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J T Epplen

Publications and source records attributed to J T Epplen.

At least 271 records · Page 15Linked to original sources

Enhanced expression of HLA-class II molecules on activated human T lymphocytes following treatment with tumor necrosis factor alpha.

Many factors induce or enhance expression of major histocompatibility complex class I and class II molecules on various cell types. Human T lymphocytes are class II negative in the resting state but show expression of class II molecules following activation. We analyzed the modulating capacity of the lymphokines recombinant interferon gamma (rIFN-gamma), interleukin-4 (IL-4), and recombinant tumor necrosis factor alpha (rTNF-alpha) on class II expression in subsets of alloactivated human T lymphocytes. The activated CD4+ T cells expressed all three class II isotypes (DR, DQ, and DP), whereas the cytotoxic CD8+ T-cell lines expressed DR and DP molecules but failed to bind DQ-specific monoclonal antibodies significantly. Treatment with rIFN-gamma and IL-4 had no effect on class II expression on any of the T-cell lines or clones, whereas rTNF-alpha enhanced class II expression in both subsets. rTNF-alpha could modulate expression of all three class II isotypes but, in principle, it appears only to affect ongoing class II synthesis as de novo synthesis of class II molecules with a resultant change in the class II phenotype from DR+ DQ- DP+ to DR+ DQ+ DP+ in the CD8+ T lymphocytes was not observed. No synergic effects of rINF-gamma and rTNF-alpha were observed; this results from the fact that activated T cells express few, if any, receptors of rIFN-gamma.

Antibodies, Monoclonal↗

DNA fingerprinting in cattle using oligonucleotide probes.

Oligonucleotide probes specific for simple tandem repeat sequences produce individual specific DNA fingerprints in man and all animal species tested so far. Here 11 different synthetic probes were hybridized to bovine genomic DNAs which had been digested with the restriction endonucleases HinfI, AluI and HaeIII. Two of these probes gave DNA fingerprint patterns which were analysed for three German breeds. Different parameters were calculated, such as the average number of bands per individual or the probability of finding identical fingerprints in two unrelated individuals. The number of polymorphic bands varies from 11 to 23 in the different breeds and the probability of finding the same banding pattern in two unrelated individuals ranges from 1.5 x 10(-7) to 2.4 x 10(-7). Hence this DNA fingerprinting procedure allows precise identification of individuals. It is also a useful additional method for paternity testing in cattle.

Animals↗

New mutation versus exclusion at the alpha-1-antitrypsin locus: a multifaceted approach in a problematical paternity case.

In a case of disputed paternity with overwhelming indications of fatherhood for the putative father, as supported by serological tests and biostatistical evaluation, a classical exclusion constellation was found at the alpha 1-antitrypsin (PI) locus: mother PI M1; child PI M1M3, and putative father PI M1M2. Additional studies included PI oligonucleotide phenotyping and DNA fingerprint analysis. Results from the entire data set led us to assume a rare genetic event at the paternal PI locus. Intracistronal crossing-over offered the most parsimonious explanation, and was compatible with the PI gene DNA sequence and the amino acid sequences of the molecule and its allelic forms, as well as with the experimental findings.

Adult↗

Individual-specific DNA fingerprinting in man using the oligonucleotide probe (GTG)5/(CAC)5.

Restriction fragment length polymorphisms (RFLPs) associated with interspersed simple repetitive DNA arise from DNA fragment lengths that contain variable numbers of the repeated motifs. Using restriction enzymes with different 4 base pair recognition sites and the simple triplet repeat hybridization probe, (GTG)5/(CAC)5, DNA multilocus fingerprints can be obtained in man. Only the DNAs of monozygous twins show indistinguishable banding patterns. Since the bands are inherited according to Mendelian laws, DNA fingerprints can be used for identification of individuals and paternity analysis. The discriminatory power in the DNA fingerprinting technique in forensic science is demonstrated and examples of paternity testing are given.

DNA Fingerprinting↗

[Induction of autoreactive T-cells by bacterial stress proteins in HLA-DR4 donors].

