Posters: Autoimmune Diseases

Abstracts •

325

Posters: Autoimmune Diseases

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Immunoelectron microscopic characterization of myoid cells (MC) with anti-desmin and anti-vimentin in normal thymus and in thymitis in myasthenia gravis (MG)

Autoimmunopathic lymphocytic hypophysitis W. Saeger

A. BQrnemann (a. G.) and Th. Kirchner PathQIQgisches Institut, Krankenhausstr. 8- 10,91054 Erlangen Aims: MC Qf the thymus are considered to. be Qf myQgenic Qrigin, and previQus investigatQrs fQund similarity to. myotubes Qr to denervated fibers. They are suggested to. play a role in intrathymic autosensitization in MG. It was Qur aim to characterize the developmental stage Qf MC as compared to. skeletal muscle development and to look fQr differences Qf MC between normal thymus and thymitis in MG. Methods: 10 cases Qf thymitis in MG and 8 nQrmal controls were immunostained with antidesmin and anti-vimentin in a pre-embedding procedure fQr light and electrQn micrQSCOpy. Results: 1. In all samples, MC are vimentinnegative. Desmin is localized at the periphery Qf MC, nQt in-between mYQfibrillar bundles. Hence, there is no. cross-striatiQn with anti-desmin staining. MC revealed desmQsQmes Qccasionally. 2. In thymitis, but nQt in normal contrQls, MC Qften fQrmed clusters. These clusters, Qr MCs in isolatiQn, were ensheathed by slender cytQplasmic processes Qf medullary epithelial cells which had a tendency to. seal MC Qff against the surrQunding cells. ApoptQsis of MC has never been Qbserved, nQr degradatiQn Qf Me within macrophages. Conclusions: 1. MC do not correspond to. myotubes, as they do not express vimentin. NQr do. they correspond to. mature Qr denervated skeletal muscle fibers, as the mYQfibrillar bundles are not interspersed with desmin at the Z-disc level. 2. There is no. degradatiQn Qf MC by apopto.sis Qr by phagQcytQsis in MG. The functiQns o.f epithelial cell sheaths is at present unknQwn. They may mediate antigen-presentatiQn in MG.

Institut fur Pathologie, Marienkrankenhaus Hamburg Aims: Lymphocytic hypophysitis is a very rare autoimmunopathic disorder. Less than 100 cases have been reported We present the morphology and immunohistochemical and clinical data of three further cases. Methods: The pituitaries (I surgical specimen, 2 post-mortem pituitaries) were embedded in paraffin and immunostained (antibodies from Dako. Hamburg) for all pituitary hormones and SIOO-protein and with antibodies against T-Iymphocytes (CD43, UCHLI) and 13lymphocytes (4KBS, BL26) and macro phages (CD68). Results: The surgical specimens of a 12 years old girl with diabetes insipidus and an intrasellar and suprasellar mass showed anterior pituitary tissue with loosened basic structure, slight interstitial fibrosis and a Iymphoplasmacytic infiltrate with macrophages and very sparse neutrophils. T -lymphocytes were more frequent than B-Iymphocytes. The first of our postmortem pituitaries revealed a Iympho.cytic infiltrate consisting mainly of T -lymphocytes in the intermediate ZQne and the adJa~enl alllellUI ano pusienul iuue. rile maie palient died due to a poly trauma and had no clinical signs of an endocrine disorder. The second postmortem pituitary was scarred and atrophic but revealed some lymphocytic infiltrates mainly of T-cell type. Especially gonadotropin- and prolactin producing cells were strongly reduced in number. The thyroid was strongly fibrotic and the adrenal cortex atrophic. The male patient showed distinct signs of hypopituitarism. Conclusions: Our 3 case reports show that lymphocytic hypophysitis can be misdiagnosed as pituitary or hypothalamic tumor (case I) or lead to a hypopituitarism (case 3) or can be an incidental finding (case 2). The autoimmunopathic nature of the disorder was suspected by demonstration of HLA antigens and of concomitant other autoimmune disorders especially the lymphocytic thyroiditis. The second autopsy case appear to suffer from an end stage of such thyroiditis.

