM patients received booster immunizations (two received dT and one received TI'); after the booster immunizations the cells of two patients still failed to proliferate in vitro. Thus, in addition to severely depressed antibody responses, patients with XHIM have a measurable T cell defect which may explain their susceptibility to pathogens such as Pneumocystis carinii and to Cryptosporidium. Interleukin (IL)-15 is a novel cytokine that induces lymphokine-activated killer (LAK) activity in neonatal cells. QH Nguyen, RL Roberts, BJ Ank, SJ Lin* CK Lau, VS Chhabra, EK Thomas,f. and ER Stiehrn. Depts of Pediatrics, UCLA Medical Center, Los Angeles, CA and *Chang Gung Children's Hospital, Linkou, Taiwan and :[:Immunex Corp, Seattle, WA. Newborn infants are more susceptible to infections due in part to defective natural killer (NK) function. We

CEM.NKR Cord Adult

35-+6(11) 5.9 -+ 2(15) 72~2(11)* 46-+3(23)* 62-+5(5)* 28-+6(11)* 53 -+ 4 (5)? 13 -+ 3 (9)* 65-+4(8)* 38-+4(22)*

12-+3(11) 38-+4(11)* 22-+3(5)* 28 -+ 5 (5)? 31-+5(8)*

Data are mean _+ SE (n); ?p < 0.05, *p < 0.01 Without cytokines, cord cytotoxicity was less than adult, but IL-15 augmented cord MNC to exceed adults. IL-15 increased cord activity 3-fold over that of cells without cytokines in K562 assays and 5- to 8-fold in CEM.NKR assays. IL-12 also enhanced both cord and adult cytotoxicity, but the combination of IL-12 + IL-15 did not increase activity as with either cytokine alone. IL-15 did slightly increase antibody-dependent cytotoxicity (in presence of anti-HIV antibody). Thus, IL-15 can potentially augment neonatal antiviral defenses.

1621 1619

Adult

Cytokine-inducing activity of Yersinia pestis. GI Vasilieva, LM Verkina, IA Ivanova, EM Doroshenko, SU Tyukavkina, Research Institute for Plague Control, Rostov-on-Don, Russia The objective of this study was to estimate the ability of Yersinia pestis EV to induce the production of cytokines that mediate macrophage-neutrophil cooperation. Monokines produced by intact and immune peritoneal macrophages and by the human U 937 monocyte-like cell line as well as neutrophilokines synthesized by intact and immune peritoneal neutrophiles were obtained. The influence of the obtained monokines on neutrophil killing and chemotactic activities, expression of Fc receptors and quantity of lysosomes in neutrophiles was studied. In addition the effect of investigated neutrophilokines on differentiation of monocytes to macrophages, their killing and chemotactic activities, and distribution of macrophage subpopulations in the total pool of these cells was investigated. The results have shown that the obtained monokines essentially enhance neutrophil functional activity were revealed. Our experiments also demonstrate that the investigated neutrophilokines promote the differentiation of monocytes o macrophages, enhance the killing and chemotactic activities and induce the redistribution of the macrophage subpopulation in the total pool.

