Ralf Wassmuth

Fcγ receptor IIa polymorphism in caucasian patients with systemic lupus erythematosus: Association with clinical symptoms

OBJECTIVE The class II human leukocyte Fcy receptor for IgG (FcgammaRIIa) occurs in 2 codominantly expressed allelic forms (R131 and H131). Cells expressing IIa-H131 interact much more effectively with complexed IgG2 and IgG3 than do cells with IIa-R131. This might be linked to variability in immune complex handling, and therefore related to disease pathogenesis. The present study examines these possibilities in a cohort of Caucasian patients with systemic lupus erythematosus (SLE). METHODS One hundred eight Caucasian patients were diagnosed with SLE according to the American College of Rheumatology criteria. The SLE patients and 187 Caucasian controls were genotyped for the FcgammaRIIa polymorphism, and associations between FcgammaRIIa genotypes, selected HLA haplotypes, and clinical as well as laboratory features were analyzed. RESULTS No significant skewing of the FcgammaRIIa polymorphism was observed in the SLE cohort. Various clinical and serologic parameters were found more frequently or at a younger age in patients homozygous for the genotype IIa-R/R131 compared with those with the genotype IIa-H/H131. In patients with the genotype IIa-R/R131, significantly higher frequencies of proteinuria, hemolytic anemia, anti-nuclear RNP antibodies, and hypocomplementemia were found. The only clinical symptom observed more frequently in patients homozygous for IIa-H/H131 was livedo. Patients with the IIa-R/R131 genotype were significantly younger at disease onset and had an earlier incidence of arthritis, sicca syndrome, nephritis, lymphadenitis, hematologic abnormalities, immunologic abnormalities, lupus anticoagulant, cryoglobulinemia, and hypocomplementemia. HLA-DR3 was found in 41.7% of SLE patients, but was not associated with clinical symptoms, serologic abnormalities, or the homozygous genotypes of the FcgammaRIIa, although an association with a significantly later onset of SLE was found. CONCLUSION The FcgammaRIIa polymorphism constitutes an additional factor that might influence the clinical manifestations and course of SLE, but does not represent a genetic risk factor for the occurrence of SLE.

Estimation of high-resolution HLA-A, -B, -C, -DRB1 allele and haplotype frequencies based on 8862 German stem cell donors and implications for strategic donor registry planning.

Human leukocyte antigen (HLA) haplotype frequency distributions in specific populations can be applied to optimize both individual stem cell donor searches and donor registry planning. We present allele and haplotype frequencies derived from a data set of 8862 German stem cell donors who were typed at high resolution for the HLA-A, HLA-B, HLA-C, and HLA-DRB1 genes upon registration. Calculated haplotype frequencies were used to estimate the probability p to find matching donors subject to donor registry size n. The impact of various matching standards on p(n) was analyzed. When high-resolution matching for HLA-A, HLA-B, HLA-C, and HLA-DRB1 is required, p(1,000,000) is 0.678. The corresponding value for n = 7,000,000 is 0.859. In a scenario with low-resolution matching and no consideration of HLA-C, p(1,000,000) is 0.863 and thus larger than p(7,000,000) in the scenario with stricter matching requirements. As recent findings support the importance of high-resolution matching of HLA-A, HLA-B, HLA-C, and HLA-DRB1 for outcomes of hematopoietic stem cell transplantation, our results are highly relevant for strategic planning and resource allocation of donor centers and registries.

