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Visceral leishmaniasis in Somalia. Significance of IgG subclasses and of IgE response
Immunology
Visceral leishmaniasis
Letters
50 (1996) 87-93
in Somalia. Significance of IgG subclasses and of IgE response
S.A. Shiddoa.b.*, G. Huldtb, L-A. Nilsson”, 0. Ouchterlony”, R. Thorstenssonb “DiLlisionof’ Parasitology. Department oJ‘ Morpho1og.v and Pathologv, Faculiy of‘ Medicine and Surgery, Somalr .Varional l,+lic~ersit~*. Mogadishu, Somalia bSwedish Institute ,ftir Injtictious Disease Control. Stockholm, Sweden ‘Department of’ Medical Microbiology and Immunologic. University qf Gothenburg, Gothenburg, .S~den Received
28 September
1995; revised
15 February
1996; accepted
26 February
1996
Abstract We have determined with clinical
visceral
the levels of IgG subclasses and IgE as well as specific antibodies of these isotypes in sera from 22 patients leishmaniasis (VL) from Somalia. The results are compared with those obtained from 30 Somali and 23
Swedish controls. We found markedly increased concentrations of IgGl in the VL sera, indicating that the pronounced increase in IgG in VL which is generally considered to be due to polyclonal B-cell activation is mainly restricted to this subclass. The IgG2 concentrations were significantly decreased. The IgG3 and IgG4 concentrations, on the other hand, did not differ between the two groups of Somali sera. The Somali control sera contained higher concentrations of IgGl and IgG3. but significantly lower concentrations of IgG2 as compared to Swedish controls. The IgG4 values, on the other hand, were not different between the two groups of control sera. Anti-leishmania antibodies belonging to all IgG subclasses, were found in the patients’ sera. There was no significant difference in total IgE between sera from VL patients and controls and specific IgE antibodies were only detected in a few patients. The Western blot assay (WB), revealed the presence of two bands corresponding to 74 kDa and 88 kDa in all patients’ sera, indicating a possible diagnostic role for WB in this particular population. Keywords: IgG subclasses;
IgE; Polyclonal
B-cell activation;
VL; Somalia
1. Introduction Visceral leishmaniasis (VL) is a systemic infection, caused by an intracellular protozoan parasite belonging to the L. donouuni complex. The parasite multiplies in macrophages preferentially in spleen, liver, lymph nodes and bone marrow, causing profound immune reactions [l]. Active stages of the disease are accompanied by high titre circulating antibodies and also by a depression both of leishmania-specific and of unrelated cell-mediated immunity [l-3]. In connection with recovery, the antibody response slowly decreases and the leishmanin test becomes positive [3]. This pattern of development of humoral and cell-mediated immunity is compatible with cytokine-mediated immune regulation
* Corresponding author. Department stitute for Infectious Disease Control.
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0 1996 Elsevier
PII SO165-‘478(96)02529-l
of Immunology, Swedish InS-105 21 Stockholm. Sweden.
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associated with activation of the murine CD4 + lymphocyte subsets Thl and Th2 [4]. Evidence has been presented to show similar mechanisms in humans [5]. An association between Thl or Th2 response and the predominant isotypes of specific antibodies during the course of infection, including VL. has been reported in studies in mice [6]. Elevated concentrations of interleukin 4 (IL-4), the main cytokine involved in Th2 activation, together with low concentration of interferon gamma (IFN-y) has been demonstrated in sera from patients with clinical VL [7]. Furthermore, it has been shown in vitro that Th2 activation results in predominantly IgG4 and IgE production [8]. We have previously studied VL in an endemic village in Somalia and in hospital patients in Mogadishu [9111. In the present study, we have investigated IgG and IgE immune responses in patients with active VL, with the aim of obtaining a better understanding of the interaction between humoral and cell-mediated immunity in this infection.
88
S.A. Shiddo et al. 1 Immunology Letters 50 (1996) 87-93
In addition to Somali controls, sera from healthy Swedes were analysed. These sera had previously been tested for total IgG, IgM and IgA [12]. The results in the present study, therefore, supplement the data on reference ranges of various immunoglobulin classes in Somalia.
2. Materials and methods
2.1. Seru Sera were obtained from 22 patients with VL, treated at hospitals in Mogadishu during the years 198991990 [9,11]. All sera had anti-leishmania antibodies at high titres and all patients but one were leishmanin negative. Twelve of the cases were parasitologically confirmed. Sera were taken before initiation of specific treatment. This group of patients has been described in a previous paper [ 111. Control sera were from 30 healthy residents of Mogadishu which is non-endemic for VL, and from 23 healthy Swedes [l 11. All the Somali control sera were negative in direct agglutination test (DAT), enzymelinked immunosorbent assay (ELISA) and immunofluorescence test (IF) against L. donouuni antigen. 2.2. Collection
and handling of sera
Blood samples were taken by venepuncture. After centrifugation, sera were kept at - 20°C until transported to Sweden, where they were stored at - 20°C. The Swedish sera were also kept at - 20°C. 2.3. Determination
of total and specljic IgGl-4
Total and leishmania-specific IgG have been determined previously [11,12]. For determination of the concentration of IgG subclasses, the single radial immunodiffusion (SRID) technique as described by Mancini et al. [13] was used. Calibrators for subclass quantification were obtained from Binding Site Ltd. (Birmingham, England). IgGl, 2, 3 and 4 antibodies against L. donovani were determined by ELISA using an antigen, prepared as follows. Stationary phase promastigotes of L. donooani, grown in RPM1 1640 (Gibco, USA) with 10% foetal calf serum, were disintegrated using a Branson sonifier cell disrupter (Branson Co, USA) at medium amplitude for 3 min in an ice bath. Extraction was performed overnight at + 4°C. The extract was centrifuged at 10000 x g for 30 min and the supernatant was used as antigen [l 11.We have previously shown that similar results were obtained in ELISA with this antigen and an antigenic preparation containing a suspension of whole parasites. The test was performed as described earlier [I l] with the follow-
