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Biomedical subjects

K Fleischhauer

Publications and source records attributed to K Fleischhauer.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cytotoxic T cells directed to tumor antigens not expressed on normal melanocytes dominate HLA-A2.1-restricted immune repertoire to melanoma.

To determine whether HLA-A21 restricted melanoma Ags exist that are not expressed on normal melanocytes, a panel of 478 T cell clones from six HLA-A21+ patients was selected for HLA-A2 restricted lysis of autologous tumor and then tested for differential recognition of HLA-A2.1+ melanomas and normal melanocytes. Four subsets of clones were identified in the panel of 107 HLA-A2-restricted CTL clones. CTL clones from three of the four subsets did not lyse melanocytes, but recognized fresh HLA-A2.1+ melanomas and defined three classes of epitopes, including unique Ags, common melanoma Ags, and Ags shared with neoplastic cells of different histologic origin. These CTL clones did not recognize any of the 10 peptides selected for specific association to HLA-A2.1 and derived from Melan-A/Mart-1, tyrosinase, gp100, or MAGE-3 proteins. By contrast, the fourth subset of HLA-A2.1-restricted CTl clones recognized both melanoma and melanocytes. These CTL clones were directed to a peptide from either Melan-A/Mart-1, tyronise, or gp100. By a limiting dilution assay, designed to evaluate the frequency of HLA-A2-restricted CTL precursors (CTLp) directed to melanoma but not to melanocytes, such precursors were found in the peripheral blood or tumor site of five of six HLA-A2.1+ melanoma patients, and their frequency was much higher than the frequency of CTLp recognizing both tumor cells and the melanocytes. These results suggest that in melanoma patients most of the HLA-A2.1-restricted immune repertoire to melanoma is directly to epitopes expressed in the neoplastic but not in the normal cells of the melanocyte lineage.

Amino Acid Sequence↗
PubMed1996

A peptide encoded by the human MAGE3 gene and presented by HLA-B44 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE3.

The human MAGE3 gene is expressed in a significant proportion of tumors of various histological types, but is silent in normal adult tissues other than testis and placenta. Antigens encoded by MAGE3 may therefore be useful targets for specific antitumor immunization. Two antigenic peptides encoded by the MAGE3 gene have been reported previously. One is presented to cytolytic T lymphocytes (CTL) by HLA-A1, the other by HLA-A2 molecules. Here we show that MAGE3 also codes for a peptide that is presented to CTL by HLA-B44. MAGE3 peptides containing the HLA-B44 peptide binding motif were synthesized. Peptide MEVDPIGHLY, which showed the strongest binding to HLA-B44, was used to stimulate blood T lymphocytes from normal HLA-B44 donors. CTL clones were obtained that recognized not only HLA-B44 cells sensitized with the peptide, but also HLA-B44 tumor cell lines expressing MAGE3. The proportion of metastatic melanomas expressing the MAGE3/HLA-B44 antigen should amount to approximately 17% in the Caucasian population, since 24% of individuals carry the HLA-B44 allele and 76% of these tumors express MAGE3.

Amino Acid Sequence↗
PubMed1996

HLA-A*02 subtype distribution in Caucasians from northern Italy: identification of A*0220.

This study describes a comprehensive easy to perform PCR-SSOP typing approach suitable for complete genomic subtyping of HLA-A*02. A single 1.6 kb PCR-amplificate spanning exons 2, 3 and 4 of the HLA-A*02 gene was used for hybridization with a panel of twenty-four SSOPs. This allowed unequivocal assignment of all so far known HLA-A2 subtypes, including A*0209 and A*0215N which differ for nucleotide substitutions in exon 4, without the need for two separate amplifications. Using this approach, HLA-A*02 subtype distribution was analyzed in 218 samples from unrelated, healthy individuals from northern Italy enrolled in the Italian Bone Marrow Registry and typed as HLA-A2 by serology or generic molecular analysis. As expected, A*0201 was found in the majority (92.6%) of samples. However, a significant number (6.8%) of individuals carried A*0205. Furthermore, A*0202, A*0208, A*0209 and A*0217, so far not described in Caucasians, were detected in a low number of samples (frequency ranging from 0.45% to 1.8%). Finally, a novel HLA-A*02 subtype, A*0220, was detected in 0.9% of the samples. As confirmed by DNA sequencing of exons 2 and 3, this allele is identical to A*0201 except for a single nucleotide substitution in codon 66 which changes the predicted amino acid sequence form Lys to Asn. The findings of this study have implications for the selection of HLA-A*02+ donors in unrelated bone marrow transplantation and of patients for specific immuno-therapy with HLA-A*02 restricted peptide vaccines.

