workshop p: pediatric immunology

WORKSHOP P

Pediatric Immunology

Klinik fur Kinder- undJugendmedizin der Ruhr-Universitat Bochum im St.-JosefHospital, Bochum, Germany

P.l Enhanced IL-10 generation induced by the cows milk protein beta-lactoglobulin in cord blood lymphocytes from children with a high risk of atopy

A. ECKHART-RINGEL, K. WENGENROT, T.JUNG, C. RIEGER, and U. SCHAUER

The first allergic symptoms in infancy are often related to cows milk allergy. In order to elucidate the role of specific T cells in the development of allergic symptoms we started a prospective study from birth throughout the first year of life. Heparinized cord blood was obtained from 8 infants with no risk of atopy (negative family history, cord bloodIgE < 0.9KU),8 infants with medium risk (one parent with manifest atopy or specific IgE) and 4 infants with high risk (both parents with atopy and/or cord blood IgE > O.9KU). In whole blood cultures the cells were incubated with 10 Ilg/ml betalactoglobulin (3rC, 8 % CO2). Beta-lactoglobulin is the protein with the highest allergenicity in cows milk. After three days cytokine production was assessed by intracellular analysis as described by JUNG et a!. O. Immuno!. Methods 1993, 159: 197). CD45 RO+ (memory) cells and CD45 RA+ (naive) cells were analyzed for interleukinto (IL-tO) and interferon-y (IFN-y) generation on a single cell niveau. IL-tO production in response to lactoglobulin by memory T cells at birth was significantly increased in high risk infants (median t5.05 % positive cells) compared to children without risk (median 2.6 %) and children with medium risk for allergy (median 4.6 %). No differences in cytokine production by CD45RA+ cells were seen and no differences of the percentage of IFN-y producing CD45 RO+ cells could be detected . At six weeks of age the children were re-examined and displayed essentially the same differences in IL-t 0 production. We conclude that cows milk sensitization is already present at birth and that IL-tO might play an important role in the development of atopy. The further follow up of the children at the age of six and twelve months will show the clinical significance of our findings.

Supported by BMFT ERG to.

25th Meeting of the Society of Immunology . 295

Clinical Immunology, Central Hospital, Tallinn, Estonia

P.2 Primary immunodeficiency in children in North-Estonia

M. GLOKMANN and S. VELBRI

'We have analyzed the cases of primary immunodeficiency in children treated in different hospitals of North-Estonia during 1986-1993. All these patients were studied in the laboratory of clinical immunology of Tallinn Central Hospital. 6107 children were studied during this 8-year period. Serum immunoglobulins were determined in all patients; in suspect of immune disorder the other immunological analyses (T and B lymphocytes, phagocytosis, immune complexes, RF etc.) were made too. Among these children there was detected one 4-year-old boy with chronic granulomatosous disease and one girl with DiGeorge syndrome at the first month of life. 5 cases of common variable hypogammaglobulinaemia were studied repeatedly, from which 3 children were detected during the analyzed period. Selective IgA deficiency was ascertained in 30 children. The main disorders in children with IgA deficiency were recurrent respiratory (16 cases) or other infections (6 cases). Juvenile arthritis was the main diagnosis in 4 cases of IgA deficiency, myasthenia - in one girl and rheumatism - in 3 cases.

Clinic for Dermatology, Clinic for Pediatric Surgery, Clinic for Pediatric Diseases, and Institute for Medical Immunology, Charite Berlin, Berlin, Germany

P.3 Immune phenomena in children with portale hypertension

S. JAHN, E. FIETZE, A. FLEGEL, C. HELLMANN, C. LIEBENTHAL, P. SIEGEL, F. KIRCHMAIR, A. LUKOWSKY, H.-D. YOLK, U. OTTING, H. MAu, and U. SPECHT

We investigated immune parameters in 22 children with portale hypertension because of a pre-hepatic block due to thrombosis in the v. portae. The main clinical symptoms were hypersplenism resulting in hemorrhagic complications, severe bloody esophageal varices and chronic infections. The aim of this study was to analyze several immune parameters reflecting immune dysregulation due to hypersplenism on the one hand side and the impaired liver function (acute phase response mechanisms) on the other side. Cytofluometric measurements showed a marked selective CD4-lymphopenia in most children. The immune state frequently reflected chronic bacterial/mycotic infections. In 8/17 patients we registered the appearance in the serum of pathogenetic ally relevant autoantibodies directed against dsDNA, ssDNA, Sm, Ro, cardiolipin or thrombocytes. Furthermore, we did not find measurable TNF-alpha levels in all sera (comparable to healthy probands). However, in only 5/20 sera low levels of IL-I-beta were seen what is rather different from what is seen in healthy controls. In 6/20 probes we found elevated IL-6 levels, normally not seen in healthies. We discuss these preliminary data in context with immune dysbalance due to hypersplenism and impaired liver function.