Fcγ receptor IIa, IIIa, and IIIb polymorphisms in German patients with systemic lupus erythematosus: association with clinical symptoms

Background: Receptors for IgG play an important part in immune complex clearance. Several studies have identified polymorphisms of receptors for the Fc fragment of IgG (FcγR) as genetic factors influencing susceptibility to disease or disease course of systemic lupus erythematosus (SLE). Objective: To examine these possibilities by evaluating a panel of clinical parameters in a cohort of 140 German patients with SLE for correlations with the FcγRIIa, IIIa, and IIIb polymorphisms in an explorative study. Methods: 140 German patients with SLE according to American College of Rheumatology (ACR) criteria and 187 German controls were genotyped for the FcγRIIa, IIIa, and IIIb polymorphisms. Associations between FcγR genotypes, combined genotypes and clinical as well as laboratory features were analysed. Results: No significant skewing of any of the three FcγR polymorphisms was seen in the German SLE cohort studied. Various clinical and serological parameters were found more frequently and at younger age in homozygous patients with the genotypes IIA-R/R131 or IIIA-F/F158 than in patients with IIA-H/H131 or IIIA-V/V158. These effects were even more pronounced in patients with the low binding combined phenotypes of the FcγRIIa, IIIa (double negative phenotypes) and FcγRIIa, IIIa, and IIIb (triple negative phenotypes). In patients with the double negative IIA and IIIA genotypes significantly higher frequencies of nephritis (63% v 33%) and proteinuria according to ACR criteria (58% v 11%), anaemia (84% v 55%), and anticardiolipin antibodies (63% v 22%) were found than in patients with the double positive genotypes. Patients with the IIA-R/R131 genotype and the double negative homozygous genotype had an earlier incidence of clinical symptoms, haematological and immunological abnormalities. Accordingly, SLE is diagnosed earlier in these patients, the difference reaching statistical significance only in the double negative v the double positive genotype (26.3 v 39.5 years) and the IIIA-F/F158 genotype v the rest (26.7 v 32.0 years). Most relevant is the fact that a higher median disease activity (ECLAM score) was demonstrated, both in the IIA-R/R131 homozygous (3.3 v 2.7) and the double negative (3.4 v 2.3) patients, reaching statistical significance in the first group. Conclusion: The results of this explorative study support the view that the FcγRIIa/IIIa and IIIb polymorphisms constitute factors influencing clinical manifestations and the disease course of SLE but do not represent genetic risk factors for the occurrence of SLE. Higher frequencies of clinical symptoms, haematological and immunological abnormalities as well as an earlier onset of clinical symptoms, haematological and immunological markers of active disease were found in patients with the IIA-R/R131 genotype and the double negative and triple negative genotypes.

Microchimerism after liver transplantation : Prevalence and methodological aspects of detection

BACKGROUND Microchimerism after liver transplantation is a readily observed phenomenon. The immunological implications, however, remain unclear. Moreover, methodological approaches and their detection limits in the study of allogeneic microchimerism have not been studied in detail. METHODS Therefore, the aim of this study was to evaluate the single-step and nested formats of the polymerase chain reaction/sequence-specific priming (PCR-SSP) approach under standardized conditions. For that purpose, a panel of recombinant plasmid clones was generated by PCR cloning. The panel contained the allelic sequences of the second exon of DRB1 covering all DR specificities on a low-resolution level. Using this panel, limiting dilution assays for various DR sequences in the presence and absence of competitor DNA were carried out to determine the minimal number of copies required for detection by single-step and nested PCR-SSP. Subsequently, 22 liver transplant recipients were analyzed in a retrospective study for the presence of allogeneic microchimerism by nested PCR-SSP. RESULTS Although at least 10 copies of template DNA could be detected by nested PCR-SSP overall, single-step PCR-SSP was on average 10(2) to 10(3) times less sensitive. Upon the addition of human competitor DNA, the detection limits decreased on average by a factor of 10. In addition, sequence-specific differences in amplification efficiency could be appreciated. Using nested PCR-SSP, peripheral blood allogeneic microchimerism could be observed in 17 of 22 HLA-DR-mismatched liver recipients. Recombinants representing recipient DRB1 specificities were used to exclude false-positive results by lack of cross-reactivities of the donor-specific primers and to evaluate negative results due to sample-related reduced amplification efficiencies in microchimerism-negative recipients. In donor/recipient combinations that differed by at least one DR specificity, allogeneic microchimerism was seen in 87.5% of the cases. In five chimerism-negative cases, sample-related problems were detected in two cases. CONCLUSION The optimization and standardization of the detection of genomic HLA sequences at low copy number may be greatly facilitated using a clonal reference system. Furthermore, a clonal reference system may be used to conduct cross-priming experiments to exclude false-positive results and may allow the determination of sample-specific detection limits for donor-derived HLA-DR specificities in chimerism-negative patients. Our evaluation of the PCR-SSP approach for the study of allogeneic microchimerism indicated that nested PCR-SSP provides the most sensitive format when HLA sequences are targeted. Yet, the detection sensitivity may vary between individual alleles and specificities. Allogeneic microchimerism in liver recipients can be observed in the majority of patients. However, the detection may be subject to the degree of mismatching, the HLA-DR alleles involved, and sample-related impaired PCR amplification efficiency.