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Die pluriglanduliire Autoimmunerkrankung im KindesaIter - eine Autopsiestudie

IMMUNOHISTOLOGICAL DETECTION AND DISTRIBUTION OF B- AND T-CELL SUBSETS IN AUTOIMMUNE TIiYROIDITIS

I. Rose uDd N. Bannert *( a. G. ) Institut furPatho.lQgie und * Zentrum fur Kinderheilkunde, Universitat Magdeburg

H. TERPE (a.G.), H. NIZZE Institute of Pathology, University of Rostock,

Aims: Lethal autQimmune disease is rarely diagnQsed in autQPsy material during childhQQd. The autho.rs present the retrospective study concerning a 15 years Qld girl with type I diabetes mellitus accompanied with o.ther Qrgan specific inflammatiQn . Methods: The autopsy was performed 24 hours postmortem with consecutive investigation of circulating antibodies against different tissues,biochemical controls of blood, cerebrospinal fluid and urine, completed by bacteriology, virology and immunhistology. Results:The adrenal glands were very small, microscopically there were islands of eosinophilic compact-type cells surrounded by lymphQcytic infiltration with T - and B-cells • a typical feature of autoimmune adrenalitis. Circulating antibodies to adrenal cortical cells were detected postmortem. Furthermore there were a moderate lymphocytic gastritis with anti-parietal cells antibodies and a severe thyreoiditis with extensive formation of lymphoid follicles mit B -cells and interfollicular T - cells lymphocytic infiltrates. Bilateral sialoadenitis Qf salivary glands, hypoplasia of endocrine pancreas and activation of the immune system were found immunohistologically. Conclusion: The autoimmune polyglandular disease with insulin-dependent diabetes mellitus and adrenal insufficiency needs early clinical diagnosis to. enable life -saving initiation of replacement therapy.

Aims: Aut.oimmune thyroiditis is particularly characterised by the formation of autoantibodies to several thyroid antigens and by germinal centers morphologically. The influence of a cellular response accompanying the humoral reaction should be investigat.ed by immunohistological approaches. Methods: A pane I of antibodies was used to detect and quanti fy CD3-, CD4-, CDS-pas i ti ve Tcells, CD20-positive B-cells, plasma cells, macrophages and the proliferation act.ivity on paraffin embedded specimens of thirty cases of thyroiditis. Results: The size and number of germinal centers correlated with surrounding CD4-positive T-cells and the proliferation activity of germinal center cell:; as well as with oncocytic metaplasia. CD8-positive T-cells outnumbered CD4-positive T-cells and B-cells in the interstitium far from germinal centers and apparently attacked the thyreocytes. The development of scars and fibrosis was obvious independent of the number of germinal centers, al though plasma cells were seen in fibrotic areas. Conclusions: Our findings suggest that both humoral and ce llular immune mechanisms partiCipate in autoimmune thyroiditis, but it seems that the different relations of lymphocytic subsets indicate different states of the immune response. CDS-positive T-cells are important for the destruction of the thyroid epithelium.

326 . Abstracts

51 Tenascin staining of activated lymphoid tissue in the thyroid and in the gastrointestinal mucosa. A comparative immunohistologic study. W. Back, Cornelia Heubner (a.G.) Pathologisches Institut, Fakultiit fur Klinische Medizin Mannheim der Universitiit Heidelberg Aims: In a previous contribution we demonstrated a characteristic coronalike localization of tenascin - a multifunctional matrix protein - around .activated lymph follicles in autoimmune lymphocytic thyroiditis. Similar .results are reported on Iymphofollicular hyperplasia of the thymus. These findings raised the question whether the expression of tenascin by perifollicular fibroblasts is a phenomenon of autoimmune response also shared by lymphoid tissue of the gastrointestinal mucosa. Methods: We studied the staining pattern of tenascin and also of collagen type III, collagen type IV and growth factors of the EGF-family in autoimmune and inflammatory lesions of the thyroid (n=40), of the gastrointestinal mucosa (n=35) as well as in lymphomas of the mucosa associated lymphoid tissue (n=1O) using immunohistochemistry on paraffin sections. Results: In benign pathologic conditions of the gastrointestinal tract - with and without autoimmune etiology - no corona-like tenascin staining around activated lymph follicles could be observed. There was a slight diffuse interstitial distribution pattern of tenascin together with enhanced expression of collagen type IV and type III in areas of mucosal repair. Only in collagenous colitis the collagen band shows a characteristic reactivity for tenascin. In lymphomas of mucosa associated lymphoid tissue there is a finely reticular staining pattern for tenascin in lymphoid infiltrates and no staining around nodular lymphoid infiltrates or residual lymph follicles. Conclusions: The expression of tenascin around activated lymph follicles is characteristic of activated lymph follicles in autoimmune thyroiditis and myasthenia gravis and is absent in gastrointestinal lymphoid tissue. The results of our study confirm various matrix compositions in activated lymphoid tissues of different organs. These differences in our study are likely to be related to the pathogenetic variety of autoimmunologic lymphoid tissue activation and to parenchymal interactions.