296 . 25th Meeting of the Society of Immunology

lUniversitatskinderklinik Gottingen; 2Universitatskinderklinik Munchen, Germany

P.4 Residual superoxide production in variant chronic granulomatous disease

v. JENDROSSEK\ B. NEUBAUER!,J. LIESE2, B. BELOHRADSKy2, and M. GAHRI

Patients with chronic granulomatous disease (CGD), an inherited disorder with deficient NADPH-oxidase-activity of phagocytes, are predisposed to severe recurrent infections. In two-thirds of the patients the X-gene coded gp91phox protein (~-chain of cytochrom bss8) is affected, the other forms with deficiency of the p22phox, p47phox or p67 phox are of autosomal inheritance.

Characterization of 34 CGD-patients showed lack or dysfunction of the gp91phox in 19 patients, defects of the p22phox, p47phox and p67phox were found in 1, 5 and 5 patients respectively. Interestingly a large number of patients was identified having residual activity of the NADPH-oxidase. Accordingly, in a high percentage of patients (10/34) diagnosis was delayed and/or the first symptoms were observed at an age over ten years. Five patients are now older than 25, two older than 40 years.

After characterization of the genetic defect by sequencing the gp91phox eDNA we were able to correlate the functional, biochemical and genetic data of the patients with residual activity and to compare them with these of patients without residual activity.

Supported by a grant from the Deutsche Forschungsgemeinschaft (Ga 148/7-2).

Klinik fur Kinder- und Jugendmedizin der Ruhruniversitat Bochum im St.-JosefHospital, Bochum, Germany

P.S Evaluation of intracellular proteins in eosinophils by flow cytometry

N. KRUG, A. M. THURAU, C. H. L. RIEGER, and U. SCHAUER

Eosinophils playa major pathophysiological role in allergic diseases. The release of their basic proteins can cause tissue damage and contribute to the allergic inflammation. We developed a method to evaluate the intracellular content of eosinophilic cationic protein (ECP) in eosinophils by flow cytometry using whole blood samples: Erythrocytes were lysed and leukocytes fixed in 0.4 % p-benzoquinone. This fixation procedure prevents unspecific binding of the fluorescent dye FITC, which is a major problem using paraformaldehyde as a fixative (FREMDEN et al. 1986. Histochemistry 84: 247-250). Cell membranes were permeabilized by 0.74 % n-octyl-beta-D-glucopyranoside (HALLDEN et al. 1989. J. Immunol. Methods 124: 103-109) and antibodies against ECP (EG 1, EG2) added. FITC conjugated goat anti-mouse IgG was used as a secondary antibody. During flow cytometric evaluation eosinophils could be easily distinguished from other leukocytes as a population with preserved high granularity (sideward scatter) after fixation and permeabilization. This population could be characterized by its high surface expression of CD9 (98 %) and low expression of CD16 (0.5 %). Additional experiments with highly enriched eosinophils (> 90 %) showed that> 90 % of the cells could be detected in the cell population, preliminary results with the described method revealed an increased level of ECP (EG2) measured as mean fluorescence, in allergic asthmatic patients (median 121,


range 99-306, n = 9) in comparison to normal controls (median 82, range n = 7) with a decrease 24 h after allergen challenge (median 88, range 58-135, n = 7). In conclusion we consider this method suitable for routine research use.

Supported by the DfG (Scha 395/3-1).

Institute of Clinical Immunology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

P.6 Primary immunodeficiencies in a population of the Czech Republic

J. LOKA]

A systematic search for primary immunodeficiencies (PI) in the out-patients from South Moravia region (2 million inhabitants) was undertaken since 1980. Thirty-eight thousand patients of any ages, of both sexes, have been examined in our department. The syndromes of PI have been classified according to WHO criteria. PI syndromes diagnosed were: selective deficiency of IgA (84 patients), common variable immunodeficiency (25 patients), X-linked agammaglobulinemia (5 patients), IgM deficiency with increased IgM (2 patients), hyper-IgE syndrome (1 male child), chronic granulomatous disease (2 boys) and hereditary angioedema (30 patients in 8 families). Our results point to a significant incidence of PI in our region, the antibody and complement defects are the most common of them.