ing modifications. Sera were tested in dilution l/1000. Monoclonal anti-human subclass antibodies, (Mabs) (anti-IgGl and anti-IgG2 from Binding Site Ltd, England and anti-IgG3 and anti-IgG4 from OXOID, BIOZAC, Jlrfalla, Sweden) were used at dilutions 1500 for IgGl and IgG2, 1: 1000 for IgG3 and 1: 10 000 for IgG4, respectively. The antigen-antibody reactions were visualized by means of a biotin-avidin system (biotinylated rabbit anti-mouse and peroxidase-conjugated avidin; DAK0 A/S, Glostrup, Denmark) according to the instructions of the manufacturer. o-Phenylenediamine/H,O, was used as substrate (T Cell Diagnostics, Cambridge, MA, USA). Incubation was performed for 15 min and the plates were read at 492 nm in a Multiscan spectrophotometer. 2.4. Western blot (WB) The antigen used for WB was a soluble extract of L. donozlani promastigotes, prepared as described above for ELISA determination. Equal amounts (v/v) of the antigen and of sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) sample buffer containing 2% SDS and 1% dithiothreitol were boiled for 5 min in a water bath. Parasite proteins were separated by gel electrophoresis on a 12% polyacrylamide resolving slab gel in the presence of sodium dodecyl sulphate according to Laemmli [14]. Approximately 200 pg of protein was loaded on the gel. Separated polypeptides were transferred to nitrocellulose (0.22 ,u, Sartorius produkter AB, Sundbyberg, Sweden) in a transfer blot cell (Bio Rad AB, Solna, Sweden) containing 20% methanol in 0.025 M Tris/O. 192 M glycine buffer and by using 100 V for 2 h. The nitrocellulose with the separated polypeptide bands was cut into 4 mm strips, each containing approximately 15 ,ug protein and blocked for 2 h at room temperature with 5% skimmed milk in PBS by gentle rocking in a rotator. Each strip was incubated for 2 h at 37°C with test serum diluted 1: 100 in the blocking solution. The strips were then washed with PBS 4 x 15 min and incubated overnight at room temperature with horseradish peroxidase-labelled rabbit anti-human IgG (Dako A/S) diluted 1:lOOO in 4% BSA. For WB analyses of IgG subclasses, the same Mabs and serum dilutions were used as for ELISA. Antibody incubation was performed for 2 h at 37°C. Strips were then washed and incubated with a 1: 10 000 dilution of peroxidase-conjugated rabbit anti-mouse immunoglobulins (DAK0 A/ S) for 1 h at room temperature. After further washing, colour reactions were developed by exposure to a solution of o-dianisidine (Sigma) (0.25 mg in 100 ml TrisHCl, pH 7.6 supplemented with 0.3 ~1 of 30% H,O,/ml buffer). The reaction was stopped by rinsing with distilled water when colour started to appear. Strips were dried and stored in the dark. Determinations of relative
S.A. Shiddo et al.
: Immunology
Lrttrrs
89
50 (1996) 87-93
.
811
. .. .
ir;n
IgG2
IgG 3
IgG 4
Fig. 1. IgG subclass levels in sera from healthy Swedes (open symbols), healthy Somalis (open bold symbols) and VL patients horizontal bars indicate the medians values.Significance levels between sera from healthy Somalis and VL patients: l&l. I’ 0.01; IgG3, not significant (ns); IgG4, ns. Significance levels between sera from healthy Swedes and healthy Somalis: IgGl. = 0.02: IgG3. P-c 0.001; IgG4, ns.
molecular weights were extrapolated, using low molecular weight calibration standards (Pharmacia, Uppsala, Sweden) 2.5. Determination leishmania-spec$c
of total serum IgE and IgE antibodies
Total serum IgE was determined by the Pharmacia CAP System, IgE FEIA (fluorescence enzyme immunoassay, Pharmacia Diagnostics AB, Uppsala, Sweden) according to the manufacturer’s instruction. Specific IgE was determined by the Pharmacia CAP system RAST FEIA (15) using ImmunoCAP, covalently coupled with a soluble extract of L. donouani promastigotes. The coupling was performed at different antigen concentrations, 10 pgg/ml and 100 p g/ml. Fifty p 1 of undiluted sera were used for the IgE determinations. The test results were obtained by comparing with standard curves. Values of 2 100 kU IgE/l and 2 0.35 kU IgE/l were considered as positive for total IgE and specific IgE, respectively [15]. 2.6. Statistical
analysis
(solid symbols).
The
< 0.001; IgG2, P = P -I 0.001: IgG2, P
patients and from Somali and Swedish controls are demonstrated in Fig. 1. The most obvious feature in the VL sera is that compared with Somali controls. there is a higher level of IgGl and a lower level of IgG2. This inverse relationship was statistically significant (P = 0.05). No difference in lgG3 or in IgG4 levels was demonstrated between sera from VL patients and Somali controls. It should be noted that significant differences in IgGl, IgG2 and lgG3 were also recorded between Swedish and Somali control sera. Compared with Swedish sera. the Somali control sera contained significantly higher levels of IgGl and IgG3 and lower levels of IgG2. The leishmania-specific antibody response of the various subclasses is demonstrated in Fig. 2. All sera from VL patients showed higher OD values of IgGl and IgG4 than any of the control sera and the majority of the VL sera also had higher values of IgG3. None of the VL sera showed IgG2 reactivity above the levels of Somali control sera. However, the median IgG2 antibody reactivity of VL patients was significantly higher than that of the control sera.
The median test and the Chi square test were used for statistical evaluations and for significance testing. 3.2. Western 3. Results 3.1. IgGl - 4 concentrations in sera from health-y Swedes, healthy Somalis and patients with VL The subclass
distribution
of IgG
in sera from
VL
blot
Sera from 12 patients with VL. from eight Somali controls and from six Swedish controls were tested in WB. The results obtained in five of the VL patients and two of the controls are shown in Fig. 3a. The immunoblot revealed 10 to 15 antigenic bands when tested for IgG antibodies. The most distinct bands corre-
S.A. Shiddo et al. / Immunology Letters 50 (1996) 87-93
90
. . . .
IgGl
lgC2
IgC3
lgG4
Fig. 2. IgG subclass antibodies against L. donouaniin sera from VL patients (solid symbols) and from healthy Somali controls (open symbols). The horizontal bars indicate the median values. Significance levels between sera from healthy Somalis and VL patients: IgGl, P < 0.001; IgG2, P I 0.01; IgG3. P I 0.001; IgG4, P 2 0.001
sponded to molecular weights of approximately 88, 74. 55, 42, 39, 27 and 24 kDa, while most of the remaining bands could not be distinctly visualised by all patient sera (lanes l-5). On the IgG subclass specific WB (Fig. 3b), almost all distinct bands were recognized by IgGl (lanes l-4) and IgG3 antibodies (lanes 9-12), while IgG2 (lanes 5-8) and IgG4 (lanes 13- 16) antibodies were either unreactive or were only weakly reactive with most of the bands. All patients sera tested reacted with two bands, corresponding to the molecular weights of 88 and 74 kDa, respectively. While sera from healthy Swedes did not react with any bands (Fig. 3a, lane 6), six of eight sera from healthy Somalis showed weak reactivity with bands corresponding to 24 and 42 kDa (Fig. 3a, lane 7).