53 ANTIGEN-PRESENTING CELLS (SYNOVIAL DENDRITIC CELLS) IN RELATION TO DIFFERENT HISTOLOGICAL TYPES OF SYNOVIAL MEMBRANES IN RHEUMATOIDARTHRITIS (RA) R BECKER (a. G.), P STIEHL Abteilung fur Immunpathologie, Institut fur Pathologie, Universitiitsklinikum Leipzig Aim: Synovial dendritic cells (SOC) are antigen presenting cells (APC) for T-lymphocytes. Our aim was to find out, whether there was a relation between SOC in different regions of synovial membranes (SM) in RA and two resp. three new own histological types (STIEHL, 1994). Type 1 is mainly B-cell-dependent, whereas type II is T -celtdependent. Type 1 has a good prognosis, but type II a worse course of disease according to our first histologic-clinical relations. Material and methods: 92 RA synovial membranes and 14 controls were investigated histo-morphometrically with enumeration of SOC in four different synovial regions (subintimal, within lymphatic follicles, perivascular and within the interstitium). The SOC were detected by S-IOO antibodies (polyclonal as well as monoclonal). . Results: Statistically, a relation of SOC was found within the type II resp. III, but not so in the type I. The main topics of detection were the subintimal as well as the perivascular regions. Conclusions: Beside earlier other relations (HLA-DR4, HLA-DRI, rheumatoid granulomas resp. hemigranulomas, inflammatory activity), also the SOC show a relation to our different histological types in RA. Therefore, the findings support our theory of possible two different formal-pathogenetic courses of rheumatoid arthritis.

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Formal Pathogenesis of LymphoepitheJial Duct-lesions in Sjogrendisease and HI V-associated Sialadenitis

MESSENGER-RNA EXPRESSION O}<' THE ALTERNATIVELY SPLICED CS-I FIBRONECTIN ISOFORM IN RHEUMATOID ARTHRITIS (RA) SYNOVIUM U. MULLER-LADNER* (a G), 1. KRIEGSMANN** (a G), DANA STRAHL***(a.G.),R.E.GAY*(a.G.), M. ELICES*** (a.G.), S.GAY* *Div. Clin. Immun. Rheum., Univ. A1ab. at Birmingham, USA, **Inst. Pathol., F.-Schiller-Univ. lena, FRG, ***Cytel Corp., San Diego, CA.

S.Ihrler (a.G.), C.Zietz, ASendelhofert (a. G.), ANerlich, U.Lohrs Pathologisches Institut der Universitlit Miinchen Aims: The formal pathogenesis of Iymphoepithelial duct-lesions in autoimmune Sjogren-disease is unclear especially with regard to participation of myoepithelial cells. An immunohistochemical investigation was addressed both to this duct-lesion and to a comparable HIV-associated salivary gland lesion, which we have described recently. Methods: Parotid specimens with Sjogren-disease (n=II), HIVassociated cystic Iymphoepitheliallesion (n=IO) and regular parenchyma (n=6) were analyzed with respect to different types of cytokeratins (pan-keratin, CKI0/13/14/1S), smooth-ml,lscle alpha actin, Ki67- and Bcl2-antigen and matrix-Ibasement membrane components (Collagen IIIIIVNINII). Results: In all cases of Sjogren- and HIV-associated lymphoepithelial lesions intense proliferation of CK 14 positive basal cells of striated ducts was proven, thereby suprabasal cell layers were additionally CK13 positive. In parallel a progressive loss of CKIS positive luminal cells as well as salivary acini was seen; myoepithelial cells were not identified immunohi~t"('hemic:>lly ~vithin the lesions. In advanced alterations the epithelial basement membrane was fragmented and partly condensed. Conclusions: Our immunohistochemical.study of Sjogren- and HIVassociated Iymphoepithelial duct-lesions indicates a metaplastic basal cell hyperplasia of striated ducts along with the inflammatory ductal infiltration. Thereby the often postulated partizipation of myoepithelial cells is not verified immunohistochemically. The confinement of the lesions to striated ducts results from the physiological distribution of basal cells. The fragmentation of the ductal basement membrane presumably is caused by the inflammatory infiltration and might induce a loss of cell polarity of proliferating basal cells with a consecutive irregular matrix deposition. Whereas the etiology both of Sjogren- and HIV-associated Iymphoepithelial sialadenitis is still unclear, om investigation exhibits a analogy of the formal pathogenesis of lymphoepithelial duct-lesions of both diseases.