Dept. of Pediatrics, University of Ulm, Ulm, Germany

P.7 Reticular Dysgenesis secondary to an intrauterine alloreaction

W. FRIEDRICH, S. MOLLER, and H. BROMMER

In patients with SCID, an intrauterine maternal-fetal transfusion may lead to persistant engraftment of maternal lymphocytes. Clinical findings of graft versus host disease in these patients are rather variable and frequently mild and even absent. Within a total group of 78 infants with SCID admitted to our institution since 1982, maternal T cells were demonstrable in 31 cases, indicating a rather high incidence of this complication. Seven patients presented with striking hematological abnormalities at birth, consistent with the diagnosis of Reticular Dysgenesis. All showed agranulocytosis and variable deficiencies of other blood cells, including global hematological failure and panytopenia in 2 cases. Abnormalities were in part reversible on immunosuppressive treatment, while laboratory abnormalities of SCID remained unchanged. Immunological reconstitution was achieved by bone marrow transplantation. Our findings indicate that Reticular Dysgenesis does not constitute a variant but rather a secondary complication of SCID, resulting from an intrauterine alloreaction mediated by maternal T cells, which leads to variable hemopoietic insufficiency.

Supported by the Deutsche Forschungsgemeinschaft (SFB 311).


Klinik fur Kinder- und Jugendmedizin der Ruhruniversitat Bochum, Bochum, Germany

P.8 Effects of lipopolysaccharide (LPS) and interferon-y (IFN-y) on the generation of I FN-y

D. POHL, V. BAUMEISTER, C. H. L. RIEGER, and U. SCHAUER

There is increasing evidence that IFN-y plays an important role in the regulation of inflammatory processes induced by allergens or infectious agents. IFN-y is increased in type IV reactions while it is decreased in delayed type I reactions. To elucidate the mechanisms involved in the differentiation of T cells secreting high or low amounts of IFN-y, human PBMC (peripheral blood mononuclear cells) were isolated by Ficollgradient-centrifugation, stimulated with SEE (staphylococcal enterotoxin E) 100 ng/ml and cultured in the presence of different costimulatory agents (IL 2, IL 10, IL 13, IFN-y, LPS, methylprednisolone, isoproterenol, theophyllin, disodiumcromoglycat). On day 7 the cells were restimulated with PMA 10 ng/ml and ionomycin 1 ~M for 5 hours. Subsequently, the amount of intracellular IFN-y was measured by FACS-analysis as described by JUNG et al. a. Immunol. Methods 1993, 159: 197). IFN-y and LPS produced the most striking modulatory effects. Exposition to IFN -y 1 ng/ml results in an increased number of cells producing IFN-y (mean increase: 90.15 % ± 25.84; n = 3). Cells cultured in presence of LPS 1 ~g/ml show a marked decrease of IFN-y-production (median: 48.54% ± 17.88; n=5). Although the percentage of cells generating IFN-y showed a wide donor dependent range from 1.14 % to 62.27 %, the modulatory effects was observed in all cultures in a concentration-dependent manner. Based on the observation that the ratio of IFN-y to IL-4 is an important regulative factor in immune responses, decreased levels of IFN-y following LPS exposition (e.g. as a consequence of bacterial colonization) could favor an allergic breakthrough in individuals at risk for atopy. In further experiments it remains to be shown whether IFN-y production following LPS-exposition varies between allergic and non-allergic individuals.

Supported by the DFG (Scha 395/2-1).

Dept. of Pediatrics,J.-W.-Goethe-University, Frankfurt a. M.; lUniversity of Mainz, Mainz; 2U niversity of Bonn, Bonn, Germany

P.9 Altered C040 ligand expression in patients with common variable immunodeficiency (CVIO)

J. RAHMIG, S. ZIELEN, F. ZEPpl, R. BIALEK2, P. SEHRT, and D. HOFMANN

We could recently demonstrate that B cells of most patients with CVID mature to plasma cells when activated by the CD40-system and IL-l 0 (1). In addition, patients who showed a defective expression of the T cell ligand for CD40 (CD40L) suffer from x-linked antibody deficiency with hyper-IgM production (HI GM). The close relationship between CVID and HIGM prompted us to study whether the expression of CD40L is altered in CVID.

T cells of 10 patients with CVID, 5 with HIGM and 10 controls were activated by phorbolester, and the expression of CD40L was studied by flow cytometry using the fusion protein (H-CD40-IgM). As expected, all patients with HIGM showed a defective expression of CD40L « 3 %). In contrast, CD40L expression was clearly demonstrable in


all patients with CVID. However, CD40L expression of CVID patients (median 31 %, range 11-60) was significantly lower than in controls (median 58 %, range 37-73, p < 0.01).