3.3. Total and speciJic IgE concentrations Fig. 4a shows total IgE concentrations in sera from VL patients and healthy Somali controls. In both groups the same number of sera had total IgE concentrations above the cut off level. The median serum concentrations of IgE was slightly higher in the control group as compared to VL patients. Leishmania-specific IgE antibodies were detected in only four of 20 sera from VL patients (Fig. 4b). All sera were reactive at low levels and one of them was borderline reactive. In the control group one serum was borderline reactive. The reactivity was similar whether
10 or 100 pg/ml ImmunoCAP.
antigen
was used for coupling
to the
4. Discussion We have found markedly increased concentrations of IgGl, reaching values above 80 g/l in VL patients. This indicates that the major part of the polyclonal activation, typical of VL, falls within this subclass. We also found a significant decrease in levels of IgG2. This inverse relationship is in agreement with findings from Brazilian [7] as well as from Sudanese patients with VL
P61. By means of ELISA, we demonstrated anti-leishmania antibodies of the IgGl and IgG4 subclasses in all of the VL patients’ sera tested, and of the IgG3 subclass in almost all cases. In IgG2-specific ELISA, the antibody reactivity of patient sera did not exceed the highest OD values found in Somali controls. The median antibody concentration was, however, significantly higher in the sera from VL patients. In one group of Sudanese patients, appreciable amounts of antibodies were found only within the IgGl and IgG3 subclasses [16], while in another report from the Sudan, specific antibodies were found in all four subclasses [17]. In another study from Venezuela, patients with active kala-azar had a predominant IgGl response to a L. chagasi antigen while the levels of IgG4 were low [18]. Our study shows that there was a significant correlation (P < 0.001) between
S.A. Shiddo et al.
/ Immunology
Letters
91
50 (1996) 87-9.1
(b) 5
6
7
Fig. 3. (a) Western blot analysis of sera from patients with VL (lanes with VL. for IgGl (lanes I -4), IgG2 (lanes 5-Q lgG3 (lanes 9-12)
total and specific IgGI values in the VL patients’ sera. In the other three subclasses, however, such a correlation was not observed, since high levels of anti-leishmania antibodies were detected without corresponding elevation in total concentrations. We could demonstrate only a weak leishmania-specific IgE response. The total IgE concentration was similar in sera from VL patients and Somali controls and remarkably low for a population in a developing country. Hagi et al. [19] found much higher values in another Somali village where, however, schistosomiasis was endemic. In our study, only five of the 18 patients came from areas endemic for Schistosoma huematobiunz. Three of the five had total IgE 2 2000 kUj1. Although several intestinal parasites are common in Somalia and a few cases of Bancroftian filariasis have reported to the hospitals of Mogadishu, schistosomiasis seems to be the most important cause of increased level
8
9 10 11 12
I-5) and controls and IgG4 (lanes
13 14 15 16
(lanes 6- 7). (b) Western 13-16) antibodies.
blot analg’si~ of bera from patients
of total and specific IgE there. In Southern Somalia, schistosomiasis exists only along the two perennial rivers, the Juba and the Shabelle [20]. On the other hand, VL is the infection in Somalia which is accompanied by the highest levels of IgG and IgM. Malaria, which also can give rise to polyclonal production of 1gG and IgM, exists only in a hypo- or-mesoendemic pattern and we have found only moderately high IgG level and no increase in IgM in sera from Somali patients with malaria [12]. African trypanosomiasis which is often accompanied by polyclonal B ccl1 activation is not endemic in Somalia and at the time of this study I-IIV infections were very scarce [21]. All scra from VL patients and controls used in this study were negative for HIV [lo]. When investigating the total concentration of the various IgG subclasses, both Somali and Swedish controls were included. A comparison between these two
92
S.A. Shiddo et al. I Immunology Letters 50 (1996) 87-93
kUL
I
1.0
0.8
. x
.
.
b 0.4 m-
-=-
_
e
-
-
Fig. 4. (a) Total IgE in VL patients (solid symbols) and healthy Somalis (open symbols). The horizontal bars indicate the median values and the dashed line the cut-off values. (b) IgE antibodies in sera from VL patients (solid symbols), healthy Somalis (open symbols) and from healthy Swedes (gray symbols) as determined by ImmunoCAP RAST FEIA coupled with 10 pg/ml of L. donovani antigen. The dashed line represents the cut-off value and the horizontal bars the median values of the groups
groups of sera showed an inverse relationship between the concentrations of IgGl and IgG2, similar to the differences between VL sera and Somali controls. This phenomenon, therefore, may be due to factors associated with the conditions of poor populations living in a tropical environment rather than specifically due to the VL infection. The difference between Swedish and Somali controls also emphasizes the importance of using local controls. If only Swedish controls had been used, such a comparison would have resulted in misinterpretation of the levels of IgG3 (Fig. 1). The WB analyses showed that antibodies of two specificities corresponding to 74 kDa and 88 kDa could be detected in all VL sera tested. This indicates a possible diagnostic role for WB in this particular population. In a study from India, Arora and Sehgal [22] found a prominent band of L. donovani promastigotes in the region of 65-66 kDa while Mary et al. [23] identified in L. infantum, two bands of 14 kDa and 16 kDa molecular weight which could be recognized by all VL sera tested, including sera from AIDS patients with proven leishmaniasis. The WB analyses of our study revealed the same two distinct bands of 74 and 88 kDa in all sera tested for all IgG subclasses as were seen in total IgG. The marked antibody production and depressed cellmediated immunity, seen in active cases of VL, is compatible with an activation of the Th2 type immune response. Furthermore, Zwingenberger et al. [7] could demonstrate endogenous production of IL-4 in all of 18 VL patients examined, whereas low levels of IFN-1, was
detected in only two of the VL patients studied. This supports the hypothesis that active VL is associated with Th2 activation. According to findings in both in vitro and in animal studies, this would lead to changes in the normal IgG subclass pattern and to increased production of IgE [8,24]. The results of our study with elevation of total and specific IgGl but lack of polyclonal activation of IgG3, IgG4 and IgE indicate that in humans, other factors than those operating in the experimental mouse systems are of importance for the regulation of the immune response in VL. For instance, the IgG4 response has been shown to be largely dependent on the length of the immunization period [25]. Of the 22 VL patients in our material, 13 had been ill for more than 1 year and this may explain the marked IgG4 response in the Somali VL patients. We have no explanation for the weak IgE response observed in our patients, but with the exception of a recent report of increased levels of IgE in malaria [26], protozoan infections generally are accompanied by only moderate IgE responses. For instance, toxoplasmosis which like VL is an intracellular protozoan infection, accompanied in its early stage by high level of antibodies together with depressed cell-mediated immunity [27], has been shown to give a relatively weak IgE response [28]. It is obvious that further studies of cytokine production in vivo and in vitro are necessary to elucidate the factors which are of importance for the expression of the cellular and humoral immune responses as well as for the immune regulation in VL.