Aims: The interaction between very late antigen-4 (VLA-4) and the fibronectin isoform CS-I may contribute to lymphocyte trafficking to RA-synovium. Therefore, we examined the mRNA expression of the CS-I isoform in the synovium of patients with RA and osteoarthritis. Methods: Snap frozen synovial tissue specimen of 7 RA and 4 OA patients were examined. The amount and location ofmRNA expressing' cells was determined by in situ hybridization using non-radioactive riboprobes for the alternatively-spliced CS-I isoform of fibronectin. CS-I 70, and CS-I 150. The signals of the in situ hybridization products were compared to mRNA expression of a heparin binding region, Hep II, of fibronectin similar in length. Double labeling was achieved by using monoclonal anti-human collagen type IV and antihuman macrophage (CD68) antibodies in an APAAP assay. Results: In all RA patients (7/7), mRNA for CS-I 70 could be detected in distinct cells in the lining layer and the sublining around terminal vessels. The CS-I 150 riboprobe was strongly expressed in cells of the same tissue areas. Double labeling revealed that CS-I mRNA expressing synoviocytes in the lining layer were CD68 negative. In OA patients only single cells expressing CS-I mRNA could be detected. The HEP II gene sequence was strongly expressed in all specimens. Conclusions' These results demonstrate for the first time mRNA for the CS-I fibronectin in RA synovium. As CS-I is the ligand for VLA4, that also binds to VCAM-I, our results support the hypothesis, that in RA CS-l may not only facilitate trafficking through synovial endothelium, but also the interaction between synovial fibroblasts and lymphocytes as well as the adhesion of synovial fibroblasts to cartilage. J. Kriegsmann and U. Miiller-Ladner were supported by a grant of the DAAD

Abstracts· 327

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55 RELATION OF A NOVEL ISOFORM OF PSEUDOGEN TO RHEUMATOID ARTHRITIS. KUHN H., STIEHL P. Institute of Pathology, University Leipzig

K-RAS

Aims: Immunhistochemical analysis of synovial tissue from patients with rheumatoid arthritis (RA) revealed a significant expression of ras oncoprotein. We want to examine if molecular alterations of k-ras oncogen are responsible for these expression. Methods: We investigated 44 synovial membranes of patients with RA for mutations in codon 12/13. The k-ras fragments was generated by nested PCR with own designed primers. The PCR products were sequenced by Taq DyeDeoxy terminator cycle sequencing (Applied Biosystems). Results: The k-ras gen shows no mutation in codon 12/13, but we found a new isoform of k-ras pseudogen. We observed in codon 7 GCG instead GGCG and in codon 13 GGC instead AGC. Only the new isoform we found in 5 synovial membranes of patients with RA and only the other form of k-ras pseudogen we found in 28 cases. In II cases we discovered both forms. In 8 controls with normal synovial membranes we observed in 6 cases both forms of k-ras pseudogen. In two cases we found one kind of both forms only. Conclusion: We found a new isoform of k-ras pseudogen in synovial membranes ofRA. In 77p.c. ofRA cases we observed only one form of pseudogen, but in controls 74p.c. of cases had both forms of k-ras pseudogen.

BONE MARROW PAmOLOGY IN RELAPSING POLYCHONDRITIS: HIGH FREQUENCY OF MYELODYSPLASTIC SYNDROMES 1. Diebold, G. Rauh (a.G.)* and U. Lohrs Pathologisches Institut and *Medizinische Poliklinik, LudwigMaximilians-Universitat Miinchen Aims: Haematopathologic changes were studied in 19 sequential cases of relapsing polychondritis (RP), a rare rheumatic disorder with presumably autoimmunologic pathogenesis which is characterized by recurrent inflammatory episodes primarily affecting auricular, nasal or laryngotracheal cartilage. Methods and Results: Anaemia was found in 8, thrombocytopenia in 2 and splenomegaly in 3 patients. A total of 18 bone marrow biopsies were obtained from 7 individuals. Bone marrow evaluation revealed myelodysplastic syndromes (MDS) with marked trilineage hyperplasia and dysplasia in three cases. Since an excess of myeloblasts or an increase of immunohistochemically detected CD34 positive progenitor cells was not seen, the disorders were designated as "refractory anaemia" or with regard to the dysplastic megakaryopoieses "MOS, unclassifiable". The three patients with MOS died after 10, 55 and 56 months offollow-up due to infectious complications. The remaining patients mostly had reactive bone marrow changes. Conclusion: Our findings support the notion that RP is a heterogenous disorder and suggest that RP may at times represent a paraneoplastic phenomenon of an underlying MOS. Since HLA typing revealed a significantly increased frequency of the antigen DR4 (10 of 17 patients positive = 59"10), we hypothesize that immunological imbalances due to the MOS in conjunction with a specific immunogenetic background may play key roles in the pathogenesis ofRP in these patients.

