Biomedical subjects

V Rebmann

Publications and source records attributed to V Rebmann.

26 records · Page 2Linked to original sources

PubMed1999

Detection of soluble HLA-G molecules in plasma and amniotic fluid.

Although the cDNA sequence of HLA-G antigens is compatible with their expression as soluble molecules (sHLA-G), the determination of native sHLA-G levels in body fluids has not yet been described. The lack of this information is likely to reflect the difficulties in developing an assay suitable to measure sHLA-G antigens in the presence of soluble HLA-A, -B and -C (sHLA-I) antigens, since most of the available anti-HLA-G mAb do not detect soluble beta2-m associated HLA-G antigens or crossreact with sHLA-I antigens. Therefore, we have developed a two-step assay which eliminates the interference of classical HLA class I antigens. In the first step, the sample is depleted of sHLA-I antigens and of HLA-E antigens with mAb TP25.99. Then, HLA-G antigens are captured with mAb W6/32 and detected with anti-beta2-m mAb in ELISA. Utilizing this assay, sHLA-G antigen levels were measured in EDTA plasma from 92 controls with known HLA types, 28 women at delivery and the corresponding cord bloods and in 50 amniotic fluids. Mean sHLA-G plasma levels did not differ between males (24.9+/-3.0 SEM ng/ml; n=42) and females (20.1+/-2.1 SEM ng/ml; n = 50). However, sHLA-G levels in HLA-A11 positive probands (mean: 13.0+/-4.4 SEM ng/ml; n=12) were significantly (P<0.05) lower than in HLA-A11 negative ones (mean: 24.5+/-2.0 SEM ng/ml; n=80). sHLA-G levels in women at delivery (mean: 22.9+/-2.2 SEM ng/ml; n=28) were in the range of controls but were significantly (P<0.001) reduced in the corresponding cord bloods (mean: 13.8+/-1.5 SEM ng/ml; n=28). sHLA-G levels in amniotic fluids (mean: 15.5 + 1.0 SEM ng/ml; n=50) were significantly (P<0.001) lower than in plasma. sHLA-G levels were 5 and 11% of those of sHLA-I antigens in plasmas and amniotic fluids, respectively. Individual sHLA-G levels were not correlated with sHLA-I levels. SDS-PAGE analysis of plasma sHLA-G antigens revealed two molecular variants with a 35 kD and a 27 kD MW corresponding to the sizes of sHLA-G1 and -G2 isoforms. In conclusion, our study has shown that the two-step assay we have developed is reliable in measuring sHLA-G antigen levels. This assay will facilitate the analysis of the biological and clinical significance of sHLA-G antigens in plasma.

Adult↗

PubMed1999

HLA-DR expression and soluble HLA-DR levels in septic patients after trauma.

OBJECTIVE: To determine if cellular and soluble HLA-DR molecules may be relevant in severely injured patients for the development of gram-positive or gram-negative sepsis. SUMMARY BACKGROUND DATA: HLA-DR molecules play a central role in the specific immune response to infection. The reduced HLA-DR expression on monocytes is considered to correlate with infectious complications and the development of sepsis. Data on the role of HLA-DR expression on T cells and soluble HLA-DR molecules are rare. METHODS: HLA-DR expression on monocytes and T cells was measured by flow cytometry. Plasma levels of soluble HLA-DR were studied by enzyme-linked immunosorbent assay. RESULTS: HLA-DR expression on circulating T cells, calculated as mean fluorescence intensity in channels, was reduced at day 1 after admission in 20 patients with subsequent severe sepsis compared with 46 patients without sepsis. The septic patients immediately after trauma had significantly lower soluble HLA-DR plasma levels than the nonseptic patients. At day 2 after admission, HLA-DR expression on monocytes was significantly lower in the severe sepsis group than in the patients without sepsis, and lasted until day 14 after injury. CONCLUSIONS: In severely injured patients, decreased levels of cellular and soluble HLA-DR appear as early indicators of an immune deviation associated with the development of severe sepsis. Moreover, immune alterations of different cell types may promote distinct kinds of septicemia.

Adolescent↗

PubMed1997

Biochemical analysis of plasma-soluble invariant chains and their complex formation with soluble HLA-DR.

The invariant chain (CD74) is preferentially localized in the cytoplasm and regulates the loading of exogenous derived peptides into HLA class II heterodimers. In addition, a small proportion of CD74:class II complexes is also expressed on the cell surface. We identified and quantified soluble CD74 (sCD74) molecules in the plasma and sCD74:sHLA-DR complexes by ELISA. EDTA plasma samples from 86 healthy probands were analyzed. sCD74 could be detected in all samples with a mean concentration of 1.14 relative units +/- 1.04 SD (range 0.17-4.31). Approximately 10% of the samples had increased amounts of sCD74 (>3.0 relative units). Complexes of sCD74 and sHLA-DR were detected in all samples and their quantities were positively correlated (r=0.83, p<0.001) with the sCD74 concentrations. SDS-PAGE analysis of plasma samples with high sCD74 concentrations (>3.0 relative units) revealed four isoforms of sCD74 with molecular weights of 45, 43, 35, 31 kDa corresponding to known sizes of intracellular CD74. However, only molecular weights of the 45 and 43 kDa isoforms of sCD74 are found complexed with sHLA-DR. Our data demonstrate, that CD74 molecules are present in their soluble form in the plasma of healthy probands and form complexes with soluble HLA-DR molecules.

Antigens, CD7↗

PubMed1996

Structural definition of the A*74 group: implications for matching in bone marrow transplantation with alternative donors.