J ALLERGYCLIN IMMUNOL

Abstracts

S399

VOLUME 99, NUMBER 1, PART 2

1622

1623

1624

Alterations of T Cell Activation and Cytokine Expression in IDDM. HN Bevhum, S Azar, and WY Almawi. Dept. Biochemistry and Internal Medicine, Faculty of Medicine, American University of Beirut, Beirut, Lebanon. Alterations in T cell activation and cytokine expression characterize many autoimmune disorders, including insulin-dependent diabetes mellitus (IDDM). The purpose of this study was to assess T cell activation & cytokine secretion in IDDM patients during the acute and chronic phase of IDDM. T cell proliferation was assessed by measuring the cellular uptake of tritiatcd thymidine, and cytokine steady-state mRNA expression was determined by RT-PCR. While T cell proliferation stimulated by mitogens, anti-CD3 + anti-CD28 mAbs, or PMA + ionomycin was significantly enhanced in chronic IDDM patients, it was reduced in acute patients. These changes in T cell proliferation were parallelled with changes in Thl (IL-2, IFN- 7, and IL-12) and Th2 (IL-4 and IL-10) cytokine mRNA expression. IDDM-induced alterations in T cell activity did not correlate with blood glucose levels, daily insulin doses, age, or sex. In essence, our results underscore the differential regulation of T cell activation in IDDM, and suggest novel immunotherapeutic approaches to manage IDDM. Pretreatment with Glucocorticoids Enhance T-Cell Activation and Cytokine Expression. WY AlmawL NA Jaff'al. Dept. Biochemistry, Faculty of Medicine, American University of Beirut, Beirut, Lebanon. Insofar as glucocorticoids (GCS) inhibit cytokine expression, we tested the effect of GCS pretreatment on cytokine/ cytokine receptor expression and on T cell proliferation. Whereas dexamethasone (DEX) and prednisonc (PRED) inhibited IL-2, IL-7, IFN-'y & IL-12 mRNA expression and T cell proliferation, pretreatment of activated T cells with DEX/PRED resulted in enhanced IL-2, IL-7, IFN-'y, and IL-12 mRNA expression upon reactivation. These changes in mRNA were paralleled by increases in IL-1R, IL-6R, & IFN-',/R expression, and augmentation of T cell proliferation. This GCS-induced rebound was specific for GCS, and was blocked by the GCS receptor antagonist, RU-486. Furthermore, GCS-induced rebound was not stimulusspecific, and was not associated with alteration in apoptosis. Our results underline the dual effects of GCS in regulating T cell activation and cytokine expression: GCS directly inhibit T cell proliferation by inhibiting cytokine expression and by enhancing cytokine receptor expression, pretreatment with GCS augments T cell proliferation. RANTES and IL-5 have different kinetic release into asthmatic airways following endobronchial allergen challenge. Teran LM, Shutte JK and ST Holgate Southampton, UK. RANTES has recently been identified as a major eosinophil chemoattractant in the broncho-alveolar lavage (BAL) fluid of asthmatic subjects (Teran et al, J Immunol 1996,157:1806-812). However, the kinetics release of this chemokine as well as that of IL-5 remains to be shown. We have studied eosinophil recruitment and its relationship to RANTES and IL-5 release into asthmatic airways following allergen challenge. 12 asthmatics underwent endobronchial allergen challenge and BAL fluid obtained 4 h (n = 6) or 24 h (n = 6) after challenge. Analysis of BAL fluid showed that eosinophil numbers obtain 4 h and 24 h after allergenchallenge were 6 and 32 times higher than numbers after saline-challenge. 4 h after challenge levels of RANTES and IL-5 were significantly increased in fluid obtained from the allergen-challenge site when compared to the saline-control (median: 135 pg/ml vs 30.5 pg/ml, p < 0.02, and 2.67 vs 0.75 pg/ml, p < 0.05, respectively [RANTES levels were 50 times higher than IL-5 concentrations]). At 24 h, there was not statistical difference in levels of RANTES at the two sites (18.5 vs 15.7). In contrast, IL-5 levels in the allergen lavage were higher than in the saline (31.1 vs 1.57 pg/ml, p > 0.02). There was a significant correlation between eosinophil numbers and RANTES concentrations, 4 h after exposure. IL-5 did not correlate with eosinophits at any time point. These findings suggest that RANTES may

regulate eosinophil recruitment during the early phase of the allergic asthmatic reaction (at 4h h) while IL-5 may predominate during the late phase of the late asthmatic reaction (at 24h).

1625

A Phase I Dose-Escalation Study of Polyclonal CD4 T Cell Ex Vivo Expansion for Immune System Restoration of HIV Infection. BL Levine, J Cotte, RG Carroll, JL Riley, CS Small, TN Francomano, OS Weislow, DC St. Louis, W Bernstein, CH June Henry M. Jackson Foundation and Naval Medical Research Institute, Bethesda, MD, and SRA Life Sciences Division, Rockville, MD HIV disease progression correlates with the loss of CD4+ T cells. Treatment strategies involving maintaining or increasing the CD4 lymphocyte levels could have beneficial effects on disease progression. This protocol involves infusion of autologous, polyclonal CD4 lymphocytes in an effort to reconstitute the immune system of infected individuals. Previously, we have successfully expanded CD4+ T cells from 10 patients in the absence of anti-retroviral drugs with a dramatic decrease in viral burden (Science 272:19391943). This occurs through an anti-HIV effect mediated by CD28 stimulation, resulting in an increase in chemokine production and a down regulation of the chemokine receptor CCR5. CD4+ T cells are purified by negative selection of a pheresis product and stimulated with immobilized anti-CD3 plus anti-CD28 mAbs. Grown in this manner, CD4+ T cells will proliferate with a population doubling time of -40hrs. No clinical toxicity has been observed following infusions of 2 • 10'~ and 11 x 10'~ cells that were >98% CD4+. Plasma viral load and long-term CD4 counts are under evaluation. Interim results of this ongoing pilot clinical study will be presented. Following the establishment of the safety and feasibility of escalating doses of CD4+ T cells, this approach may be combined with the infnsion of antigen-specific or gene-modified cells.