From the peripheral blood of HLA-DR4+ healthy donors or patients with rheumatoid arthritis autoreactive T-cell lines were obtained after stimulation with bacterial heat-shock protein. All lines obtained express an alpha/beta-T-cell receptor and they are predominantly CD4+. The determination of the fine specificity of those autoreactive T cells showed, that either amino-acid position #71 or #86 of the HLA-DR beta 1 chain was critical for recognition by the antigen receptor.

Blood Donors↗

DNA fingerprinting of eukaryote genomes by synthetic oligodeoxyribonucleotide probes.

An extreme level of DNA sequence polymorphism, the basis of DNA fingerprinting, was first demonstrated using genome derived cloned probes. Subsequently, it was shown that DNA fingerprinting can also be carried out using short synthetic oligodeoxyribonucleotide probes specific for simple repetitive sequences. Further, in addition to radioactively labeled probes, non-radioactive oligonucleotides generate equally informative hybridization patterns. We discuss the development in the area of DNA fingerprinting and its future scope with respect to plant, animal and the human DNA.

Animals↗

Rapid method based on reversed-phase high-performance liquid chromatography for purification of human myelin basic protein and its thrombic and endoproteinase Lys-C peptides.

Reversed-phase high-performance liquid chromatography was applied to isolate myelin basic protein from human brain, followed by separation of proteolytic peptides thereof on the same chromatographic system. Brain tissue was delipidated under conditions that keep copurifying proteases inactive. The crude brain protein fraction was applied directly to a C4 column. The homogeneous protein obtained in this way was digested with thrombin and endoproteinase Lys-C in order to produce short defined myelin basic protein peptides. The purified peptides were used to determine the antigen fine specificity of myelin basic protein recognizing T lymphocyte lines isolated from multiple sclerosis patients.

Brain Chemistry↗

Heterogeneities in the distribution of (GACA)n simple repeats in the karyotypes of primates and mouse.

Tandemly organized simple repetitive sequences are widespread in all eukaryotes. The organization of the simple tetrameric (GACA)n sequences at chromosomal loci has been investigated using in situ hybridization with chemically pure oligonucleotide probes. Both biotin- and digoxigenin-attached (GACA)4 probes reveal specific hybridization signals over the short arms of all acrocentric autosomes in man. In the other examined primates the NOR-bearing autosomes could be detected by in situ hybridization with (GACA)4, and a major concentration of the GACA simple repeats could be observed on the Y chromosome in the gibbon and mouse: the hybridization site in the gibbon Y chromosome coincides particularly with the silver-stainable NOR. In the past, accumulations of (GACA)n sequences were demonstrated mainly on vertebrate sex chromosomes. Therefore, the organization of GACA simple sequences is discussed in the context of their evolutionary potential accumulation and the possible linkage with the primate rDNA loci.

Animals↗

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes.

We have investigated the extent of DNA variability in intronic simple (gt)n(ga)m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)n(ga)m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.

Amino Acid Sequence↗

Stain analysis using oligonucleotide probes specific for simple repetitive DNA sequences.

The use of oligonucleotide fingerprinting is evaluated in practical forensic work, using both artificially and systematically produced stains as well as actual case work material. The probes (CAC5/(GTG)5 are superior because of their individualizing potential in comparatively fresh specimens with little DNA degradation, whereas (GACA)4, still produces substantial information when high molecular weight DNA is lacking. The overall limitations and the advantages of this technology are discussed in detail and compared to the classical minisatellite probes.

Blood Stains↗

Organization of DYZ2 repetitive DNA on the human Y chromosome.

The location of the human Y-specific repetitive DNA sequence DYZ2 with HaeIII cleavage sites spaced at 2.1 kb was reexamined. Previous reports had mapped the 2000 DYZ2 copies to the very distal end of the heterochromatic Yq12 band. In the present study, a cloned DYZ2 fragment (pHY2.1) was used for Southern and slot blot analyses of male DNA as well as for nonradioactive in situ hybridization to chromosomes. DNA and metaphase preparations from 79 individuals with polymorphic or aberrant Y chromosomes were examined. DYZ2 repeats are not confined to the distal tip of Yq12, but extend through the entire heterochromatin of Yq12. In the naturally occurring length polymorphisms of Yq, the amount of DYZ2 sequence varies in proportion to the measured sizes of band Yq12. Explanations are presented for the fact that previous studies restricted the location of DYZ2 to the telomeric end of Yq12.

