Nucleotide sequence analysis of an HLA-B47 variant (HLA-B*4702).
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Alleles↗
Biomedical subjects
G F Fischer
Publications and source records attributed to G F Fischer.
Peripheral blood dendritic cells express Fc epsilon RI as a complex composed of Fc epsilon RI alpha- and Fc epsilon RI gamma-chains and can use this receptor for IgE-mediated allergen presentation.
Originally limited to basophils and mast cells, the spectrum of high affinity IgE receptor (Fc epsilon RI-bearing cells has expanded recently to include Langerhans cells, dermal dendritic cells (DC), monocytes, and eosinophils. As a result of studies on the distribution, structure, and function of Fc epsilon RI on APCs, we discovered a minor nonbasophil, nonmonocyte PBMC population that can bind IgE via Fc epsilon RI. This receptor occurs on the surface of these cells as a multimeric structure containing Fc epsilon RI alpha- and Fc epsilon RI gamma-chains but, unlike its counterpart on basophils, lacking Fc epsilon RI beta. Further experiments revealed that these Fc epsilon RI alpha gamma-expressing cells closely resemble peripheral blood DC by immunophenotype (HLA-DRhigh, HLA-DQhhigh; CD4+, CD11a+, CD32+, CD33+, B7/2 (CD86)+; CD11blow, CD14low, CD40low, CD54low, CD64low) and cell morphology. These features allowed us to isolate Fc epsilon RI-expressing DC from the peripheral blood and to investigate their immunostimulatory properties. We found Fc epsilon RI-positive DC to be efficient stimulators of both primary (allogeneic MLR) and Fc epsilon RI/IgE-dependent, secondary T cell responses at low cell numbers. Thus, Fc epsilon RI-expressing DC may not only amplify established type I allergic immune reactions but, unlike Fc epsilon RI-positive semiprofessional APCs, may be able to prime naive T cells to common and/or cryptic epitopes of IgE-reactive Ags.
Allergens↗
HLA-B27 determination using serological methods. A comparison of enzyme immunoassay and a microlymphocytotoxic test with flow cytometry and a molecular biological assay.
Typing for HLA-B27 is routinely performed in patients with seronegative spondarthritides. Besides the microlymphocytotoxic test (MLCT), other serological techniques have been developed such as enzyme immunoassays (EIA) using serum or plasma as a source for the determination of soluble HLA-B27 (sHLA-B27) and flow cytometric (FC) methods. The aim of the present study was to check the accuracy and reliability of the EIA for sHLA-B27 in comparison to the MLCT using antibodies against HLA-B27 and cross-reacting specificities (CRS), as well as an FC method and a molecular biological method. Any discrepant results should be typed with the MLCT using a complete panel of anti-HLA-class I antibodies, with FC and with a molecular biological technique. The EIA should also be repeated in those patients, using serum and plasma from a new venipuncture. In 81 patients with rheumatic disorders, the EIA and the MLCT using antibodies against HLA-B27 and CRS were performed. Based on the MLCT with a complete panel of anti-HLA-class I antibodies as a standard, discrepant test results were obtained for 9 out of 81 patients with the MLCT using antibodies against HLA-B27 and CRS and with the EIA. The following wrong results occurred: in the MLCT with anti-HLA-B27 and CRS, there were two false-negative results; in the EIA there were four false-negative and one false-positive results; one sample was undeterminable. In comparison with the MLCT, including the complete panel of HLA-class I antibodies, as well as with a molecular biological technique, typing with FC showed a complete concordance. Our investigations demonstrated that for routine typing for HLA-B27 the MLCT cannot be replaced by EIA because of a significant number of mistypings. The MLCT performed only with antibodies against HLA-B27 and CRS may also lead to typing errors. No errors were detected using flow cytometry. If only serological methods can be performed in a laboratory a combination of flow cytometry and MLCT could therefore enhance the safety of HLA-B27 typing.