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DISTRIBUTION OF TGFALPHA IN PSORIATIC AND RHEUMATOID SYNOVIUM

PERACUTE FATAL ASPERGILLUS SEPSIS AS A COMPLICATION OF SYSTEMIC LUPUS ERYTHEMATOSUS AND RHEUMATIC DISEASES P. Nenoff"* (a.G.), L.-C. Hom*, M. Mierzwa*(a.G.}, hena Lehmano***(a.G.), H. Weidenbach*(a.G.), P. Caffier*(a.G.) * Institut fur Pathologie, ** Universitiits-Hautklinik, *** Institut fur K1inische Immunologie, Universitiit Leipzig

A. KONIG (a.G.)*; V. KREN.N (a.G.)**, F. GOHLKE (a.G.)*, J. EULERT (a.G.)*, H.K. MULLER-HERMELINK**

*Orthopadische Univ. Klinik Konig-Ludwig-Haus Wiirzburg **Institut flir Pathologie der Univ. Wiirzburg Psoriatic arthritis (PA) is an autoimmune arthropathy of uncertain pathogenesis occuring in 5 to 7% of patients with psoriasis. TGFalpha plays a key role in the pathogenesis of the skin lesion We were therefore interested to see whether TGFalpha is expressed within the psoriatic synovium and whether the expression shows differences to rheumatoid synovium (RA). Methods: The expression of TGFalpha within the synovial tissue was studied by indirect immmunofluorescence and immunoperoxidase techniques within cryosections from patients with definite psoriatic arthropathy (n~5). TGFalpha was detected by a monoclonal antibody using an amplification step with a biotinylated secondary antibody followed by detection with CY-3avidin. Results: TGFalpha could be detected within the synovial lining cells, multinucleated cells, macrophages and within the inflammatory infiltrate which were located in the vicinity to blood vessels. When compared to the RA synovium TGFalpha showed a different pattern staining macrophages and synovial lining cells to a lesser extent. Conclusions: The different expression of TGFalpha in RA and PA synovium suggests a different functional role of this mediator in the local autoimmune response. Since TGFalpha was shown to playa central role in the psoriatic skin lesion TGFalpha mediated mechanisms may also playa pathogenetic role in the inflammatory process of psoriatic arthritis. ~

Aims: Invasive aspergilloses are not uncommon in patients with immunoproliferative disorders or after bone marrow or organ transplantation. However, it has been rarely occured in patients with autoimmune or rheumatic disease. Methods: Between 1992 and 1994 at Leipzig University 20 invasive mould infections were detected. Among them three aspergilloses were observed in patients with autoimmune or rheumatic disease. Results: A 39-years old women suffering from SLE treated by antibiotics and high-dose corticoids developed a disseminated fatal Aspergillus (Asp.) fumigatus infection. The autopsy showed mycotic abscesses in the lungs, liver, spleen, thyroid gland, renal parenchyma, heart muscle, in the submucosa of jejunum and colon and a focal mycotic leptomeningitis and encephalitis. The second patient, a 56-years old man suffering from rheumatic disease developed a wound infection after ablatio cruris with consecutive sepsis. The autopsy showed a Asp. infection of the spleen and a septic alteration of the parenchymatous organs. The third patient, a 67-years old man with treated polymyalgia rheumatica developed a peripher arterial occlusive syndrome. An ablatio femoris was done. Postoperative a septic mould infection by Asp. occured. On autopsy Asp. was found in the lung, the liver and arterial system Conclusions: The present cases demonstrate, that invasive aspergilloses may occur in patients with autoimmune and rheumatic diseases, receiving high doses corticosteroids and antirheumatic drugs. Physicians should alert to this possible complications.

328 . Abstracts

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FETAL ATRIAL-AXIS-DlSCONNECfION ASSOCIATED WITH CLINICALLY A CASE REPORT INAPPARENT MATERNAL COLLAGENOSIS

NOVEL AUTOANTmODIES AGAINST PODOCYTES IN RENAL DISEASE. W.B.STORCH University of Heidelberg, 69469 Weinheim, Netztal 32