S.A. Shiddo et al.
I Immunology Letters 50 (1996) 87-9-T
Acknowledgements We are grateful to Pharmacia Diagnostics for allowing us to use their facilities for the determination of IgE immunoglobulins.This work was supported by grants from UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), the Swedish Agency for Research Co-operation with Developing Countries (SAREC), and the Somali Academy for Science and Arts (SOMAC).
References P.E.C. (1961) Immunity in kala-azar. Trans. R. [ll Manson-Bahr, Sot. Trop. Med. Hyg. 55. 550-555. VI Ho. M.. Koech, D.K., Iha, D. and Bryceson, A.D.M. (1983) Immunosuppression in Kenyan visceral leishmaniasis. Clin. Exp. Immunol. 51, 207-214. P.E.C., Heish. R.B. and Gamham, P.C.C. (1959) [31 Manson-Bahr, Studies in leishmaniasis in East Africa. IV. The Montenegro test in kala-azar in Kenya. Trans. R. Sot. Trop. Med. Hyg. 53, 380-383 T.R., Cherwinski. H., Bond, M.W.. Gieldlin, M.A. [41 Mosmann, and Coffman, R.L. (1986) Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136, 2348-2357. S. (1991) Human Thl and Th2 subsets: doubt no [51 Romagnani, more. Immunol. Today 12, 256-257. [61 Kaye, P.M.. Curry, A.J. and Blackwell, J.M. (1991) Differential production of Thl-and Th2-derived cytokines does not determine the genetically controlled or vaccine-induced rate of cure in murine visceral leishmaniasis. J. Immunol. 146, 2763-2770. K.. Harms, G.. Pedrosa, C., Omena, S., Sand[71 Zwingenberger, kamp. B. and Neifer, S. (1990) Determinants of immune response in visceral leishmaniasis: evidence for endogeneous interleukin 4 over interferon-y production. Clin. Immunol. Immunopathol. 57. 242-249. Y.. Nakanishi, N., Tomizava, K.. Os[81 Ishizaka. A., Sakayama, hika, E., Kojima, K. and Taguchi, Y. (1990) The inductive effect of interleukin 4 on IgG4 and IgE synthesis in human peripheral blood lymphocytes. Clin. Exp. Immunol. 79. 392-396. A. Aden, Huldt, G.. [91 Shiddo. S.A.. AkufTo. H.O., Mohamed. Nilsson, L-A., Ouchterlony, 0. and Thorstensson, R. (1995) Visceral leishmaniasis in Somalia. Prevalence of leishmanin-positive and sero-positive inhabitants in an endemic area. Trans. R. Sot. Trop. Med. Hyg. 89. 21-24. K.A.. [lOI Shiddo, S.A., Mohamed, A. Aden, Akuffo, H., Mohamud, Herzi, A.A.. Huldt, G., Nilsson. L-A., Ouchterlony, 0. and Thorstensson. R. (1995) Visceral leishmaniasis in Somalia. Prevalence of markers of infection and disease manifestations in a village in an endemic area. Trans. R. Sot. Trop. Med. Hyg. 89. 361-365. A. Aden, Huldt, G., Loftenius. A.. [l ‘I Shiddo. S.A., Mohamed, Nilsson, L-A., Ouchterlony, i). and Thorstensson, R. (1995) Visceral leishmaniasis in Somalia. Circulating antibodies as measured by DAT, immunofluorescence and ELISA. Trop. Geogr. Med. 47. 68-73.
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[I21 Shiddo. S.A., Huldt, G., Jama, H., Nilsson. L-j)\.. Ouchterlony. 0.. Warsame. M. and Jonsson, J. (1094) Reference ranges for IgG. IgM and IgA in the serum of urban and rural Somalis. Trop. Geogr. Med. 46, 27.-31. [I31 Mancini, G., Carbonara, A.O. and Hcrcmans. J.F. (1965) lmmunochemical quantitation of antigens by radial immunodiffusion. Immunochemistry 2, 235-254. [I41 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. L. (1988) In vitro assay for [I51 Johansson, S.G.O. and Yman, Immunogobulin E. Clin. Rev. Allerg) 6. Y?’ 139. [I61 El Amin, E.R.M., Wright, E.P. and Vlug. A. (1986) Characterization of the humoral immune response in Sudanese leishmaniasis: specific antibody detected by class- and subclass-specific reagents. Clin. Exp. Immunol. 64, I4 IY. [17] Elassad, A.M.S., Younis. S.A.. Siddig. M.. Grayxon. J., Petersen, E. and Ghalib, H.W. (1994) The significance of blood levels of IgM, IgA. IgG and IgG subclasses in Sudanese visceral leishmaniasis patients. Clin. Exp. Immunol. 95. 29-l 299. [I81 Ulrich, M.. Rodriguez, V., Centeno. M. and Convit, J. (1995) Differing antibody IgG isotypes in rhc polar forms of leprosy and cutaneous leishmaniasis charactcrizcd by antigen-specific T cell anergy. Clin. Exp. Immunol. 100. 54 58. A. and Schrodcr. H. (1990) U91 Hagi. HT.- Huldt, GI. Loftenius, Antibody response in schistosomiasia hacmatoblum in Somalia. Relation to age and infection intensity. Ann. ‘I‘rop. Med. Parasitol. 84. 171-179. [201 Cahill, K.M. (1969) Health on the Horn of Africa. A study of the major diseases of Somalia. Spottiswood-Bellantyne. London. L.. Biberfeld, G.. Osman. S.. Isse. A. and 1211 Jama. H.. Grillner, Abdiraham. M. Bygdeman (1987) Scxuallh transmitted viral infections in various population groups in hlogadishu, Somalia. Genitourin. Med. 63, 329-332. of major 1221 Arora, Sunil K. and Sehgal. S. (19XY) ldcntification antigens of Leishmania donorani using kala-azar sera. Med. Microbial. Immunol. 178, 8 I - 88. D.. Dunan, S. and Quilici. M. (1992) 1231 Mary. C., Lamouroux, Western blot analysis of antibodies IO I.ur.shmturia infimtum antigens: potential of the l4-kD and Ih-kD antigen5 for diagnosis and epidemiologic purposes. Am. Sot. Trop. I lg. 47, 764771. F.T., Schell, R.S., Nau, G. and Fitch. W.F. (1989) [241 Gajewski, Regulation of T-cell activation: difference among T-cell subsets. lmmunol. Rev. 111, 79.-- 110. R.C.. van der Gadg, R. and Van Lceucn. J. (1983) [251 Aalberse, Serologic aspects of IgG4 antibodies. Prolonged Immunization results in an IgG4 restricted response. J. Immunol. 130. 722 726. H., Helmby, H.. Hagstedt. M.. C‘arlson. J., Larsson. 1261 Perlmann. P.H.. Troye-Blomberg. M. and Perlmann. P. (1994) IgE elevation and IgE anti-malaria antibodies in Pla.wwdiunr ,fhlcipantm malaria: association of high IgE lecels uirh cerebral malaria. Clin. Exp. Immunol. 97, 284-292. in experimental toxo~271 Huldt, G. (1967) Studies on pathogenesis plasmosis (thesis). Tryckeri Balder AB. Stochholm. [281 Wong, S.Y.. Hajdu. M.P., Ramirez. R.. Thullicz. P.. McLeod. R. and Remington, J.S. (1993) Role of qccific immunoglobulin in diagnosis of acute toxoplasma infection and toxoplasmosis. J. Clin. Microbial. 31, 2952- 2959.