Taken together, our data gave additional evidence that important T cell signals are defective in patients with CVID.

1. LANCET 1993, 342: 750-751.

!Abt. Rheumatologie und Klinische Immunologie, Medizinische Universitats-Klinik, Freiburg; 2 Abt. Immunologie und Transfusionsmedizin, Medizinische Hochschule, Hannover, Germany

P.10 Common variable immunodeficiency syndrome (CVIO) and MxA-protein expression in blood leukocytes

A.J. RUMp!, D.JAKSCH!ES2, U. A. WALKER!, M. SCHLESIER 1

, P. VONWUSSOW2,

and H. H. PETER!

Common variable immunodeficiency (CVID) has repeatedly been suspected to involve persistent viral infections or an autoimmune condition either as underlying immunopathogenic mechanism or as associated phenomena. However, formal proof of a viral participation in the pathogenesis of CVID is lacking. Measurement of MxA-protein in leukocyte lysates has become a sensitive test for evaluating the activation of the host's type-I interferon system. Both in viral infections and autoimmune diseases such as SLE MxA-protein positive peripheral leukocytes are common. We therefore examined the PBL of 15 patients with longlasting hypogammaglobulinemia for their MxA-protein content: 13 patients suffered from CVID, one from hyper-IgM syndrome and one patient had chronic B lymphocytic leukemia associated with immunoglobulin deficiency and chronic papilloma virus infection (condylomata accuminata). Only the latter patient exhibited a strong MxA-protein expression. 2 CVID patients were borderline positive, the remaining 12 CVID patients including the hyper-IgM syndrome were MxA-protein negative. There was no relationship to low CD4/CD8 ratios and increased CDS/CD5r T cell counts, a condition often seen in CVID as well as in chronic viral infections. The results argue against a viral or autoimmune pathogenesis of CVID.

!Division of Rheumatology and Clin. Immunology, Dept. Medicine, and 2Dept. Pediatrics, University Hospital, Freiburg, Germany

P.ll Recombinant IL2 therapy followed by syngeneic bone marrow transplantation of an IL2 deficient infant

J. A. RUMPi, C. NIEMEYER2, M. SCHLESIERi, T. TOCHELMANN2, S. FESKEi, R. DRACERi, A. JAHKElSi, M. BRANDIS2, and H. I I. PETLK 1

The study describes a turkish male infant (VE) with a T cell activation defect resulting in IL2 deficiency. The boy is the second child of consanguinous parents; their first boy died at the age of 11 months of BCGitis due to a similar SCID-syndrome with IL2 deficiency. After an uncomplicated 41 week gestation period, VE suffered S days after birth from


stomatitis aphthosa and fever. The diagnosis of scm was rapidly established by cellular in vitro testing. VE's blood lymphocytes exhibited a strongly impaired proliferative T cell response to anti-CD3/anti-CD28 and ConA and were unable to secrete significant amounts of IL2 and IFN-gamma. Addition of exogeneous IL2 improved the mitogenic T cell response and IFN gamma production in vitro. On the basis of these data the child was treated with increasing doses of recombinant IL2 (Proleukin®, Cetus Corp., Emeryville). The therapy was started with 104 U/kg body weight by subcutaneous injections for 9 days. The dose was increased to 2 x 104 U/kg for another 9 days and then continued for 15 days with 3 x 104 U/kg. The patient tolerated the therapy very well. During rIL2 therapy the lymphoproliferative responses to ConA (10 ug/ml) and antiCD3/anti-CD28 as well as the IFN-gamma synthesis in vitro were improved. The IgG and IgM synthesis in vitro after SAC and IL2 stimulation was normalized. The clinical course of the child was stable allowing an uncomplicated syngeneic bone marrow transplantation (BMT) 5 weeks later. Although maternal T cells could be detected in VE's peripheral blood 4 weeks after BMT T cell functions did not improve so far. Thus, a continuation of the successful rIL2 treatment may represent an alternative therapy in this case of SCID syndrome.