Genes↗
PubMed1995

The peptide-binding motif for the human transporter associated with antigen processing.

Presentation of antigenic peptides by human leukocyte antigen class I molecules is dependent on peptide transport into the endoplasmic reticulum by the transporters associated with antigen processing (TAP) (Germain, R. N. 1994. Cell. 76:287-299). This translocation step is currently regarded as permissive for all peptides with COOH-terminal residues capable of binding to HLA class I molecules (Momburg, F., J. Roelse, J. C. Howard, G. W. Butcher, G.J. Hämmerling, and J.J. Neefjes. 1994. Nature (Lond.). 367:648-651). In this report, we show that the human transporter selects peptides according to a binding motif based on the strong effects on peptide affinity of the three NH2-terminal positions and the COOH-terminal residues. TAP favors strongly hydrophobic residues in position 3 (P3) and hydrophobic or charged residues in P2, whereas aromatic or acidic residues in P1, as well as Pro in P1 and P2, have strong deleterious effects. Selection of naturally presented peptides by the transporter is suggested by their higher average affinity for TAP, as compared to nonselected peptides. The TAP preferences in the three NH2-terminal positions correspond to those of the vast majority of human leukocyte antigen class I alleles, but they represent an obstacle for peptide supply to some alleles, e.g., the B7-like group. We propose that peptides binding to these alleles, and in general, peptides with TAP affinities below a certain threshold, may be transported as extended precursors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Biotinylation of biotinidase following incubation with biocytin.

Human serum biotinidase, purified to homogeneity (1920 units/mg protein), was incubated with biocytin prior to electrophoresis and transblotting with avidin-peroxidase. Avidin reacted with biotinidase maximally when incubated at pH 7.5-9, less at pH 7 and none below pH 7. No avidin reactivity occurred when biotinidase was incubated with biotin or in the absence of biocytin. Inclusion of the nucleophilic acceptors, ethanolamine or hydroxylamine, to the incubation mixture with biocytin and biotinidase resulted in loss of avidin reactivity. High concentrations of mercaptoethanol also prevented avidin reactivity. These results suggest that biotinidase can be biotinylated in the presence of biocytin at neutral to alkaline pH probably through a thioester bond formed with a cysteine residue in the active site of the enzyme. Biotinidase may then function as a biotinylating enzyme when incubated with appropriate nucleophilic acceptors.

Amidohydrolases↗
PubMed1995

Biotinylation of histones by human serum biotinidase: assessment of biotinyl-transferase activity in sera from normal individuals and children with biotinidase deficiency.

Serum biotinidase has biotinyl-transferase activity in addition to biocytin hydrolase activity. A sensitive assay for biotinyl-transferase activity was developed based on the transfer of biotin from biocytin to histones. Biotinidase biotinyl-transferase occurs at physiological and alkaline pHs, whereas hydrolysis of biocytin occurs optimally at pH 4.5 to 6.0. Measurement of hydrolysis requires micromolar concentrations of biocytin, whereas biotinylation of histones can be detected readily at 1.5 nM biocytin. Because polylysine is readily biotinylated by biotinidase in the presence of biocytin, whereas polyarginine is not, the enzyme likely transfers biotin to the epsilon-amino group of lysyl residues. To determine if patients who are deficient in biocytin hydrolase activity are also deficient in biotinyl-transferase activity, serum from 103 children (25 identified by exhibiting clinical symptoms and 78 detected by newborn screening) with profound biotinidase deficiency (less than 10% of mean normal biotinyl-p-aminobenzoate hydrolyzing activity) were assessed for biotinyl-transferase activity and for the presence of cross-reacting material (CRM) to antibodies prepared against purified serum biotinidase. Sera from all symptomatic patients, both CRM-negative and CRM-positive, had no biotinyl-transferase activity. Sera that was CRM-negative from children ascertained by newborn screening also had no biotinyl-transferase activity, whereas sera from 67% of the CRM-positive children identified by newborn screening had varying degrees of biotinyl-transferase activity. These results indicate that there is a large group of enzyme-deficient children detected by newborn screening who are different biochemically from those who are symptomatic. The clinical relevance of having some degree of biotinyl-transferase activity for individuals with biotinidase deficiency remains to be determined. In addition, it is important to determine if biotinyl-transferase activity, especially biotinylation of histones, is a physiological function of biotinidase.