1626

Identification and Purification of a Novel Superantigen from Mycobacterium Tuberculosis. C.K. Doyle and R.R. Rich. Department of Microbiology and Immunology, Baylor College of Medicine, Houston, TX. Due to the well documented profound T cell response to antigens from Mvcobacterium tuberculosis (MTB) as well as a reported V[~ skewing viewed in human tuberculosis patients, this bacterium was tested for the production of a novel superantigen (SAg). Heat killed and dessicated MTB of the strain H37Ra were utilized to make cell extracts to test for SAg activity. Using the human EBV transformed B cell line Raji and the MHC class II-deficient mutant thereof, a titratable, class II-dependent T cell proliferation was demonstrated in response to a factor(s) present in the soluble fraction of the cell extracts. T cell proliferation assays on the cytoplasmic, cell wall, and culture filtrate fractions of the MTB strain H37Rv revealed that the SAg is likely derived from the cell wall and secreted into the culture. Analyses of T cells from multiple donors using mAbs demonstrated an increase in the expression of Vl33, 5.1, 8, 12, and 21 T cell subsets. The crude MTB cell extracts were applied to a gel filtration column and subsequently an anion exchange affinity matrix column and eluted, partially purifying the SAg activity. A novel SAg in MTB has thus been identified and partially purified. Further biochemical characterization and functional analysis of the MTB SAg will aid in gaining more knowledge of SAg for a better understanding of why these proteins are expressed and have been retained through evolution in such a diverse array of microorganisms.

S400

1627

Abstracts

Grass pollen immunotherapy: A 3 year double-blind placebo-controlled withdrawal study. SM Walker, MR Jacobson, SR Durham Imperial College School of Medicine at the National Heart and Lung Institute, London, UK. We previously showed that in 40 adults with severe summer hayfever, grass pollen immunotherapy (Alutard SQ, ALK-AbelIo, Denmark) was effective (Varney V, BMJ 1991;302:265-269) and efficacy was maintained for a further 3 years (1990-1992) with continued treatment (Walker Set at, Allergy 1995;50:405-413). 32 remaining patients were then randomised for a further 3 years (1993-1995) to receive monthly treatment with either Alutard SQ or matched placebo (histamine containing) injections. Results were compared with untreated (immunotherapy-naive) hayfever sufferers. There was a highly significant decrease in seasonal symptoms (p=0.001), rescue medication (p=0.007) and visual analogue scores (p=0.002) in favour of both immunotherapy treated groups compared to the untreated group. Median scores for the immunotherapy treated groups were virtually identical whether or not immunotherapy had been discontinued over the previous 3 years. Maintained clinical improvement for both groups was accompanied by a persistent decrease in immediate conjunctival sensitivity to grass pollen (both p=<0.02). The immediate skin test response to allergen paralleled the clinical data. Weal sizes remained small throughout the study period showing no statistical difference between the active and placebo treated groups (p = 1.0), but a significant difference (p=0.001) from the untreated rhinitic group. The late cutaneous response to intradermal allergen (LPR) at 24hrs decreased in size down to zero by 1993 and remained low in both groups compared to the rhinitic control group who showed large LPRs throughout the study (p=0.0001). We conclude that in selected patients with severe hayfever grass pollen immunotherapy is effective. Efficacy is well-maintained 3 years following discontinuation of treatment.

J ALLERGYCLINIMMUNOL JANUARY1997

amino acid sequence. We investigated the T cell response to Der p 2 using recombinant Der p 2 (rDer p 2) variants with reduced reactivity for IgE Ab (Mol lmmunol 33:399. 1996). The substitution of Arg for Cys at position 73 (Cys73Arg) reduced in vitro IgE Ab reactivity by 60% to 85%, and in vivo, Cys73Arg required 10 to > 100 -fold more antigen to give a comparable skin test response to Der p 2. T cell proliferative responses to D. pteronyssinus extract (D.pt), purified Der p 2, rDer p 2, and Cys73Arg were measured using PBMC from 6 D.pt. skin test (+) patients and 6 skin test ( - ) subjects. All patients had a positive proliferative response to D.pt., with stimulation indices (SI) of 6-16. Four of six patients responded to the purified allergens: Der p 2 (SI: 6-22.5), rDer p 2 (SI: 3-10.5), Cys73Arg (SI: 3-16.3). Among the skin test ( - ) subjects, 1/6 showed proliferative responses to D.pt. and purified allergens. All individuals tested had a strong proliferative response to PHA and tetanus toxoid (SI: 6-34). No correlation was seen between IgE Ab titers to D.pt. or Der p 2, or disease state (asthma or allergic rhinitis), and the magnitude of the T cell response. These results suggest that the Arg substitution, while markedly reducing IgE Ab reactivity, does not alter a dominant T cell epitope. Modified rDer p 2, such as Cys73Arg and other site specific variants, could provide an alternative immunogen for allergen immunotherapy. These well defined variants retain the full complement of T cell epitopes and the reduced Ab reactivity would decrease the risk of anaphylaxis. This strategy could potentially be applied to any cloned allergen.