1 Kinderklinik and 2Institut fiir Pathologie der Universitat Wiirzburg, Wiirzburg; 3Institut fiir Pathologie, Kreiskrankenhaus Liidenscheid, Liidenscheid, Germany

P.12 Immunohistological studies in a male fetus carrying the defective gene for X-linked Iymphoproliferative disease (XLP)

XLP is characterized by a selective immunodeficiency to the Epstein-Barr virus (EBV). EBV infection in males with the defective XLP gene leads to fatal infectious mononucleosis, hypogammaglobulinaemia and/or B cell lymphoma. The condition is uniformly lethal by the age of 40 years. Prior EBV infection most males with the defective XLP gene are clinically healthy. In a pregnant female carrier of a large XLP family we performed a prenatal diagnosis by multiplex PCR using two polymorphic markers flanking the XLP gene locus (Xq25-q26). The male fetus was found to carry the defective XLP gene. At the 16th week of gestation a therapeutic abortion was performed. Using immunohistochemistry on paraffin and cryostat sections (T, B cell and macrophage subpopulations, activation/ differentiation/apoptosis markers) we examined the fetus to gain further insights into the development of the immune system in XLP patients. We found normal morphological markers in all organs. Immunoreactive tissues, like spleen, thymus and lymph nodes displayed normal lymphoid and macrophage subpopulations. This is the first report on a immuno-histopathological examination of a fetus carrying the defective XLP gene. Our findings indicate, that the development of the immune system in the fetus seems to be normal until birth. Taken together with post mortem findings from older children with XLP we suggest, that the essential defect in XLP may involve the regulation of apoptosis in activated (auto reactive) T cells.


Kinderklinik der Universitat Wurzburg, Wurzburg, Germany

P.13 Carrier detection in families with X-linked Iymphoproliferative disease (XLP) using haplotype segregation analysis

V. SCHUSTER, S. SEIDENSPINNER, and H. W. KRETH

X-linked lymphoproliferative disease (XLP) is a rare worldwide occurring inherited immunodeficiency which is triggered by Epstein-Barr virus infection. We and others have recently mapped the XLP gene locus to Xq25-q26 using two point and multipoint linkage analysis. The primary defect in XLP is still unknown.

We examined five German XLP-families with 21 affected males. Clinical and laboratory phenotypes ranged from severe and fatal infectious mononucleosis (57°;\») to acquired hypogammaglobulinaemia (28 'Yc,), malignant lymphoma (28 %), aplastic anaemia (19 %) and hypergammaglobulinaemia M (19 %).

Genomic DNA of affected and unaffected family members was examined by Southern blot and polymerase chain reaction (PCR) analysis using different polymorphic Xchromosomal DNA markers which flank the XLP gene locus (Xq25-q26) proximally and distally. In most cases haplotype segregation analysis enabled the determination of female carrier status with an accuracy of > 99 %. Haplotype analysis was also useful for recognizing males not at risk for XLP. Since stem cell or bone marrow transplantation may cure the underlying immunodeficiency, these XLP-negative subjects are potential stem cell and bone marrow donors if they are histocompatible with their affected relatives.

In conclusion, molecular genetic haplotype segregation analysis is a fast and reliable method for the determination of the XLP status in males and females of affected families.

'Sektion Molekularbiologie and 2Kinderheilkunde II, Universitat Ulm, Ulm, Germany; } Abteilung Kinderheilkunde, Universitat Zurich, Zurich, Switzerland

P.14 Mutationen in den rekombinationsaktivierenden Genen 1 und 2 (RAGll2) bei SCID-Patienten

K. SCHWARZl,2, L. LUDWIG 1, D. LINDNER I , W. FRIEDRICH2, R. SEGER}, T. E. HANSEN

HAGGEl, E. KLEIHAUER2, and C. R. BARTRAM 1,2

SCID ist eine heterogene Erkrankung mit unterschiedlichen Defekten der Lymphozytendifferenzierung und -aktivierung. In dieser Studie haben wir SCID-Patienten und die entsprechenden Familien auf Mutationen in den RAG 1/2-Genen untersucht. Diese Genen reprasentieren gegenwartig eine der fruhesten Schlusselstellen in der Lymphozytendifferenzierung. Sie kontrollieren oder interagieren selbst im V (D)J -Rearrangement-ProzeB der Ig- oder TCR-Gene.

30 SCID-Patienten wurden auf genomischer Ebene auf einen «Single Stranded Conformation Polymorphism» (SSCP) als Indikator fur mogliche Mutation untersucht. Die Mutationen wurden sequenziert. Familienanalysen bestatigten den autosomal rezessiven Erbgang dieser SCID-Gruppe.

Vier Kinder in vier Familien hatten RAG1-Mutationen, drei Kinder in zwei Familien hatten RAG2-Mutationen. Ein Kind hatte Veranderungen sowohl im RAG1- wie im RAG2-Gen. Eine Deletion, Missense und Nonsense-Mutationen konnten nachgewiesen


werden. Die funktionelle Konsequenz dieser Mutationen wird gegenwartig in einem Invitro-Rekombinationstest uberpruft.