Blotting, Southern↗

Myelin autoreactivity in multiple sclerosis: recognition of myelin basic protein in the context of HLA-DR2 products by T lymphocytes of multiple-sclerosis patients and healthy donors.

A panel of 20 human myelin basic protein (hMBP)-specific T-lymphocyte lines was generated from the peripheral blood of eight multiple sclerosis (MS) patients and two healthy donors, most of them expressing the HLA-DR2 haplotype, which is associated with an increased susceptibility to MS. Using HLA-DR gene-transfected mouse L-cell lines as antigen-presenting cells, we established that of the 20 hMBP-specific T-lymphocyte lines, 7 were restricted by the DR2a gene products of the DR2Dw2 haplotype. Four T-cell lines recognized hMBP in the context of the DR2b products of the DR2Dw2 haplotype. DR2b-restricted T-cell responses were demonstrable only in T-cell lines derived from MS patients. The hMBP epitopes presented by the DR2a heterodimer were mapped to peptides covering amino acid residues 1-44, 76-91, 131-145, or 139-153 and to a region spanning the thrombin-cleaved bond at Arg130-Ala131. DR2b-restricted T-cell lines recognized epitopes within amino acids 80-99 and 148-162. Peptide 139-153 was also presented in the context of HLA-DR1 molecules. Our data show that (i) in MS patients both the DR2a and DR2b products of the DR2Dw2 haplotype function as restriction elements for the myelin autoantigen hMBP, (ii) the DR2a molecule presents at least five different epitopes to hMBP-specific T lymphocytes, and (iii) anti-hMBP T-cell lines derived from individual donors can differ in their antigen fine specificity as well as in their HLA restriction.

Adult↗

T cell receptor beta chain usage in myelin basic protein-specific rat T lymphocytes.

Myelin basic protein (MBP)-specific T cell lines and clones have been established from rats of the major histocompatibility complex (MHC)-compatible Lewis and BS strains. All lines and clones are MHC class II restricted and share the CD4+ phenotype. The cells proliferate specifically in response to either a peptide representing amino acids #68-88 of guinea pig MBP, to residues #47-67 or to an unidentified myelin antigen which is distinct from MBP. All lines and clones specific for MBP express the same T cell receptor (TcR) variable (V) beta chain element, which is homologous to the mouse V beta 8.2 gene segment. Three lines/clones with the same antigen fine specificity have identical V beta D beta J beta junctions on the protein level, a region which represents part of the potential antigen-binding portion of the TcR; two of the lines express members of the V alpha 2 family. These results suggest biased usage of TcR V beta elements in rat T cells specific for MBP. Our findings broaden the basis for a rational therapeutic strategy to specifically intervene in the rodent model system of experimental allergic encephalomyelitis.

Amino Acid Sequence↗

Increased detectability of somatic changes in the DNA from human tumours after probing with "synthetic" and "genome-derived" hypervariable multilocus probes.

DNA fingerprinting with two minisatellite (33.15, M13) and two simple repeat probes [(GACA)4, (CAC)5/(GTG)s] was performed to screen for somatic changes in the DNA from various solid human tumours in comparison with constitutional DNA from the same patient. Loss of bands or changes in band intensities were observed. Together the probes 33.15 and (CAC)5/(GTG)5 detected deviating fingerprint patterns in 63% of the colorectal carcinomas investigated. In mammary and stomach carcinomas, only 1/11 and 2/11 tumours, respectively, showed differences with either of the three probes, 33.15, (GACA)4 and (CAC)5/(GTG)5.

Breast Neoplasms↗

DNA fingerprinting with the oligonucleotide probe (CAC)5/(GTG)5: somatic stability and germline mutations.

DNA fingerprints were generated from various human somatic tissues and from peripheral blood of 179 children and their 80 parents using (CAC)5/(GTG)5 oligonucleotide probes. Whereas somatic stability of the fingerprint patterns was demonstrated, the average rate for germline mutations was estimated to be approximately 0.001 per DNA locus and gamete, with the three different restriction enzymes used. Seven out of eight mutations observed appeared to be of paternal origin.

Base Sequence↗