D. - D. DITIERT (a. G.), I. NAWRACALA (a. G.f, I. SCI:INEIDER""

Institut filr Pathologie,Universitlit Gottingen; Kinderklinik", Universitlit Aims: Determination of the frequency of circulating novel human Gottingen; Institut filr K1inische Pathologie, Universitlit Zurich"" autoantibodies against podocytes (lmmun. Infekt. 23, 25-26, 1995). Methods: Sera of about 8000 patients with various diseases and of The pregnancy of a primigravida showed no signs of abnonnality until the 100 healthy controls were studied for antibodies against podocytes by 27th gestational week when fetal bradycardia led to an emergency Cesar- standard indirect immunofluorescence and confocal laser scanning ean section. ECG immediately after birth showed a third degree atrio-ven- fluorescence microscopy using cryotome sections of a block tricular block. A respiratory distress syndrome of rn° was diagnosed. consisting of rat kidney, stomach, and liver as antigen substrate. A Echography showed no signs of a vitium cordis but revealed densities in anti-human-IgG serum marked with fluoresceinisothiocyanate and the posterior papillary muscle. A chest x-ray displayed an opacity in pro- further substituted with Evans blue served as the conjugate Cojection of the left ventricle of the heart. In spite of maximal therapy the localization studies to show podocyte structure were performed by double labelling using mouse monoclonal antibodies against the 44 kd cardio-respiratory problems could only be partly influenced, and the child podocyte protein O. Histochem. Cytochem. 39,1047-1056,1991) and died 40 hours after birth. a Cy 3-marked anti-mouse-immunoglobulin. At autopsy a prematurely born male was found. Outer examination re- Results: Antibodies against podocytes were found in the serum of 10 vealed no signs of malfonnation. The inner examination was restricted to patients (3 with renal disease, 2 with liver disease, 2 with rheumatic the lungs and heart. The lungs had a firm consistency, and histology disease, I with achalasia, I with carcinoma of the pancreas, and I with showed signs of a severe acute respiratory distress syndrome. The prepa- anemia), but not in healthy controls. The clinical diagnosis of patients ration of the heart revealed an anterior defect of the membranaceous atrial with renal disease were: renal cirrhosis, nephrotic syndrome due to septum, open ductus Botalli, nonnal valves, and normal ventricular wall IgA nephritis and mesangioproliferative glomerulonephritis as well as idiopathic membraneous glomerulonephritis. Co-localization studies thickness. Slices cut for examination of the conduction system of the heart using monoclonal antibodies against the 44 kd podocyte protein did not show myocardial cells in the basal part of the atrial septum. Instead, showed a similar fluorescence pattern for both antibodies. partly removed necrosis with discrete mononuclear infiltrations, scars, and Conclusions: The novel autoantibodies against podocytes are rare and calcifications were seen. probably not specific for renal disease. The target antigens remain This muscular discontinuity within the right atrium, defined as atrial-axis- unclear. Nevertheless, the observations suggest that autoimmunity to disconnection, has been described in cases of maternal SS-A I SS-B podocytes maybe plays a role in the pathogenesis of renal disease. The antibodies, which can often be detected in collagenosis. The 22 year-old mechanism of action is unknown. mother showed no symptoms of a collagenosis. Screening for extractable nuclear antibodies, however, resulted in strongly elevated concentration of antibodies against SS-A and SS-B. Based on these antibodies and other elevated laboratory parameters a collagenosis has been diagnosed. The risk of repeated fetal involvement in a next pregnancy is expected to be lowerable by corticoid therapy.

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FUSION OF VERTEBRAL BODIES AND THE SACROLILIAC JOINT BY CARTILAGINOUS TISSUE IN HLA-B27 TRANSGENIC RATS 1. KRIEGSMANN*(a.G), B. N. FRANKLIN**(a.G.), R. E. GAY" (a.G.), 1. D. TAUROG***(aG.), R. E. HAMMER***(a.G.), R. BRAUER*(a.G.), D. KATENKAMP*, S. GAY** *Inst. Pathol., Friedrich-Schiller-Univ. Jena, **Div. Clin. Immunol. Rheumalol., Univ. of Alabama at Binningham, Binningham, AL, USA, ***Univ. Texas Southwestern Med. Ctr., Dallas, TX, USA

In situ Expression of Regulatory Peptides in Lupus-Glomerulonephritis (SLE-GN) H. Stein (a. G.), M. Ondracek (a. G.), I.L. Noronha (a. G.), Z. Niemir (a.G.), R. Waldherr, Pathologisches Institut der Universitlit Heidelberg Regulatory peptides (cytokines, growth factors, chemokines) playa critical role as mediators of inflammation in various immunological processes and determine the extent of pathological changes. In recent years our group has demol)§trated that there is a complex pattern of cytokine production in various fonns of inflammatory renal diseases. In the present study we analyzed kidney biopsies from 20 patients with SLE-GN (WHO classes II - V; different activity and chronicity scores). The in situ production of IL-Ia, IL-I~, TNF-a, IL-4, 1L-4 receptor (IL4R), IL-IO, M-CSF, IL-S and Mep-I was investigated at the protein (immunocytochemistry, APAAP-method) and the mRNA-level (in situ hybridization using digoxigenin labeled cRNA-probes). Normal renal tissue from tumor nephrectomies served as control. The proinflammatory cytokines IL-la, IL-l~ and TNF-a are mainly produced in crescent formations by infiltrating mononuclear cells and proliferating capsular epithelial cells. In addition, TNF-a is present in mesangial areas in cases with mesangial proliferation. The production of these cytokines in infiltrating interstitial cells correlates with the activity of interstitial inflammation. The THz cytokines 1L-4 and IL-IO are predominantly produced in endothelial cells and glomerular and interstitial infiltrates. IL-4R production is upregulated in endothelial cells. The chemokines IL-S and MCP-I are strongly positive in crescent fonnations and infiltrating mononuclear cells. An increased production ofM-CSF is observed in endothelial cells, some mesangial areas and interstitial infiltrates. When comparing different classes of SLE-GN, the marked increase in regulatory peptides correlates with the severity of the morphological changes and the activity of the inflammatory process. A good correlation between immunocytochemistry (protein) and in situ hybridization (mRNA) is observed. Our results demonstrate that the local production of regulatory peptides is an ' important event in the scenario of SLE-GN.