20 % der getesteten SCID-Patienten hatten somit Mutationen in einem der RAGGene. Der Phanotyp der Patienten ist augerst variabel. Allen gemeinsam ist das Fehlen von peripheren B-Zellen, wahrend T -Zellen nicht oder in geringer Zahl nachweis bar sind. Die T-Zellen sind entweder durch maternofotale Transfusion erworben oder Patienten eigen. 1m zweiten Fall konnen sie aufgrund der «ieakiness» der entsprechenden RAG-Mutation entstanden sein.

U niversitatskinderklinik Freiburg, Germany

P.1S B cell epitope mapping of bovine asl-casein

P. SPUERGIN, M. WALTER, H. MUELLER, and]. FORSTER

We want to identify the B cell epitopes of asl-casein, one of the major allergenes of cow's milk. Furthermore we want to know whether different cow's milk allergic patients react to common or individual epitopes. For this purpose a set of decapeptides shifted by one amino acid were synthesized according to the known amino acid sequence of bovine a s lcasein. The peptides were synthesized on polyethylene pins (Cambridge Research Biochemicals) using the Fmoc-method. Peptides were screened with sera from twenty cow's milk allergic patients using an ELISA to human IgE, comprising successive incubation with three antibodies, the last one labeled with horseradish peroxidase. It could be shown that the individual patient'S sera react with different regions of the a s ]

casein sequence (individual epitopes). This could be proposed based on the disordered conformation of the protein. But there are also some regions showing comparable reactions with different sera. In conclusion> sera from cow's milk allergic patients reacted with different epitopes on bovine as [-casein, but showed also a common reaction pattern.

Universitatskinderklinik, Freiburg, Germany

P.16 Evaluation of an IgE-ELISA specific for bovine a-caseins

P. SPUERGIN, M. WALTER, H. MUELLER, and]. FORSTER

ELISA was done by coating microtiter plates (Greiner) with highly purified a-casein and incubation with diluted human sera. Binding of specific IgE was detected by successive incubation with three antibodies, the last one conjugated with horseradish peroxidase. The ELISA results were compared to CAP-RAST data (code f78, Pharmacia). As a qualitative criterion and to distinguish between immunreactions to the different caseins and other cow's milk proteins IgE-immunoblots were made. Sera from twelve cow's milk allergic patients (age Y2 to 4 years) with positive provocation and skin prick tests to bovine caseins were compared with twelve sera from children (age l;2 to 5 years) with negative tests. 92 % of the allergic children were positive in the ELISA (OD > 0.1),67 % had positive RAST (class> 1) and 83 % showed distinct reactions to a-casein in the immunoblot. None of the control group gave positive rea<:tions in the ELISA or RAST and only one sho,,"ed positive

reactions to a-casein in the immunoblot. In conclusion, our ELISA proved to have a higher sensitivity than the RAST compared with clinical allergy of the children studied.


Institute of Immunology, University of Vienna, and 1 Pediatric Department, Wilhelminenspital der Stadt Wien, Vienna, Austria

P.17 A new phenotype of MHC-c1ass II deficiency

H. M. WOLF, 1. HAUBER, H. GULLE, V. THON, H. EGGENBAUER, M. B. fISCHER, S. FIALA 1, and M. M. ElIlL

Major histocompatibility complex (MHC) class II deficiency is an inherited disease characterized by defective expression of MHC class II antigens on human leukocytes and a cellular and humoral immunodeficiency. We report on twin brothers who showed a defect in MHC class II surface expression on B cells, monocytes and activated T cells. In contrast to previously reported patients, mRNA levels for HLA-DRa in the infants' MNC were normal, but mRNA levels for HLA-DR~ were significantly decreased. Low levels of intracellular HLA-DR alpha and beta chain protein were detected and, most surprisingly, cellular and humoral immune responses were induced in both infants following tetanus vaccination. The two boys presented with a benign clinical course of the disease and appear to represent a new, previously unrecognized phenotype of MHC class II deficiency.