Adult↗
Genotyping of the human platelet antigen systems 1 through 5 by multiplex polymerase chain reaction and ligation-based typing.
BACKGROUND: Platelet-specific antibodies may be involved in refractoriness to platelet transfusions, disorders such as neonatal alloimmune thrombocytopenia, and post-transfusion purpura. Genotyping for the major human platelet antigen (HPA) systems HPA-1 through HPA-5 is of considerable help in establishing the diagnoses of these diseases. STUDY DESIGN AND METHODS: A new genotyping method is described. Alleles of all five systems are amplified in a multiplex polymerase chain reaction. Subsequently, aliquots of the amplification products are thermocycled in the presence of a pair of allele-specific oligonucleotide probes and a heat-stable ligase. After heat denaturation, the probes hybridize adjacent to complementary sequences of the amplification product. In a perfect match, the two probes become covalently joined. Detection of the ligation product is performed with an enzyme-linked immunosorbent assay. RESULTS: Complete concordance of genotypes between the ligation-based typing and established genotyping methods was determined in 54 Austrian (HPA-1, -2, -3, and -5) and 56 Japanese (HPA-4) individuals. Ligation-based genotyping of HPA-1 polymorphism using platelet-derived RNA as starting material gave concordant results in all 15 cases tested. CONCLUSION: Multiplex polymerase chain reaction in combination with ligation-based typing allows fast typing of large numbers of platelet donors and screening for critical antigens in pregnant women.
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Ligation based HLA-B*27 typing.
We have established a ligation based typing method to detect HLA-B*27 alleles at the DNA level. The method requires amplification of exon 2 of the HLA-B locus from genomic DNA by the polymerase catalyzed chain reaction (PCR) using group specific primers. An aliquot of the PCR amplification product, heat stable ligase and a pair of oligonucleotide probes, designed to hybridize adjacently to HLA-B*27 specific sequences of the amplified DNA are subsequently thermocycled. If the probes are perfectly complementary they become ligated otherwise they stay separated. The ligation of probes can be detected through their different labels by an enzyme linked immunosorbent assay (ELISA). Ligation based detection of beta-actin sequences which have been co-amplified serves as positive control for each PCR reaction. We observed complete concordance when typing 76 HLA-B*27 positive and 107 HLA-B*27 negative individuals either by serology or by the ligation based approach. We conclude that ligation based typing is a reliable tool for the DNA based detection of HLA-B*27 alleles. The procedure allows automation to a large extent and should be easily applicable to the typing of other HLA-class I alleles.
Base Sequence↗
Conservation of a maternal-specific methylation signal at the human IGF2R locus.
The human IGF2R gene has been reported to be either biallelically or very rarely monoallelically expressed, in contrast to the maternally expressed mouse counterpart. We describe here an analysis of the 5' portion of the human IGF2R gene and show that it contains a maternally methylated CpG island in the second intron. A similar maternally methylated intronic element has been proposed to be the imprinting box for the mouse gene and although the relevance of this element has yet to be directly demonstrated, methylation has been reported to be essential to maintain allele-specific expression of imprinted genes. Allelic expression analysis of human IGF2R in 70 lymphoblastoid cell lines identified only one line showing monoallelic expression. Thus, in this tissue monoparental methylation of the IGF2R gene does not correlate with allele-specific expression. We also confirm here that the human IGF2R gene is located in an asynchronously replicating chromosomal region, as are all other imprinted genes so far analyzed. The mouse and human IGF2R intronic CpG islands both contain numerous large direct repeats that are methylated following maternal, but not paternal, transmittance. Thus features that attract maternal-specific methylation are conserved between the mouse and human genes. Since these intronic CpG islands share organizational rather than sequence homology, this suggests that secondary DNA structure may play a role in attracting a maternal methylation imprint.
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An HLA-DR11 variant (HLA-DRB1*1115) segregating in a family of Turkish origin.
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Alleles↗
A combination of two distinct in vitro amplification procedures for DNA typing of HLA-DRB and -DQB 1 alleles.