Aims: Information of the histopathology of the spine characteristic of ankylosing spondylitis (AS) is very limited. Therefore, the purpose of the present study was to evaluate the pathology of the spine and other joints in HLA-B27 transgenic rats, which might serve as a useful model for this disease. Methods: 12 rats, 6 of each sex, from the 21-4H line were sacrified at 7 month of age. The spine was divided into four parts from the neck to the pelvis and examined along with the knees and feet by H&E staining. Results: Most prominent, all animals (\2/12) revealed cartilaginous fusions of vertebrae (synchondrosis) and formation of syndesmophytes in AS. 6/12 rats had also synchondroses of the sacroiliac joint, in part associated with pannus-like lesions. In addition, 4 animals exhibited foci with an invasive pannus-like tissue in small vertebral joints. 5 rats had pannus-like tissue in the knee joints, and 3 showed deformities of the feet with immature cartilage and the fonnation of a pannus-like tissue. Furthermore, cartilagineous metaplasia could be detected in the patellar ligament. Conclusions: The present study reveals for the first time the fusion of vertebral bodies by cartilaginous tissue in an animal model. The data suggest that cartilage fusion is the initial process in ancylosing spondylitis and that the HLA-B27 transgenic rat is a most useful model . to study the pathology of this disease. 1. Kriegsmann was supported by a grant of the DAAD

Abstracts . 329

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ARTERITIS OF THE PANARTERITIS NODOSA TYPE IN THE TESTIS AND EPIDIDYMIS OF THE BEAGLE-DOG P. Schneider Hermann-Volz-Str. 77, 88400 Biberach

EPIDEMIOWGY AND DIAGNOSIS OF INVASIVE MOULD INFECTIONS IN IMMUNCOMPROMISED PATIENTS: RESULTS OF A 3 YEAR STUDY AT THE LEIPZIG UNIVERSITY L.-C. Hom*, P. Nenoff **(a.G.), M. Mierzwa**(a.G.), R Schwenke***(a.G.), T. Friedrich*, U.-F. Haustein**(a.G.) *Institut fur Pathologie, ** Universitiits-Hautklinik, *** KIinik fur Innere Mediz!!! II, Universitiit Leipzig

Aims: A panarteritis, which occurred in Beagle dogs, seems to be suitable to draw a comparison with the very same lesion in man. Thus, it is worthwhile to document this alteration. Methods: In the course of 19 subacute or chronic toxicologic studies (13 up to 26 weeks) 564 Beagle dogs (strain: Chbb: Beagle) were evaluated clinically. At the end an autopsy on the animals was done and all organs were examined macroscopically and histologically. The histologic slides were stained with haematoxylin-eosin stain routinely. Results: An elective occurrence of a panarteritis in the testis and epididymis was observed some years ago. Twenty cases (3.7%) of this lesion were proved. Typical are alterations which are wedge-shaped or involve the whole vessel wall with fibrinoid necroses of the media, proliferative reactions of the intima, leucocytic, Iymphoplasmocytic and histiocytic infiltrates as well as the development of extensive granulation tissue. Conclusions: The results presented fulfil the aims mentioned above. The panarteritis of dogs is comparable to panarteritis of man. This arteritis of different animal species develops from deposits of immune complexes in the wall of arteries (Drommer, 1991; Trautwein, 1991). Drommer, W (1991): Pathologie der Haustiere, Teil I: Organveriinderungen, 78-81. Hrsg.: Schulz, L.-Cl., Gustav Fischer Verlag Jena Trauwein, G. (1991): Pathologie der Haustiere, Teil II: Krankheiten und Syndrome, 175-176. Hrsg.: Schulz, L.-Cl., Gustav Fischer Verlag lena