Amidohydrolases↗
PubMed1995

Complete generic and extensive fine-specificity typing of the HLA-B locus by the PCR-SSOP method.

This study describes sequence specific oligonucleotide probe (SSOP) typing of hypervariable regions in exons 2 and 3 of HLA-B locus genes. A single HLA-B specific PCR-product spanning from bp 84 in exon 2 to bp 241 in exon 3 was used for dot blot hybridization to forty-seven chemiluminescent labeled oligonucleotide probes. Thirty-one of these probes were derived from four hypervariability zones in exon 3 of HLA-B genes and covered most known sequence polymorphisms within these regions. In addition, sixteen probes derived from polymorphic regions in exon 2 were used to discriminate alleles not unequivocally characterized by the exon 3 based probes. This SSOP panel gave rise to eighty-six distinct hybridization patterns that could be used to unequivocally define all WHO-designated serological HLA-B specificities except for HLA-B54 in all homo- and heterozygous combinations. Furthermore, sixty-six out of ninety-seven molecularly defined HLA-B subtypes were characterized by unique hybridization patterns in all homozygous and most (possibly all) heterozygous combinations. The reproducibility of these results was confirmed by analysis of forty-four Workshop reference cell lines and of seventy-eight randomly chosen samples (one-hundred forty-seven alleles) from unrelated individuals serologically typed in the laboratory. For sixty-five samples (one-hundred-thirty-three alleles), molecular typing confirmed the results obtained by serology and allowed molecular subtype assignment for ninety-one alleles tested. A serologically blank allele could be defined by molecular analysis in three cases. The method presented here for molecular typing of the HLA-B locus can be used as an alternative to biochemical methods such as one-dimensional isoelectric focusing for assignment of serologically cross-reacting HLA-B molecules as well as for subtype characterization of a large variety of HLA-B alleles.

Alleles↗
PubMed1994

Characterization of natural peptide ligands for HLA-B*4402 and -B*4403: implications for peptide involvement in allorecognition of a single amino acid change in the HLA-B44 heavy chain.

This study describes the characterization of endogenous peptides associated with the two major subtypes of HLA-B44. The two subtypes differ for a single amino acid substitution from Asp (HLA-B*4402) to Leu (HLA-B*4403) in position 156 of the alpha 2 domain, causing strong alloreactivity in vivo. In order to study the involvement of peptides in this phenomenon, the peptide motifs of the two subtypes were determined from natural peptide pools using Edman degradation. The motif was found to be essentially identical for HLA-B*4402 and -B*4403, with a strong predominance for Glu at position 2, Tyr or Phe at positions 9 and 10 and hydrophobic residues, especially Met, at position 3. Two individual naturally processed ligands of HLA-B*4403 were sequenced and shown to be derived from intracellularly expressed proteins found in protein sequence databases. The sequence of these natural peptide ligands conform well to the determined motif. These data will allow the prediction of HLA-B44 restricted peptide epitopes from viral and tumor antigens of known amino acid sequences. Moreover, they indicate that the peptide repertoire presented by HLA-B*4402 and -B*4403 is very similar, suggesting that the strong alloresponse between these two subtypes is not due to presentation of a different set of self peptides.

Amino Acid Sequence↗
PubMed1993

Cloning and chromosome mapping of human retinoid X receptor beta: selective amino acid sequence conservation of a nuclear hormone receptor in mammals.

The murine retinoid X receptor beta (mRXR beta) is a nuclear hormone receptor that activates transcription of murine major histocompatibility complex (MHC) class I genes in response to retinoic acid. In this study, the human RXR beta gene was mapped onto the short arm or centromeric region of chromosome 6 (6pter-q13), which also harbors the MHC. Chromosomal localization was performed by Southern hybridization of genomic DNA from human rodent cell hybrids with the mRXR beta gene as a probe. In addition, a full-length cDNA clone encoding a human RXR beta was isolated by nucleic acid screening of a human cDNA library with a fragment of the mRXR beta gene as a probe. Comparison of the nucleotide-coding sequences of the human and the murine RXR beta revealed a predominance of third base substitutions, resulting in selective conservation of the predicted amino acid sequence of the proteins. The overall sequence homology was 97.6% on the amino acid level as opposed to 91.6% on the nucleotide level. In Northern hybridization experiments with the human cDNA as a probe, RXR beta gene transcripts were detected in a variety of human tumor cell lines, regardless of whether these cell lines expressed MHC class I genes.