Visceral leishmaniasis
Letters
50 (1996) 87-93
in Somalia. Significance of IgG subclasses and of IgE response
S.A. Shiddoa.b.*, G. Huldtb, L-A. Nilsson”, 0. Ouchterlony”, R. Thorstenssonb “DiLlisionof’ Parasitology. Department oJ‘ Morpho1og.v and Pathologv, Faculiy of‘ Medicine and Surgery, Somalr .Varional l,+lic~ersit~*. Mogadishu, Somalia bSwedish Institute ,ftir Injtictious Disease Control. Stockholm, Sweden ‘Department of’ Medical Microbiology and Immunologic. University qf Gothenburg, Gothenburg, .S~den Received
28 September
1995; revised
15 February
1996; accepted
26 February
1996
Abstract We have determined with clinical
visceral
the levels of IgG subclasses and IgE as well as specific antibodies of these isotypes in sera from 22 patients leishmaniasis (VL) from Somalia. The results are compared with those obtained from 30 Somali and 23
Swedish controls. We found markedly increased concentrations of IgGl in the VL sera, indicating that the pronounced increase in IgG in VL which is generally considered to be due to polyclonal B-cell activation is mainly restricted to this subclass. The IgG2 concentrations were significantly decreased. The IgG3 and IgG4 concentrations, on the other hand, did not differ between the two groups of Somali sera. The Somali control sera contained higher concentrations of IgGl and IgG3. but significantly lower concentrations of IgG2 as compared to Swedish controls. The IgG4 values, on the other hand, were not different between the two groups of control sera. Anti-leishmania antibodies belonging to all IgG subclasses, were found in the patients’ sera. There was no significant difference in total IgE between sera from VL patients and controls and specific IgE antibodies were only detected in a few patients. The Western blot assay (WB), revealed the presence of two bands corresponding to 74 kDa and 88 kDa in all patients’ sera, indicating a possible diagnostic role for WB in this particular population. Keywords: IgG subclasses;
IgE; Polyclonal
B-cell activation;
VL; Somalia
1. Introduction Visceral leishmaniasis (VL) is a systemic infection, caused by an intracellular protozoan parasite belonging to the L. donouuni complex. The parasite multiplies in macrophages preferentially in spleen, liver, lymph nodes and bone marrow, causing profound immune reactions [l]. Active stages of the disease are accompanied by high titre circulating antibodies and also by a depression both of leishmania-specific and of unrelated cell-mediated immunity [l-3]. In connection with recovery, the antibody response slowly decreases and the leishmanin test becomes positive [3]. This pattern of development of humoral and cell-mediated immunity is compatible with cytokine-mediated immune regulation
* Corresponding author. Department stitute for Infectious Disease Control.
0165-2478/96/$12.00
0 1996 Elsevier
PII SO165-‘478(96)02529-l
of Immunology, Swedish InS-105 21 Stockholm. Sweden.
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associated with activation of the murine CD4 + lymphocyte subsets Thl and Th2 [4]. Evidence has been presented to show similar mechanisms in humans [5]. An association between Thl or Th2 response and the predominant isotypes of specific antibodies during the course of infection, including VL. has been reported in studies in mice [6]. Elevated concentrations of interleukin 4 (IL-4), the main cytokine involved in Th2 activation, together with low concentration of interferon gamma (IFN-y) has been demonstrated in sera from patients with clinical VL [7]. Furthermore, it has been shown in vitro that Th2 activation results in predominantly IgG4 and IgE production [8]. We have previously studied VL in an endemic village in Somalia and in hospital patients in Mogadishu [9111. In the present study, we have investigated IgG and IgE immune responses in patients with active VL, with the aim of obtaining a better understanding of the interaction between humoral and cell-mediated immunity in this infection.
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In addition to Somali controls, sera from healthy Swedes were analysed. These sera had previously been tested for total IgG, IgM and IgA [12]. The results in the present study, therefore, supplement the data on reference ranges of various immunoglobulin classes in Somalia.