1630

Clinical and Immunological Effects of a Long-term Sublingual-oral Immunotherapy to Mite: A Double Blind Study. G. Passalacqua, M Albano, P Falagiani* AM

Riccio, C Pronzato, N Fiorino, S Ruffoni, GW Canonica. 1628

Ultra-rush Bee Venom Immunotherapy Results in Decrease of Histamine Receptor Expression on Peripheral Blood CD4+ Lymphocytes. M Jutel, T Zak-Nejmark, M Wrzyszcz, J Malolepszy. Wroclaw School of Medicine, Wroclaw, Poland A cumulative dose of bee venom (BV) exceeding the quantity contained in one bee sting is administered during 3.5 hours of ultra-rush bee venom immunotherapy (VIT). The mechanisms of rapid clinical tolerance induced by VIT are largely unknown. We investigated whether VIT is accompanied by long-term down-regulation of histamine receptors on peripheral blood CD4+ lymphocytes. Longterm (several hours) down-regulation is characterized by loss of total receptor number and recovery which requires de novo protein synthesis. Eight bee venom allergic patients with a history of severe systemic reactions to bee stings were investigated. A cumulative dose of 111.1 Ixg BV was administered s.c. over 3.5 hours under intensive care conditions according to an ultra-rush protocol. Blood samples were obtained just before the initiation of VIT and 30 min. after the last injection on the same day. Percentage of CD4+ lymphocytes binding histamine fluorescein was determined in unstimulated as well as specific allergen (phospholipase A:-PLA) stimulated ceil samples using fluorescence microscope. In all subjects we observed significant reduction of histamine receptor expression on unstimulated as well as PLA stimulated cells (p < 0.001). Interestingly, histamine receptor expression after VIT was similar to the values observed before VIT in the presence of HI-blocker clemastine. We conclude, that CD4+ lymphocytes are a target for VIT, which induces long-term downregulation of histamine receptors and possibly other G protein-coupled receptors.

1629

rDer p 2 variants with decreased IgE Ab reactivity retain T cell epitopes. A M Smith, EA Taketomi, SSJ Sung, TAE Platts-Mills, MD Chapman, Charlottesville, VA. USA. Der p 2, a major dust mite allergen, stimulates Ab and T cell responses in the majority of mite allergic individuals. T cell epitopes have been shown to span the entire primary

Allergy and Clinical Immunology Service, Genoa University and *Laboratorio Farmaceutico Lofarma, Milan. ITALY The present study investigated the clinical and immunological effects of an oral immunotherapy to dust mite in a long-term study. Twenty patients suffering from rhinoconjunctivitis and/or mild intermittent asthma with single senzitization to mite were enrolled. Both symptom/medication scores (diary card) and nasal allergic inflammation (by means of allergen specific nasal challenge) were evaluated. After a 3-month run in period, during which clinical and immunological parameters were assessed, the patients were randomly allocated to two groups receiving either placebo or active treatment (LAIS, Laboratorio Farmaceutico Lofarma, Milan), prepared as tablets to be dissolved sublingually and swallowed. The study was prolonged up to 2 years in double blind fashion. Nineteen patients completed the study and no relevant side effect was recorded. The actively treated group showed a significant clinical improvement (run-in vs 1st y: p = .004; run-in vs 2nd y: p = .008; 1st y vs 2nd y: p = .01); the improvement was also significant vs the placebo group (1st y: p = .05; 2nd y: p = .01). Concerning the specific nasal challenge, a significant reduction of the celrular infiltration (eosinophils arid neutrophils) and [CAM-1 expression on nasal epithelium was observed in the active group after 12 months of treatment (p < .05 for cells and ICAM-1 expression). Thus, the investigated immunotherapy appeared clinically effective and able to reduce the allergic inflammation.