Amino Acid Sequence↗
PubMed1993

Isoelectric focusing subtypes of HLA-A can be defined by oligonucleotide typing.

This study describes a simple and direct method for sequence-specific oligonucleotide probe (SSOP) typing of the A locus of HLA class I genes. Genomic DNA from a panel of over 200 cells which have been characterized by the methods of serology and isoelectric focusing (IEF) for the HLA class I antigens was used for locus-specific PCR amplification of HLA-A sequences. Dot blot hybridization of the amplified products was performed with 28 SSOPs derived from hypervariable regions in exon 2 and 3. Co-amplification of three alleles of HLA-H pseudogene in apparent linkage disequilibrium with HLA-A2 and A10 was observed but did not interfere with the typing of HLA-A alleles. Using short SSOPs (15 nucleotides each) in single temperature tetramethylammonium chloride (TMAC) hybridization and washing steps, 30 IEF-definable isotypes of HLA-A antigens could be unambiguously defined by their hybridization patterns. Moreover, comparison of the typing results with available nucleotide sequences of HLA-A alleles showed that the conditions used allowed faithful detection of single codon mismatches between probe and template. Thus, these alleles can be identified by their unique hybridization patterns generated by the SSOPs. Nucleotide sequence analysis of any new HLA-A allele will further permit its rapid and unambiguous characterization by SSOP typing.

Amino Acid Sequence↗
PubMed1989

[The surgical anatomy of the genital nerves of the male and their preservation in excision of the rectum].

Studies of the fetus and new-born were undertaken to determine the exact localization of the nn. erigentes in the lower pelvis. The particular topography was visualized using histological slides and a special digital imaging technique. The nn. erigentes were found to be bundled along the diaphragmatic part of the urethra before entering the two cavernous bodies of the penis. These findings suggest that a small anterior patch of rectal muscle covering the diaphragmatic part of the urethra should be left behind in male patients undergoing total proctocolectomy in order to preserve potency. This operative technique cannot be employed for carcinomas extending to the anterior rectal wall. Since 1980, the new technique has been used in 18 male patients with rectal carcinoma and sexual function was preserved in all cases.

Adult↗
PubMed1984

Morphological characteristics of neocortical laminae when studied in tangential semithin sections through the visual cortex of the rabbit.

In semithin sections cut tangentially with respect to the surface of the cerebral cortex and with a side length of at most a few mm, it is extremely difficult to be certain which lamina has been sectioned, for instance lamina III or upper lamina IV. In order to provide criteria to overcome this difficulty, a systematic study was carried out in which 1 micron semithin sections through the visual cortex of the rabbit were cut in both the frontal and tangential planes and compared with 8-12 micron paraffin sections stained for cells and/or myelin. It was found that the arrangement of thick dendrites, of myelinated fibres and of unmyelinated profiles which may be either dendrites or neurites, permits subdivision of the visual cortex into three characteristic horizontal zones which are termed zones A, B and C. The relation between these three zones and the cytoarchitectonic pattern is as follows: Zone A corresponds to the cytoarchitectonic lamina I; zone B comprises lamina II/III, lamina IV and the upper half of lamina V; zone C corresponds to the lower half of lamina V and all of lamina VI. Zones A and B can further be subdivided: Zone A consists of two layers, whereas zone B can be divided into three tiers, each of which is characterized by a particular arrangement of the myelinated and unmyelinated profiles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

An improved method for perfusion of the isolated brain of the rat-influence of perfusion conditions and application of analeptic and anticonvulsant drugs.

A method for the perfusion of the isolated brain of the rat with synthetic fluorocarbon emulsion is described. The functional states of the brains were investigated using biochemical, biophysical, and histological methods. After 4.5 h of perfusion, all brains were in excellent condition and comparable to the in vivo state. The influence of perfusion conditions on the viability of the brains was studied. Deep hypothermia was well tolerated; at 13 degrees the EEG exhibited a mean frequency of about 8 Hz, while the mean amplitude was about 40% lower than at 28 degrees or 36 degrees. Constant-pressure perfusion did not affect brain function, provided that the pressure amplitude was very slowly decreased. During application of pentylenetetrazole (PTZ) it was found that the threshold concentration of PTZ in the perfusion medium to induce epileptic seizure discharges was 0.13 mg/ml. However, after repeated application of PTZ, the threshold concentration decreased exponentially, indicating a kindling phenomenon. It could be shown that not PTZ as such, but the spike potentials provoked by PTZ, induced the kindling effect. This was inhibited by application of primidone or phenobarbital.

Adenosine Triphosphate↗