The differential hybridisation of oligonucleotide probes to polymerase chain reaction (PCR)-amplified DNA has become a standard procedure for tissue typing. We describe a typing method in which differential ligation replaces differential hybridisation, which is a significant simplification of this strategy. After amplification by the PCR two labelled, sequence-specific oligonucleotides hybridise, in the fluid phase, to one strand of heat-denatured amplification product in juxtaposition. In the case of perfectly complementary sequences surrounding the gap, a thermostable ligase catalyses the ligation of the two oligonucleotides, otherwise they stay separated. The use of heat-resistant ligase enables easy repetition of the denaturation-annealing-ligation cycle in a thermocycler. The ligation products are detected by an enzyme linked immunosorbent assay. We tested this typing approach in a model system, the characterisation of three functional alleles of HLA-DRB3 using three probe pairs. No discrepancies were observed in typing 100 individuals of known genotypes. A total of 33 probe pairs combined with generic and group-specific amplification allowed the typing of alleles of HLA-DRB and -DQB1 loci at low resolution. We confirmed ligation-based typing results of 259 individuals with sequence-based HLA-DRB1 typing and HLA-DQB1 typing using PCR with sequence-specific primers (SSPs). In addition, more than 1,500 ligation-based HLA-DRB1 typings were concordant with SSP typing. Excellent signal-to-noise ratios in the enzyme-linked immunosorbent assay make ligation-based typing remarkably robust. The time requirement of 2.5 h post-PCR enables practicable typing of putative organ donors. The whole procedure is more easily amenable to automation than methods based on differential hybridisation requiring additional incubators and extra handling for hybridisation and washing.
Alleles↗
Association between chronic cutaneous lupus erythematosus and HLA class II alleles.
CCLE, a disease entity at the benign end of the lupus spectrum, is characterized by marked photosensitivity and skin lesions in sun-exposed areas. The histopathology of lesions resembles hypersensitivity type IV reactions. We have asked whether an association between class II alleles and CCLE exists. RFLP analysis of HLA-DQA genes revealed a Taq I HLA-DQA1 allelic restriction fragment overrepresented in a group consisting of 26 patients as compared to healthy control individuals. This result was corroborated by typing with oligonucleotide probes. The presence of the DQA1*0102 allele in the patients' group led to a relative risk of 4.57, with a statistical significance of p < 0.05 after correction for 36 comparisons. Although not statistically significant, it is interesting that all patients possess in at least one of their HLA-DQA1 alleles a nucleotide sequence coding for the amino acid glutamine at position 34 of the DQ alpha molecule. The expected frequency of these alleles in the control population amounts to 82%. The HLA-DRB1*16 allele, which is found in linkage disequilibrium with the HLA-DQA1*0102 allele, is also observed at an increased frequency in the patient's group, though the association was not significant after correction for the number of comparisons. However, no associations of CCLE with alleles at the HLA-DPB1 locus was found. The association of CCLE with certain HLA class II alleles points to an involvement of HLA-DQ and/or -DR molecules in the pathogenesis of the disease. Alternatively, genetic loci in linkage disequilibrium may code for elements which contribute to the development of CCLE.(ABSTRACT TRUNCATED AT 250 WORDS)
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The HLA-DRB6*0201 allele of a pseudogene commonly associated with HLA-DR2 specificities is present in an HLA-DRB1*0101-DRB5*0101 haplotype.
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Adult↗
HLA-DR1-positive patients suffering from rheumatoid arthritis are at high risk for developing mucocutaneous side effects upon gold therapy.
Population studies suggest an association between RA and, depending on the ethnic background, HLA-DR1 and/or -DR4. One standard regimen for the treatment of RA is the use of gold compounds like SATM to arrest progression of the disease. In the present study, the immunogenetic background of RA patients developing side effects upon SATM treatment was determined. A total of 53 patients under SATM therapy were tested for their HLA-DRB and -DQ alleles by DNA typing; a significantly higher frequency of HLA-DR1 (p < 0.004, uncorrected) was observed in patients presenting with mucocutaneous side effects (MCT) when compared with patients without MCT. The RR was 6.85. Thus, HLA-DR1 seems to be a marker for the susceptibility of gold adverse reactions.