Differential inhibitory effects of intravenous immunoglobulin preparations on HLA-alloantibodies in vitro.

Background. Treatment of allosensitized patients with intravenouslyadministered pooled immunoglobulin preparations (IVIG) may lead to along-lasting reduction of anti-HLA alloantibody titers. An inhibitory responseof IVIG preparations on lymphocytotoxicity is suggested to depend on IgG andto predict a successful reduction of anti-HLA alloantibodies upon theadministration of high-dose IVIG invivo. Methods. In this study, we evaluated different IVIG preparations fortheir in vitro inhibitory capacity on lymphocytotoxicity and binding ofanti-HLA alloantibodies to purified HLA antigens. For that purpose sera from24 highly sensitized patients awaiting kidney transplantation and serologicalHLA testing reagents were used. Panel-reactive antibody (PRA) determinationsusing standard complement-dependent cytotoxicity testing and anti-HLAalloantibody determination by ELISA were carried out in the presence andabsence of 50% (v/v)IVIG. Results. The addition of IgG-containing IVIG preparations gave onlya moderate inhibitory response judging from the average decrease of PRA levels(absolute &Dgr;PRA range: −2% to 16%), whereas the largest inhibition oflymphocytotoxicity was seen after the addition of IgM/IgA-containing IVIGpreparations (absolute &Dgr;PRA range: 19% to 44%). For both IgG andIgM/IgA-containing IVIG preparations, the reduction of lymphocytotoxicityoccurred in a dose-dependent fashion without a preference for particularanti-HLA class I antibody specificities. Significantly lower inhibitoryeffects on anti-HLA antibody reactivity were observed when the effects of IVIGpreparations were monitored by ELISA (absolute &Dgr;PRA range: 7% to22%). Conclusions. Our data suggest that the immunomodulatory capacity islargely caused by the IgM/IgA fraction of IVIG when analyzed bylymphocytotoxicity. The different effect on ELISA versus complement-dependentcytotoxicity testing suggests that interactions of IVIG with complement ratherthan anti-idiotypic antibodies may contribute to the inhibitory effects ofIVIG invitro.

Multiple and High-Titer Single Autoantibodies in Schoolchildren Reflecting the Genetic Predisposition for Type 1 Diabetes