2. Materials and methods
2.1. Seru Sera were obtained from 22 patients with VL, treated at hospitals in Mogadishu during the years 198991990 [9,11]. All sera had anti-leishmania antibodies at high titres and all patients but one were leishmanin negative. Twelve of the cases were parasitologically confirmed. Sera were taken before initiation of specific treatment. This group of patients has been described in a previous paper [ 111. Control sera were from 30 healthy residents of Mogadishu which is non-endemic for VL, and from 23 healthy Swedes [l 11. All the Somali control sera were negative in direct agglutination test (DAT), enzymelinked immunosorbent assay (ELISA) and immunofluorescence test (IF) against L. donouuni antigen. 2.2. Collection
and handling of sera
Blood samples were taken by venepuncture. After centrifugation, sera were kept at - 20°C until transported to Sweden, where they were stored at - 20°C. The Swedish sera were also kept at - 20°C. 2.3. Determination
of total and specljic IgGl-4
Total and leishmania-specific IgG have been determined previously [11,12]. For determination of the concentration of IgG subclasses, the single radial immunodiffusion (SRID) technique as described by Mancini et al. [13] was used. Calibrators for subclass quantification were obtained from Binding Site Ltd. (Birmingham, England). IgGl, 2, 3 and 4 antibodies against L. donovani were determined by ELISA using an antigen, prepared as follows. Stationary phase promastigotes of L. donooani, grown in RPM1 1640 (Gibco, USA) with 10% foetal calf serum, were disintegrated using a Branson sonifier cell disrupter (Branson Co, USA) at medium amplitude for 3 min in an ice bath. Extraction was performed overnight at + 4°C. The extract was centrifuged at 10000 x g for 30 min and the supernatant was used as antigen [l 11.We have previously shown that similar results were obtained in ELISA with this antigen and an antigenic preparation containing a suspension of whole parasites. The test was performed as described earlier [I l] with the follow-
ing modifications. Sera were tested in dilution l/1000. Monoclonal anti-human subclass antibodies, (Mabs) (anti-IgGl and anti-IgG2 from Binding Site Ltd, England and anti-IgG3 and anti-IgG4 from OXOID, BIOZAC, Jlrfalla, Sweden) were used at dilutions 1500 for IgGl and IgG2, 1: 1000 for IgG3 and 1: 10 000 for IgG4, respectively. The antigen-antibody reactions were visualized by means of a biotin-avidin system (biotinylated rabbit anti-mouse and peroxidase-conjugated avidin; DAK0 A/S, Glostrup, Denmark) according to the instructions of the manufacturer. o-Phenylenediamine/H,O, was used as substrate (T Cell Diagnostics, Cambridge, MA, USA). Incubation was performed for 15 min and the plates were read at 492 nm in a Multiscan spectrophotometer. 2.4. Western blot (WB) The antigen used for WB was a soluble extract of L. donozlani promastigotes, prepared as described above for ELISA determination. Equal amounts (v/v) of the antigen and of sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) sample buffer containing 2% SDS and 1% dithiothreitol were boiled for 5 min in a water bath. Parasite proteins were separated by gel electrophoresis on a 12% polyacrylamide resolving slab gel in the presence of sodium dodecyl sulphate according to Laemmli [14]. Approximately 200 pg of protein was loaded on the gel. Separated polypeptides were transferred to nitrocellulose (0.22 ,u, Sartorius produkter AB, Sundbyberg, Sweden) in a transfer blot cell (Bio Rad AB, Solna, Sweden) containing 20% methanol in 0.025 M Tris/O. 192 M glycine buffer and by using 100 V for 2 h. The nitrocellulose with the separated polypeptide bands was cut into 4 mm strips, each containing approximately 15 ,ug protein and blocked for 2 h at room temperature with 5% skimmed milk in PBS by gentle rocking in a rotator. Each strip was incubated for 2 h at 37°C with test serum diluted 1: 100 in the blocking solution. The strips were then washed with PBS 4 x 15 min and incubated overnight at room temperature with horseradish peroxidase-labelled rabbit anti-human IgG (Dako A/S) diluted 1:lOOO in 4% BSA. For WB analyses of IgG subclasses, the same Mabs and serum dilutions were used as for ELISA. Antibody incubation was performed for 2 h at 37°C. Strips were then washed and incubated with a 1: 10 000 dilution of peroxidase-conjugated rabbit anti-mouse immunoglobulins (DAK0 A/ S) for 1 h at room temperature. After further washing, colour reactions were developed by exposure to a solution of o-dianisidine (Sigma) (0.25 mg in 100 ml TrisHCl, pH 7.6 supplemented with 0.3 ~1 of 30% H,O,/ml buffer). The reaction was stopped by rinsing with distilled water when colour started to appear. Strips were dried and stored in the dark. Determinations of relative
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.
811
. .. .
ir;n
IgG2
IgG 3
IgG 4
Fig. 1. IgG subclass levels in sera from healthy Swedes (open symbols), healthy Somalis (open bold symbols) and VL patients horizontal bars indicate the medians values.Significance levels between sera from healthy Somalis and VL patients: l&l. I’ 0.01; IgG3, not significant (ns); IgG4, ns. Significance levels between sera from healthy Swedes and healthy Somalis: IgGl. = 0.02: IgG3. P-c 0.001; IgG4, ns.
molecular weights were extrapolated, using low molecular weight calibration standards (Pharmacia, Uppsala, Sweden) 2.5. Determination leishmania-spec$c
of total serum IgE and IgE antibodies
Total serum IgE was determined by the Pharmacia CAP System, IgE FEIA (fluorescence enzyme immunoassay, Pharmacia Diagnostics AB, Uppsala, Sweden) according to the manufacturer’s instruction. Specific IgE was determined by the Pharmacia CAP system RAST FEIA (15) using ImmunoCAP, covalently coupled with a soluble extract of L. donouani promastigotes. The coupling was performed at different antigen concentrations, 10 pgg/ml and 100 p g/ml. Fifty p 1 of undiluted sera were used for the IgE determinations. The test results were obtained by comparing with standard curves. Values of 2 100 kU IgE/l and 2 0.35 kU IgE/l were considered as positive for total IgE and specific IgE, respectively [15]. 2.6. Statistical
analysis
(solid symbols).
The
< 0.001; IgG2, P = P -I 0.001: IgG2, P
patients and from Somali and Swedish controls are demonstrated in Fig. 1. The most obvious feature in the VL sera is that compared with Somali controls. there is a higher level of IgGl and a lower level of IgG2. This inverse relationship was statistically significant (P = 0.05). No difference in lgG3 or in IgG4 levels was demonstrated between sera from VL patients and Somali controls. It should be noted that significant differences in IgGl, IgG2 and lgG3 were also recorded between Swedish and Somali control sera. Compared with Swedish sera. the Somali control sera contained significantly higher levels of IgGl and IgG3 and lower levels of IgG2. The leishmania-specific antibody response of the various subclasses is demonstrated in Fig. 2. All sera from VL patients showed higher OD values of IgGl and IgG4 than any of the control sera and the majority of the VL sera also had higher values of IgG3. None of the VL sera showed IgG2 reactivity above the levels of Somali control sera. However, the median IgG2 antibody reactivity of VL patients was significantly higher than that of the control sera.