Aims: . Invasive aspergillosis is a common infection in patients who are immunocompromised. Invasive mould infection, e.g. aspergillosis, is difficult to diagnose in the absence of confirmation by tissue biopsy and histological studies. Methods: The prevalence of invasive mould infections at the Leipzig University between 1992 and 1994 was investigated. The clinical, mycological, and radiological diagnostic findings were evaluated. Definitive diagnosis was obtained by identification of mould hyphae on stained smears, and/or positive culture of bronchial secretion and BAL, and/or detection of Aspergillus (Asp.) antigen (Pastorex, SanofilPasteur, France) in serum, bronchial secretion or BAL, and confirmed by histology. Results:In 20 patients a mould infection was recorded, among them 19 invasive aspergilloses. Underlying diseases were leukaemia (n= 11), aplstic aneamia (n=2), NHL (n=I), systemic lupus erythematosus (n=I), kidney transplantaion (n= I), peritonitis after Billroth II resection (n= 1), AIDS (n=l), glioblastoma (n=l) and subarachnoideal hemorrhage (n=I). The causative fungi were Asp. fumigatus (n=13), Asp. terreus (n=I), Asp. flavus as simultaneous infection with the Basidiomycete Coprinus spec. (n= I), and the Dermatiaceae Scedosporium prolificans. In four patients the infection was confirmed by morphology alone. Conclusions: Fungal infections still cause major problems in clinical diagnosis and some cases will be documented only if post mortem examination is performed. In the recent years more aspergilloses are found, may be by selection of Aspergillus as a result of antimycotic therapy in Candida infections.

Posters: Experimental Pathology

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64 AlTIDn.MJNE PHl'lOIENA DUE

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INTRAVASAL LYMPlD4A

E.W. HERBST', J. v. KEMPIS (a.G.)", G. KOHLER', H.H. PETER (a.G.)"

Pathologisches Institut' und Abteilung fUr Klinische munologie und Rheumatologie", Universitat Freiburg

Im-

We report on three cases of intravasal lymphoma of B cell type (angiotropic large cell lymphoma) clinically presenting as an autoimmune disease: (1) 41 years old female with the diagnosis of an acute rheumatoid arthritis (rheumatoid factor-negative) meeting the criteria of the "American College of Rheumatology"(ACR). The disease could be elucidated by the post-mortem finding of organized vascular occlusions of synovial blood vessels, obviously due to obstructive accumulations of lymphoid blasts of the intravasal lymphoma. (2) 47 years old female with the clinical diagnosis of p-ANCA-positive panarteritis nodosa meeting 5 criteria of the ACR. The histology of a muscle biopsy was compatible with panarteritis. Post-mortem explanation of the disease by demonstration of intravascular occlusive blasts of the B cell type. (3) 62 years old female with generalized teleangiectatic skin rash. Diagnosis of a pronounced lympho-histiocytic vasculitis with vascular occlusions on repeated skin biopsies, no clear demonstration of the intravasal lymphoma of B cell type that was not demonstrated until post-mortem examination. The preferential intravasal localization of the lymphoma may be explained pathogenetically as a result of disturbed migration of the blasts through the vessel wall, resulting in intravasal accumulation of the lymphoma cells with secondary thrombotic and reactive perivascular inflammatory processes. Immunohistologically, some vessels with accumulations of blasts showed no reactivity of the endothelial marker CD 34, that could be hypothetically related with a local lack of adhesion molecules.

FORMAM IDE FREE mRNA IN SITU HYBRIDIZATION - IS THERE AN UNIVERSAL PROTOCOL? A. BERNDT (a.G.), H. KOSMEHL, D. CELEDA (a.G.)"', D. KATENKAMP Institute of Pathology, Friedrich Schiller University, lena; Institute of Molecular Biotechnology, Jena, Germany* Aims: To define conditions for a standardized and sensitive non radioactiv~ mRNA in situ hybridization on cryostat and paraffin sections the influence offormamide content and hybridization temperature was studied using digoxigenin (Dig) labelled oligodeoxynucleotide probes. Methods: Hybridizations were performed using a basic protocol unless the formamide content was varied between 0% and 25% , the hybridization temperature was set at room temperature (RT), 37°C or 45°C. Dig-Probes: 200nglml of poly d(T) and B actin sense / antisense (both Dianova, Germany), PDGF-A and PDGF-B probe cocktails, insulin and preproglucagon probe cocktails (all from R&D System, UK). Tissue material: cryostat sections of Dupuytren's fibromatosis nodules, paraffin sections of a neuroendocrine tumor of the pancreas. The results were evaluated by sense probe hybridization, RNase predigestion and immunohistochemistry. Detection: DIG- Nucleic Acid Detection Kit (Boehringer Mannheim, Germany). Results: Using poly d(T), B actin sense / antisense probes and PDGF probes b~st hybridization results on cryostat sections of Dupuytren's disease were achieved under formamide free and RT conditions. Formamide free insulin and pre-proglucagon hybridization on paraffin sections at RT generates also good results. An increase in formamide content (25%) and hybridization temperature 37°C prevents glucagon hybridization but improves the hybridization signal for insulin. Conclusion: In contrast to the general accepted rules ofmRNA in situ hybridization formamide free conditions and low hybridization temperature could result in an optimal hybridization signal. An appropriate hybridization protocol is suggested. Nevertheless, it is necessary to test the individual optimal hybridization conditions for each single probe.