Abstract: The study aimed to compare the HLA specificities of AAb‐positive healthy schoolchildren with those of patients with type 1 diabetes (T1D). HLA‐DRB1 and DQB1 alleles were determined in 178 AAb‐positive and 339 AAb‐negative schoolchildren aged 6‐17 years without first‐degree relatives with T1D and in 274 patients with T1D. AAbs against glutamic acid decarboxylase (GADA), protein tyrosine phosphatase (IA‐2A), and insulin (IAA) were determined by 125I‐antigen binding, and islet cell cytoplasmic antibodies (ICAs) immunohistochemically. Here, 82.6% (147/178) of AAb‐positive schoolchildren had single AAbs and 17.4% (31/178) had multiple AAbs. In both groups, GADA occurred with highest and IAA with lowest frequency. In children with single AAbs at levels between the 99th and 99.9th percentile, frequencies of the diabetes‐associated DRB1 (*03, *04) and DQB1 (*02, *0302) alleles and the protective DRB1 (*15) and DQB1 (*0602) alleles did not differ from those of controls. In patients, the positive associations were confirmed for DRB1*04 (OR = 5.39) and DQB1*0302 (OR = 9.05), whereas DRB1*15 (OR = 0.05) and DQB1*0602 (OR = 0.06) were negative‐associated (p < 0.001). The same association was found in schoolchildren with multiple AAbs for DRB1*04 (OR = 3.84), DQB1*0302 (OR = 4.95), and DRB1*15 (OR = 0.1; p < 0.001‐0.014), and with high‐titer single AAbs (≥99.9th percentile), but none of them had DQB1*0602. The highest risk genotype DQB1*02/*0302 occurred in 36.5% of patients (OR = 21.07) and in 19.3% of children with multiple AAbs (OR = 8.8; p<0.001). It is concluded that probands with multiple and high‐titer single AAbs in the general population have the same genetic predisposition for T1D as patients and are therefore at highest risk for the disease.

DQB1 promoter sequence variability and linkage in caucasoids.

Sequence variability in the upstream regulatory regions (URR) of HLA class II genes has been described as an additional mechanism of diversity in these polymorphic genes. For HLA-DQB1, 12 URR variants have been identified previously by sequence analysis of approx. 600 bp located immediately upstream of the first exon of the DQB1 gene. To investigate the distribution of these promoter alleles and their linkage with the structural portion of the DQB1 gene, a population-based study was carried out. Sequence information was utilized to develop 25 sequence-specific oligonucleotide probes to analyze enzymatically amplified locus-specific DNA. Supplemented with one sequence-specific primer pair to differentiate QBP1-6.2 from -6.3, all known 12 QBP1 alleles could be identified. Subsequently, 215 healthy, unrelated German controls were investigated for the distribution and linkage of DQB1 and QBP1 alleles. A total of 10 out of 12 known QBP1 alleles were observed. Since there was tight linkage between the promoter region and exon 2 of DQB1, the phenotype and genotype frequencies of the promoter alleles corresponded by and large to the frequencies observed for their linked DQB1 alleles. Exceptions were mainly seen for DQ5 and DQ6 haplotypes, as single DQB1 alleles could be linked to different, however, closely related QBP1 alleles and vice versa. Interestingly, for each DQB1 allele a single DQB1/QBP1 haplotype dominated (75.9 to 96.4%) the distribution. It is concluded that promoter and coding region variability are tightly linked by linkage disequilibrium. Exceptions are restricted to DQB1 DQ5 and DQ6 haplotypes. Since functional differences between different QBP1 alleles exist, the maintenance of haplotypic integrity may be of functional importance.

Gliadin Antibodies in Adult Insulin-Dependent Diabetes - Autoimmune and Immunogenetic Correlates