The median test and the Chi square test were used for statistical evaluations and for significance testing. 3.2. Western 3. Results 3.1. IgGl - 4 concentrations in sera from health-y Swedes, healthy Somalis and patients with VL The subclass
distribution
of IgG
in sera from
VL
blot
Sera from 12 patients with VL. from eight Somali controls and from six Swedish controls were tested in WB. The results obtained in five of the VL patients and two of the controls are shown in Fig. 3a. The immunoblot revealed 10 to 15 antigenic bands when tested for IgG antibodies. The most distinct bands corre-
S.A. Shiddo et al. / Immunology Letters 50 (1996) 87-93
90
. . . .
IgGl
lgC2
IgC3
lgG4
Fig. 2. IgG subclass antibodies against L. donouaniin sera from VL patients (solid symbols) and from healthy Somali controls (open symbols). The horizontal bars indicate the median values. Significance levels between sera from healthy Somalis and VL patients: IgGl, P < 0.001; IgG2, P I 0.01; IgG3. P I 0.001; IgG4, P 2 0.001
sponded to molecular weights of approximately 88, 74. 55, 42, 39, 27 and 24 kDa, while most of the remaining bands could not be distinctly visualised by all patient sera (lanes l-5). On the IgG subclass specific WB (Fig. 3b), almost all distinct bands were recognized by IgGl (lanes l-4) and IgG3 antibodies (lanes 9-12), while IgG2 (lanes 5-8) and IgG4 (lanes 13- 16) antibodies were either unreactive or were only weakly reactive with most of the bands. All patients sera tested reacted with two bands, corresponding to the molecular weights of 88 and 74 kDa, respectively. While sera from healthy Swedes did not react with any bands (Fig. 3a, lane 6), six of eight sera from healthy Somalis showed weak reactivity with bands corresponding to 24 and 42 kDa (Fig. 3a, lane 7).
3.3. Total and speciJic IgE concentrations Fig. 4a shows total IgE concentrations in sera from VL patients and healthy Somali controls. In both groups the same number of sera had total IgE concentrations above the cut off level. The median serum concentrations of IgE was slightly higher in the control group as compared to VL patients. Leishmania-specific IgE antibodies were detected in only four of 20 sera from VL patients (Fig. 4b). All sera were reactive at low levels and one of them was borderline reactive. In the control group one serum was borderline reactive. The reactivity was similar whether
10 or 100 pg/ml ImmunoCAP.
antigen
was used for coupling
to the
4. Discussion We have found markedly increased concentrations of IgGl, reaching values above 80 g/l in VL patients. This indicates that the major part of the polyclonal activation, typical of VL, falls within this subclass. We also found a significant decrease in levels of IgG2. This inverse relationship is in agreement with findings from Brazilian [7] as well as from Sudanese patients with VL
P61. By means of ELISA, we demonstrated anti-leishmania antibodies of the IgGl and IgG4 subclasses in all of the VL patients’ sera tested, and of the IgG3 subclass in almost all cases. In IgG2-specific ELISA, the antibody reactivity of patient sera did not exceed the highest OD values found in Somali controls. The median antibody concentration was, however, significantly higher in the sera from VL patients. In one group of Sudanese patients, appreciable amounts of antibodies were found only within the IgGl and IgG3 subclasses [16], while in another report from the Sudan, specific antibodies were found in all four subclasses [17]. In another study from Venezuela, patients with active kala-azar had a predominant IgGl response to a L. chagasi antigen while the levels of IgG4 were low [18]. Our study shows that there was a significant correlation (P < 0.001) between
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(b) 5
6
7
Fig. 3. (a) Western blot analysis of sera from patients with VL (lanes with VL. for IgGl (lanes I -4), IgG2 (lanes 5-Q lgG3 (lanes 9-12)
total and specific IgGI values in the VL patients’ sera. In the other three subclasses, however, such a correlation was not observed, since high levels of anti-leishmania antibodies were detected without corresponding elevation in total concentrations. We could demonstrate only a weak leishmania-specific IgE response. The total IgE concentration was similar in sera from VL patients and Somali controls and remarkably low for a population in a developing country. Hagi et al. [19] found much higher values in another Somali village where, however, schistosomiasis was endemic. In our study, only five of the 18 patients came from areas endemic for Schistosoma huematobiunz. Three of the five had total IgE 2 2000 kUj1. Although several intestinal parasites are common in Somalia and a few cases of Bancroftian filariasis have reported to the hospitals of Mogadishu, schistosomiasis seems to be the most important cause of increased level
8
9 10 11 12
I-5) and controls and IgG4 (lanes
13 14 15 16
(lanes 6- 7). (b) Western 13-16) antibodies.
blot analg’si~ of bera from patients
of total and specific IgE there. In Southern Somalia, schistosomiasis exists only along the two perennial rivers, the Juba and the Shabelle [20]. On the other hand, VL is the infection in Somalia which is accompanied by the highest levels of IgG and IgM. Malaria, which also can give rise to polyclonal production of 1gG and IgM, exists only in a hypo- or-mesoendemic pattern and we have found only moderately high IgG level and no increase in IgM in sera from Somali patients with malaria [12]. African trypanosomiasis which is often accompanied by polyclonal B ccl1 activation is not endemic in Somalia and at the time of this study I-IIV infections were very scarce [21]. All scra from VL patients and controls used in this study were negative for HIV [lo]. When investigating the total concentration of the various IgG subclasses, both Somali and Swedish controls were included. A comparison between these two
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kUL
I
1.0
0.8
. x
.
.