University Hospital, Dept. of Pediatrics, Pediatric Immunology, Mainz, Germany

P.1S Phenotype analysis and clonal specificity of autoreactive T cells in peripheral blood (PB) and synovial fluid (SF) from patients with Juvenile Rheumatoid Arthritis

F. ZEPI', w. MANNHARlrr, P. HABER~IEHL, N. MICHELS, and M. OTHMER

Juvenile Rheumatoide Arthritis ORA) is defined as a non-infectious chronic arthritis of childhood. The etiology and pathogenesis of the disease remain unknown. To test the hypothesis that an autoreactive immune response is involved in the development of JRA cellular immunity was analyzed in children with active disease. - Lymphocyte phenotype, T cell-receptor (TCR) usage and activation pattern of T cells in the PB of 61 patients with JRA (37 pauci-, 17 polyarticular and 7 systemic) and in Sf of 7 children with polyarticular JRA were analyzed using flow-cytometry (FCM). Age-matched healthy children served as controls. Autoreactive T cells with specificity for human cartilage-antigens were cloned under limiting dilution conditions. Clone-phenotype and TCR-usage were determined by fCM, antigen-specificity and MHC-restriction pattern were analyzsed. - In comparison to children with pauciarticular JRA and healthy probands, children with active polyarticular JRA exhibited elevated levels of circulating CD25+/HLADR+ T cells but reduction of CD45RO+ T cells in PB. This was associated with a significant accumulation of activated (CD25-CD45RO-HLADR-positive) T cells in the SF from affected joints. While up to 35 01<, of T cells derived from SF expressed yoTCR initially, cloned -auto reactive T cells without exception carried a~-TCR. The majority of isolated TCC represented HLADR-restricted CD4+ T helper-clones. TCC recognized cartilage-antigens (fetal cartilage, collagen II) in the context of autologous target-cells. Interestingly, 5 TCC exhibited crossreactivity with viral antigens (EBV). The


lack of a predominant TCR-V~-usage argues in favor of a clonally heterogeneous autoimmune response. - JRA is characterized by activation of T cellular immunity and homing of auto reactive T cells at the site of the inflammation. The proof of cartilagespecific autoreactive T cells in SF strongly indicated that a cellular autoimmune response presumably triggered by exogenous antigens plays an important role in the pathogenesis of ]RA.

University Hospital, Dept. of Pediatrics, Mainz, Germany; ISmithKline Beecham, Rixensart, Belgium

P.19 Specific cell-mediated immunity after vaccination with an acellular (DTPa) pertussis vaccine and after natural disease

F. ZEPP, H.]. SCHMITT, M. KNUf, P. HABERMEHL, and M. SLAOUI 1

Pertussis is a two stage disease in which bacterial attachment to respiratory mucosa is followed by pharmacological effects mediated by various toxins. Currently, acellular pertussis vaccines containing adhesins and detoxified toxins are intensively studied, with the objective of achieving protection while minimizing adverse effects attributed to conventional whole-cell vaccines. The aim of this study was the investigation of the specific cellular immune responses induced by DTPa-vaccination in comparison to immune responses after natural disease. - The capacity of peripheral blood T lymphocytes to respond to the pertussis related antigens pertussis-toxin (PT) and the adhesins FHA and 69kDa-protein was investigated in children before and after vaccination with DTPa by measurement of antigen-specific proliferation, lymphocyte phenotype, cytokine production and expression of activation markers (CD25, CD45RO, CD7I, HLADR). The results were compared to findings in children 4-6 weeks after recovery from pertussis infections. - Before vaccination only PT possess unspecific mitogenic properties. Vaccination with the DTPa created a specific T-cellular response to PT, FHA and 69kDa, that was shown to increase continually, depending on the progress of the vaccination schedule. The presence of a definite PT -specific response was proven by testing a non-mitogenic recombinant PT. Phenotype analysis revealed predominant activation of CD4+ -T helper-cells. Measurement of cytokine-pattern showed preferentially induction of IL2/IFNy - producing T helper I-cells. Flowcytometric studies of intracellular cytokine-synthesis pattern further supported these observations.

Our data indicate that DTPa-vaccination induces a potent immune response to PT, FHA and 69kDa equivalent to the specific cellular immune response in the sequel of natural infection.


Universitats-Kinderkliniken 1 Frankfurt a. M., 3Mainz, 4Bonn; 2Institut fur klinische Immunologie, J ena, Germany

P.20 Defective IL-10-production in patients with humoral immunodeficiency

The biological role of IL-10 is still unclear. IL-10 acts as a potent inhibitor of cytokine synthesis by human monocytes and prevents antigen specific T cell proliferation. On the other hand IL-10 is a powerful growth and differentiation factor for activated human B lymphocytes. In addition, B cells of patients with X-linked immunodeficiency with hyper-IgM (HIGM) and common variable immunodeficiency (CVID) produce IgG when B cells are activated through CD40-system in the presence of IL-10. This prompted us to investigate whether failure of immunoglobulin production in CVID and HIGM is in part due to defective IL-10 production.