Adult↗
Detection of established and novel alleles of the HLA-DPB1 locus by PCR-SSO.
The polymorphism at the HLA-DPB1 locus was investigated in 490 Austrian individuals. Exon 2 sequences of the HLA-DPB1 genes were amplified by polymerase chain reaction and probed with a panel of sequence-specific oligonucleotide probes. HLA-DPB1 genotypes were deduced from the hybridization pattern of the probes. HLA-DPB1 allele frequencies and Hardy-Weinberg equilibrium were calculated. Linkage disequilibria between HLA-DPB1 and HLA-DQA1 alleles were determined. Four novel hybridization patterns indicating new alleles were found.
Alleles↗
IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset.
In a recent study we reported that CD27 is expressed on a subpopulation of human B lymphocytes and presented circumstantial phenotypic evidence that CD27 expression may be acquired late during B cell differentiation. Here we present functional data showing that, after in vitro stimulation, CD27+ but not CD27- B cells secrete large amounts of both IgM and IgG. Using double immunofluorescence staining of CD27 and IgD, three functionally different B cell subsets representing distinct stages of B cell differentiation can be isolated: 1) the CD27- IgD+ B cells, which do not secrete appreciable Ig; 2) the CD27+IgD+ B cells, which exclusively secrete IgM; and 3) the CD27+IgD- B cells, which comprise the IgG-producing cells. Furthermore, costimulation of CD27- B cells with low m.w. B cell growth factor, in the presence or in the absence of a CD40 mAb, does not induce these cells to become Ig-secreting cells. Although CD27- B cells hardly secrete Ig of any isotype in response to Staphylococcus aureus+IL-2, these cells proliferate vigorously and express the IL-2R alpha chain (CD25) under these stimulatory conditions. Furthermore, both CD27- and CD27+ B cells are capable of producing similar amounts of IL-6 and TNF-alpha. Taken together, these findings indicate that CD27 is a unique non-Ig surface marker discriminating naive from primed B lymphocytes. Furthermore, the capacity to proliferate and to secrete the B cell differentiation factors IL-6 and TNF-alpha already exists at an early B cell differentiation stage at which the cells lack CD27 expression and are not induced to produce Ig.
Antigens, CD↗
Association between IgE response against Bet v I, the major allergen of birch pollen, and HLA-DRB alleles.
The association of the human IgE response against Bet v I, the major allergen of birch pollen, and the HLA-DR and DQ phenotype was studied. Birch pollen allergic patients showed a typical case history, positive skin-prick test, and positive RAST with birch pollen extracts. They were divided into two groups. Group I (n = 37) consisted of individuals generating IgE antibodies that selectively reacted with Bet v I. Their serum IgE did not react with minor allergens from birch pollen as tested by immunoblot analysis, nor did they show a response against allergens from a panel of grass and other tree pollen or perennial allergens from animals and fungi as determined by skin-prick test. Patients belonging to group II (n = 34) possessed IgE reacting with Bet v I plus one or more additional allergens. The control group consisted of 637 healthy blood donors. Comparison of the frequencies of RFLP-defined HLA-DR and DQ alleles in patients and the control group revealed that the distribution of DRB3 alleles in group I patients differed significantly from that in the control group: A higher frequency of the DRw52a/c alleles in comparison to the control group (pcorr less than 0.02) was observed. In addition, alleles defined by nucleotide sequences coding for the amino acid sequence tyrosine-phenylalanine-histidine at positions 30-32 of the beta chain of DR molecules were found with a higher frequency in patient group I (pcorr less than 0.02), too. These alleles comprise DRw52a/c and some DRB1 alleles.(ABSTRACT TRUNCATED AT 250 WORDS)
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Analysis of the gene polymorphism of ABO blood group specific transferases helps diagnosis of acquired B status.