b 0.4 m-
-=-
_
e
-
-
Fig. 4. (a) Total IgE in VL patients (solid symbols) and healthy Somalis (open symbols). The horizontal bars indicate the median values and the dashed line the cut-off values. (b) IgE antibodies in sera from VL patients (solid symbols), healthy Somalis (open symbols) and from healthy Swedes (gray symbols) as determined by ImmunoCAP RAST FEIA coupled with 10 pg/ml of L. donovani antigen. The dashed line represents the cut-off value and the horizontal bars the median values of the groups
groups of sera showed an inverse relationship between the concentrations of IgGl and IgG2, similar to the differences between VL sera and Somali controls. This phenomenon, therefore, may be due to factors associated with the conditions of poor populations living in a tropical environment rather than specifically due to the VL infection. The difference between Swedish and Somali controls also emphasizes the importance of using local controls. If only Swedish controls had been used, such a comparison would have resulted in misinterpretation of the levels of IgG3 (Fig. 1). The WB analyses showed that antibodies of two specificities corresponding to 74 kDa and 88 kDa could be detected in all VL sera tested. This indicates a possible diagnostic role for WB in this particular population. In a study from India, Arora and Sehgal [22] found a prominent band of L. donovani promastigotes in the region of 65-66 kDa while Mary et al. [23] identified in L. infantum, two bands of 14 kDa and 16 kDa molecular weight which could be recognized by all VL sera tested, including sera from AIDS patients with proven leishmaniasis. The WB analyses of our study revealed the same two distinct bands of 74 and 88 kDa in all sera tested for all IgG subclasses as were seen in total IgG. The marked antibody production and depressed cellmediated immunity, seen in active cases of VL, is compatible with an activation of the Th2 type immune response. Furthermore, Zwingenberger et al. [7] could demonstrate endogenous production of IL-4 in all of 18 VL patients examined, whereas low levels of IFN-1, was
detected in only two of the VL patients studied. This supports the hypothesis that active VL is associated with Th2 activation. According to findings in both in vitro and in animal studies, this would lead to changes in the normal IgG subclass pattern and to increased production of IgE [8,24]. The results of our study with elevation of total and specific IgGl but lack of polyclonal activation of IgG3, IgG4 and IgE indicate that in humans, other factors than those operating in the experimental mouse systems are of importance for the regulation of the immune response in VL. For instance, the IgG4 response has been shown to be largely dependent on the length of the immunization period [25]. Of the 22 VL patients in our material, 13 had been ill for more than 1 year and this may explain the marked IgG4 response in the Somali VL patients. We have no explanation for the weak IgE response observed in our patients, but with the exception of a recent report of increased levels of IgE in malaria [26], protozoan infections generally are accompanied by only moderate IgE responses. For instance, toxoplasmosis which like VL is an intracellular protozoan infection, accompanied in its early stage by high level of antibodies together with depressed cell-mediated immunity [27], has been shown to give a relatively weak IgE response [28]. It is obvious that further studies of cytokine production in vivo and in vitro are necessary to elucidate the factors which are of importance for the expression of the cellular and humoral immune responses as well as for the immune regulation in VL.
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Acknowledgements We are grateful to Pharmacia Diagnostics for allowing us to use their facilities for the determination of IgE immunoglobulins.This work was supported by grants from UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), the Swedish Agency for Research Co-operation with Developing Countries (SAREC), and the Somali Academy for Science and Arts (SOMAC).
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[I21 Shiddo. S.A., Huldt, G., Jama, H., Nilsson. L-j)\.. Ouchterlony. 0.. Warsame. M. and Jonsson, J. (1094) Reference ranges for IgG. IgM and IgA in the serum of urban and rural Somalis. Trop. Geogr. Med. 46, 27.-31. [I31 Mancini, G., Carbonara, A.O. and Hcrcmans. J.F. (1965) lmmunochemical quantitation of antigens by radial immunodiffusion. Immunochemistry 2, 235-254. [I41 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. L. (1988) In vitro assay for [I51 Johansson, S.G.O. and Yman, Immunogobulin E. Clin. Rev. Allerg) 6. Y?’ 139. [I61 El Amin, E.R.M., Wright, E.P. and Vlug. A. (1986) Characterization of the humoral immune response in Sudanese leishmaniasis: specific antibody detected by class- and subclass-specific reagents. Clin. Exp. Immunol. 64, I4 IY. [17] Elassad, A.M.S., Younis. S.A.. Siddig. M.. Grayxon. J., Petersen, E. and Ghalib, H.W. (1994) The significance of blood levels of IgM, IgA. IgG and IgG subclasses in Sudanese visceral leishmaniasis patients. Clin. Exp. Immunol. 95. 29-l 299. [I81 Ulrich, M.. Rodriguez, V., Centeno. M. and Convit, J. (1995) Differing antibody IgG isotypes in rhc polar forms of leprosy and cutaneous leishmaniasis charactcrizcd by antigen-specific T cell anergy. Clin. Exp. Immunol. 100. 54 58. A. and Schrodcr. H. (1990) U91 Hagi. HT.- Huldt, GI. Loftenius, Antibody response in schistosomiasia hacmatoblum in Somalia. Relation to age and infection intensity. Ann. ‘I‘rop. Med. Parasitol. 84. 171-179. [201 Cahill, K.M. (1969) Health on the Horn of Africa. A study of the major diseases of Somalia. Spottiswood-Bellantyne. London. L.. Biberfeld, G.. Osman. S.. Isse. A. and 1211 Jama. H.. Grillner, Abdiraham. M. Bygdeman (1987) Scxuallh transmitted viral infections in various population groups in hlogadishu, Somalia. Genitourin. Med. 63, 329-332. of major 1221 Arora, Sunil K. and Sehgal. S. (19XY) ldcntification antigens of Leishmania donorani using kala-azar sera. Med. Microbial. Immunol. 178, 8 I - 88. D.. Dunan, S. and Quilici. M. (1992) 1231 Mary. C., Lamouroux, Western blot analysis of antibodies IO I.ur.shmturia infimtum antigens: potential of the l4-kD and Ih-kD antigen5 for diagnosis and epidemiologic purposes. Am. Sot. Trop. I lg. 47, 764771. F.T., Schell, R.S., Nau, G. and Fitch. W.F. (1989) [241 Gajewski, Regulation of T-cell activation: difference among T-cell subsets. lmmunol. Rev. 111, 79.-- 110. R.C.. van der Gadg, R. and Van Lceucn. J. (1983) [251 Aalberse, Serologic aspects of IgG4 antibodies. Prolonged Immunization results in an IgG4 restricted response. J. Immunol. 130. 722 726. H., Helmby, H.. Hagstedt. M.. C‘arlson. J., Larsson. 1261 Perlmann. P.H.. Troye-Blomberg. M. and Perlmann. P. (1994) IgE elevation and IgE anti-malaria antibodies in Pla.wwdiunr ,fhlcipantm malaria: association of high IgE lecels uirh cerebral malaria. Clin. Exp. Immunol. 97, 284-292. in experimental toxo~271 Huldt, G. (1967) Studies on pathogenesis plasmosis (thesis). Tryckeri Balder AB. Stochholm. [281 Wong, S.Y.. Hajdu. M.P., Ramirez. R.. Thullicz. P.. McLeod. R. and Remington, J.S. (1993) Role of qccific immunoglobulin in diagnosis of acute toxoplasma infection and toxoplasmosis. J. Clin. Microbial. 31, 2952- 2959.