We have identified two new A*74 alleles (A*7402 and 7403) in two unrelated individuals. A*7402 differs from A*7401 by a single amino acid substitution in the signal peptide and may be the result of a gene conversion event at the 3' end of exon 1. A*7403 differs from A*7401 by a single amino acid exchange in the alpha 1 domain and is most likely due to a point mutation in exon 2, since no HLA class I donor allele has been found. Since A*7402 appears to be the ancestor of the other two A*74 alleles, it is possible that A*7401 and 7403 have been created by successive point mutations. The sequences of the expressed proteins of A*7401 and 7402 are identical. The heavy chain sequence of A*7403 differs from these alleles at the crucial residue 79 which is located in the sequence stretch of the alpha 1 alpha-Helix where the Bw4/Bw6 determinants have been identified and which probably affects TCR interaction. This variation can therefore be expected to stimulate alloreactive T cells, graft rejection and graft versus host disease emphasizing the relevance for matching in bone marrow transplantation with alternative donors.

Alleles↗

PubMed1995

Biochemical analysis of HLA-DP gene products by isoelectric focusing and comparison with cellular and molecular genetic typing results.

The polymorphism of HLA-DPA1 and HLA-DPB1 genes of 39 lymphoblastoid cell lines, representing 3 DPA1 and 14 DPB1 alleles, was studied after immunoprecipitation of DP molecules, subsequent isoelectric focusing (1D-IEF) and immunoblotting by a polyclonal DP antibody. Two 1D-IEF variants for DPA1 and 7 for DPB1 gene products were identified, including 2 new variants associated with the DPB1*1401 and DPB1*1901 alleles. The 1D-IEF polymorphism correlated clearly with molecular-genetic-defined alleles and primed lymphocyte or T cell line (PLT) specificities with exceptions observed in DUCAF (DPB1*0202) and ARBO (DPB1*0101). Both exceptional cases may reflect amino acid variations outside the hypervariable regions (HVRs) at the second exon of DPB1. The findings were supported by a comparison of HVR amino acid sequences with estimated net charges to the isoelectric points of the DPB1 molecules. 1D-IEF seems to be a valuable method for studying the expression of DPA1 and DPB1 alleles provided the molecules differ in their isoelectric points.

Amino Acid Sequence↗

PubMed1992

Quantitation of the human component C4: definition of C4 Q0 alleles and C4A duplications.

We determined the C4 plasma concentrations of 48 genotypically CA4- and C4B-defined unrelated individuals from 34 families with a total of 196 members by an enzyme-linked immunosorbent assay using Rg:1,2 (C4A) and Ch:1 (C4B) specific monoclonal antibodies. The results obtained allowed the establishment of rules for the detection of C4 Q0 alleles in the heterozygous form and of C4A gene duplications. In the present study seven homoduplications of the C4A 3 allotype were defined which had not been detected by allotyping. This procedure allows the simple, reliable, and quick determination of Rg:1,2 and Ch:1 plasma levels which are not influenced by daily rhythms of C4 production.

Alleles↗

PubMed1985

Polymorphism of the fourth complement component in the dog.

The polymorphism of the fourth complement component was studied in 118 unrelated dogs from different breeds and 73 unrelated beagles. In the mixed breed population, five different variants were observed with the following gene frequencies: C4*1 = 3.9%; C4*2 = 13.6%; C4*21 = 6.0%; C4*3 = 9.0% and C4*4 = 51.1%. In beagles, only four variants could be identified with gene frequencies of C4*2 = 24.2%; C4*21 = 1.4%; C4*3 = 20.6% and C4*4 = 46.3%. In both populations analyzed positive linkage disequilibria between certain DLA antigens and C4 variants were recognized. The biochemical analysis of the canine C4 revealed a double-banding pattern for the C4 alpha chain which gives preliminary evidence for two gene loci encoding for the fourth complement component in the dog.

Animals↗

PubMedDate not supplied

The genetically-engineered secretory B27/Q10 chimeric molecule inhibits HLA-B27 restricted alloreactive T-lymphocytes.

OBJECTIVES: Intracellularly persisting bacterial infections and high association with HLA-B27 are the hallmarks of reactive arthritis. Soluble HLA-B27 molecules are induced by bacterial infection; however their biological role in arthritis is unknown. It was the aim of this study to generate soluble HLA-B27 molecule and to analyze its effect on cytotoxic HLA-B27 alloreactive CD8+ T-lymphocytes in order to better understand potential functional links between persistent infection and HLA-B27 association. METHODS: Using PCR Exons 1 through 4 of HLA-B*2705 were fused to Exon 5 of the soluble murine MHC class I variant Q10 and stably transfected into Hela-cells. Transfectants were analyzed using specific PCR, RT-PCR and intracellular and extracellular staining with anti-HLA-B27 monoclonal antibody ME1. Secretion of B27Q10 in the supernatant was examined by isoelectric focusing (IEF). The effect of B27Q10 on T-cells was analyzed using either HLA-B27- or HLA-A2-restricted alloreactive T-cells in a standard 51Cr-release assay. RESULTS: PCR and RT-PCR demonstrated the DNA and mRNA of B27Q10 in the transfectants. By intracellular and extracellular staining with ME1 B27Q10-molecule was detected intracellularly but was not expressed in the cell membrane. Using IEF soluble B27Q10-molecules were found in supernatants of transfectants in a concentration of up to 1.342 microg/ml. Soluble B27QJO-molecule inhibited specifically the cytotoxicity of HLA-B27-restricted alloreactive T-cells by about 30%. CONCLUSION: The secretory non-membrane-expressed molecule B27Q10 inhibits HLA-B27 specific T-cells. The inhibition of cytotoxic T-cells by bacteria induced soluble HLA-B27 may thus enable bacterial persistence.

Animals↗