Mononuclear cells of 8 patients with HIGM, 10 with CVID and 10 controls were activated via different T cell pathways (PHA 5 [!g/ml, CD3+CD28, CD2 and CD2+CD28) and IL-10 production was measured after 48 hours by ELISA. IL-10 production of patients with CVID did not differ from controls except after CD2 activation (median 239 pg/ml, range 34-886 in CVID vs 841 pg/ml, range 296-2073 in controls, p < 0.01). In contrast, independent from mode of T cell activation IL-10 production was hardly detectable in patients with HIGM.

Taken together, our data gave additional evidence that T cell activation via TCR and CD28 is normal in CVID opposed to a defect of the CD2 pathway. However, in patients with HIGM both defective CD40 ligand expression and a defect of IL-10 production may contribute to the failure of B cells to mature to IgG- and IgM-secreting cells.

Universitats-Kinderklinik, Frankfurt a. M., Germany

P.21 A new ELISA technique to measure anti-pneumococcal antibodies

S. ZIELEN, N. STRNAD, P. BAUSCHER, G. WAGENER, and D. HOFMANN

Immunization with polyvalent pneumococcal capsular vaccine followed by measurement of type specific immunity is used in infection prone individuals to exclude an underlying polysaccharide specific immunodeficiency. Previous assays developed for this purpose have shown many limitations because either radioactive antigens or antigens conjugated to poly-L-Iysine or tyramine were required to circumvent poor binding of polysaccharides (PS) to polystyrene ELISA plates.

We developed a new ELISA technique using Nunc CovaL ink NH microtiter plates to measure pneumococcal type specific antibodies. CovaLink NH are modified plates with secondary amino groups bound to their surface, which in the presence of a water-soluble carbodiimide as coupling reagent, facilitate direct binding of polysaccharides.

We compared binding characteristics of pneumococcal PS antigens types 3, 6, 8, 14, 18, 19, 23 and PRP for CovaLink NH and a conventional polystyrene ELISA plate.


Checkerboard titration technique of PS antigens between 0.04-30 [!g/ml clearly demonstrate that with Covalink NH optimal binding of a pooled serum from immunized donors was achieved for all PS antigens tested at a concentration of 1 [!g/ml. As expected, binding of PS to the conventional plate was rather poor even at concentrations of 30 [!g/ml. The CVs for the ELISA ranged from 2 to 6 % for intra-assay and from 5-12 % for inter-assay.

In conclusion, with these new techniques preparation of PS conjugated is unnecessary and measurement of type specific immunity to pneumococcal immunization can be simply achieved.

Institute of Immunology and Division of Neonatology, University of Heidelberg, Heidelberg, Germany

P.22 Complement factor C9 deficiency in term and preterm neonates

G. ZILOW, J. BRUSSAU, W. HAUCK, and E. P. ZILOW

The conventional assay for the quantitation of serum complement factor C9 is radial immunodiffusion using polyclonal antisera to C9. Plasma concentrations of C9 in healthy newborn infants are below the detection limit of about 40 [!g/ml of this method. We therefore established a sensitive ELISA system which allows quantitation of C9 in plasma of term and preterm newborn infants. In this assay polyclonal goat anti-human C9 was immobilized to microtiter plates followed by incubation with the C9 containing sample. Binding of C9 was detected with a peroxidase-labelled second anti-C9 antibody. The test provides a sensitivity of < 1 [!g/ml purified C9. The EDTA plasma concentrations of complement factor C9 were 100 ± 19 [!g/ml in healthy blood donors (n = 20). In all newborns EDTA-plasma samples were obtained within the first 6 hours of life. C9 concentrations in plasma samples of term newborn infants were 13 ± 13 [!g/ml (gestational age > 36 weeks, n = 38). There were no significant differences between healthy neonates, neonates with mothers colonized with group B streptococcus (GBS) (15 ± 13 [!g/ml, n = 22) and GBS-colonized neonates (14 ± 13 [!g/ml, n = 11). In preterm newborn infants (gestational age < 36 weeks, n = 20) significantly diminished C9 concentrations were observed (1.5 ± 2.6 [!g/ml). Compared to healthy preterm babies preterm infants with early onset infection (n = 19) had only slightly elevated C9 concentrations (4.4 ± 5.7 [!g/ml). Complement C9 is known to be required for efficient complement-mediated cytolysis of bacteria, e.g. E. coli. E. coli is one of the major pathogens for neonatal sepsis. The increased incidence of infection in newborn infants due to this germ may be related to these relative C9 deficiency.

workshop p: pediatric immunology

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