Blood group typing of an aged patient suffering from ileus provided evidence for an acquired B. As a parameter independent of cell membrane molecules or secreted blood group substances, the nucleotide sequence polymorphism of A and B transferases was investigated. Restriction fragment length polymorphism of DNA chains amplified in a polymerase chain reaction from the coding region of the glycosyltransferase indicated that no gene for B transferase was present in the patient's genome. We conclude that the assessment of polymorphism of AB0 blood group transferase can be used as a marker independent of blood group molecules for confirming a suspected acquired B.
ABO Blood-Group System↗
Distribution of polymorphic HLA-DR and -DQ alleles as determined by restriction fragment length polymorphism analysis in an Austrian population.
The restriction fragment length polymorphism (RFLP) of HLA-DR beta, -DQ alpha and -DQ beta genes was analyzed in 637 unrelated individuals from the Austrian population. The restriction enzyme was Taq I, and three exon-specific probes were applied. The gene frequencies, the Hardy-Weinberg equilibrium in DR beta and DQ loci, linkage disequilibria between the loci and haplotype frequencies are calculated. Rare associations between DR and DQ loci are described. Two RFLP patterns are demonstrated which were unique in the overall 1,000 individuals tested so far.
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Ratio of complement receptor over Fc-receptor III expression: a sensitive parameter to monitor granulocyte-macrophage colony-stimulating factor effects on neutrophils.
In vitro activation of human granulocytes leads to altered expression of distinct surface antigens. Compared with the changes observed with classic activating reagents such as the phorbol ester PMA similar, but less pronounced alterations of surface antigen expression were observed upon granulocyte activation with human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF). In particular, stimulation with hrGM-CSF is followed by an enhanced expression of the complement receptors CD35 (CR1) and CD11b (CR3) while the low affinity Fc-gamma receptor CD16 (FcRIII) is downregulated. In order to investigate whether there are similar effects under in vivo conditions, we studied the granulocytes from patients undergoing rhGM-CSF therapy before, during, and after treatment. We found a marked increase in CD35 (CR1) and CD11b (CR3) expression and a substantial decrease or even loss of CD16 (FcRIII) on these granulocytes. These changes correlated well with our in vitro data and occurred extremely rapidly after therapy onset. Furthermore, therapy monitoring using ratios calculated by the mean fluorescence channel numbers of CR and FcRIII stainings may combine the advantage of high sensitivity with high reproducibility as a result of the contrasting change in CR and FcRIII expression during granulocyte activation. Being nonparametric values, such ratios are not influenced by individual flow cytometry standardization. Taken together, these activation-associated changes of surface receptor expression and especially of CR over FcRIII ratios are useful parameters for monitoring the in vivo effects of rhGM-CSF.
Antigens, CD↗
HLA-DRB3 typing by restriction digestion of locus-specific amplified DNA.
Locus HLA-DRB3 codes for the serologically defined supertypic specificity DRw52 in HLA-DR3, -5 and -w6 haplotypes. Three specificities of DRw52 (DRw52a, -b and -c) can further be distinguished by cellular techniques or by DNA typing with allele-specific oligonucleotide probes. These specificities were recently reported to have significant importance in antigen presentation. To avoid a time-consuming hybridization procedure, we have developed a simple typing system using PCR and subsequent digestion by allele-specific restriction endonucleases. A system was established with locus-specific amplification of HLA-DRB3 and digestion by the enzymes KpnI, ScaI and HinfI which recognize unique restriction sites within the amplified region. This allowed HLA-DRB3 typing on agarose gel by determining whether the amplification product has been digested or not. This typing system was compared to conventional oligotyping by analyzing 145 RFLP-typed individuals for their DRw52 specificity using both methods. Agarose typing correlated well with oligotyping and was shown to be more simple and practical even in heterozygous individuals.
Base Sequence↗