Gliadin antibody (GA) tests used in screening for coeliac disease (CD) frequently yield positive GA results without accompanying CD in cases of diabetes mellitus type 1 (DM-1). To enlighten this phenomenon we screened 848 DM-1 patients for IgA- and IgG-GA. Subsequently, 16 out of 19 high titre GA patients (6 with CD) were compared with 37 low titre DM-1 patients matched for sex, age and disease duration, for autoimmune and immunogenetic markers. Chronic thyroiditis and thyroid peroxidase (TPO) antibody positivity were more frequent in the GA-positive than in the GA-negative sub-group (38 vs. 2.7%, p=0.003, and 69 vs. 27%, p<0.001, respectively). The tissue transglutaminase (tTg) IgA titres correlated with CD but not with GA. tTg IgG titres were lower in GA-positive individuals (p = 0.0012). GA-positivity correlated with a higher titre of factor XIII IgA antibodies (p < 0.001). GA-positive DM-1 patients were characterised by a distinct immunogenetic profile; the risk of HLA DQB1*02 was lower among GA-positive patients than among GA-negatives (OR 0.4, preventive fraction 0.43). All CD patients were HLA DRBl*03-DQBl*02-positive, but none of the five patients with normal biopsies. GA-positive patients instead had HLA DRB1 * 13 in 37.5% as compared to 8.6% in GA-negative (OR 6.4, etiologic fraction 0.32). Thus, the occurrence of positive GA in DM-1 is correlated to TPO antibody positivity, thyroiditis and factor XIII IgA antibodies, but inversely correlated to tTg IgG, and seems to be associated with another HLA haplotype than that previously found to be associated with CD

Cyclosporine A sensitivity in vitro and P-glycoprotein expression in patients on dialysis and after kidney transplantation.

BACKGROUND In allogeneic kidney transplantation the response to cyclosporine A (CsA) is important for graft outcome. Although CsA therapy is controlled by drug monitoring to ensure therapeutic CsA levels, the sensitivity to the effects of CsA varies among individuals. Since CsA is an antagonist of cytostatic drugs in P-glycoprotein (Pgp)-mediated transport, increased Pgp expression might contribute to an increased resistance to CsA. METHODS The sensitivity of lymphocytes at three different concentrations of CsA was tested in a non-radioactive lymphocyte-transformation test and related to Pgp expression as determined by flow cytometry on mononuclear cells. Five groups, including healthy donors (CON; n = 25), patients on dialysis (DIAL; n = 25), patients before transplantation (PTX; n = 5) and after transplantation [short-term (ATX; n = 5) and long-term (LTX; n = 25)] were investigated. RESULTS In LTX, the sensitivity to CsA at 400 and 1000 ng/ml was significantly different from CON and DIAL. Overall a higher sensitivity to CsA was seen in patients after transplantation. In ATX, sensitivity to CsA was significantly higher than in PTX at a concentration of 1000 ng/ml CsA. However, comparing all groups no significant changes in Pgp expression were noted. Analysing the relationship between CsA sensitivity and Pgp expression, no significant heterogeneity could be observed between the different groups. CONCLUSION In conclusion, our data suggest that in vitro testing of CsA sensitivity prior transplantation and Pgp expression monitoring yield independent results and cannot substitute for each other as predictors of graft outcome. The differential role of each test for the evaluation of CsA sensitivity or resistance remains to be determined.

NK-KIR Gene Repertoire and Outcome of Patients with Acute MyeloidLeukemia after Allogeneic Hematopoietic Cell Transplantation from UnrelatedDonors

In this retrospective study, the influence of donor and recipient KIR-gene content and their respective ligands including clinical parameters as potential confounding variables on the outcome of 150 acute myeloid leukemia (AML) patients undergoing allogenic hematopoietic cell transplantation (HCT) from unrelated donors was systematically investi- gated. There was no significant influence of KIR ligand mismatching and of donor/recipient KIR haplotype combinations on overall survival (OS), disease free survival (DSF), non-relapse mortality (NRM) and relapse. Isolated effects of KIR haplotypes, were detected for acute, chronic Graft versus Host Disease (aGvHD and cGvHD) as well as for the cumula- tive incidence of non-relapse mortality and relapse. The incidence of non-relapse mortality was evaluated in donor and re- cipient pairs harbouring KIR AA homozygosity (AA/Bx: p=0.038, HR=0.73, 95% CI=0.35-1.46 and AA/AA: p=0.043, HR=0.64, 95% CI 0.53-1-17). Our data suggest that KIR genotyping may be useful in patients in whom several HLA- identical unrelated donors can be identified but is probably not necessary for the primary donor selection algorithm.