PubMed Health⌕ Search

Biomedical subjects

E P Cowan

Publications and source records attributed to E P Cowan.

At least 19 recordsLinked to original sources
PubMed1999

Absence of human T-lymphotropic virus type I tax sequences in a population of normal blood donors in the Baltimore, MD/Washington, DC, area: results from a multicenter study.

BACKGROUND: It was reported recently that sequences corresponding to the human T-lymphotropic virus type I (HTLV-I) tax gene were detected in peripheral blood mononuclear cells from 8 to 11 percent of healthy blood donors without detectable antibodies to HTLV-I. A multicenter blind study was conducted to determine if these results could be independently confirmed. STUDY DESIGN AND METHODS: Specimens were collected from 100 anti-HTLV-I-negative healthy blood donors and from 11 anti-HTLV-I- or anti-HTLV-II-positive individuals. All samples were coded and distributed to each of four independent testing laboratories for polymerase chain reaction analysis to detect sequences of the HTLV-I or HTLV-II tax gene, using detailed procedures specified by the laboratory reporting the original observation. Each laboratory also tested a dilution panel of a plasmid containing HTLV-I tax to determine the analytical sensitivity of the procedure. RESULTS: The analytical sensitivity of the screening methods permitted detection of as few as 1 to 10 copies of the tax gene. However, HTLV-I tax sequences could not be detected in any of the anti-HTLV-I-negative blood donors at more than one test site. CONCLUSION: HTLV-I tax sequences appear not to be present in this population of 100 blood donors negative for anti-HTLV-I.

Adult↗
PubMed1999

Atopic dermatitis and HTLV-1-associated myelopathy: associated or coincidental disorders?

Reports from Jamaica have indicated that some patients with infective dermatitis or atopic dermatitis (AD) are seropositive for antibodies to human T-lymphotropic virus type 1 (HTLV-1). We describe a 32-year-old Israeli woman with long-term AD and paresthesia in the distal parts of the extremities. Neurological examination revealed a positive Babinski's sign. HLA typing demonstrated that this patient has the common HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and infective dermatitis haplotype for DRB1* DQB1*. The presence of HTLV-1 was demonstrated with polymerase chain reaction; HTLV-1-antibodies were detected by the Western blot method and by inoculation of the patient's peripheral blood mononuclear cells into F344 rats. This study confirms the presence of HTLV-1 antibodies and proviral genome in a patient with AD which later evolved into HAM/TSP. We cannot yet conclude whether these two diseases are associated or coincidental disorders.

Adult↗
PubMed1998

Cytokine gene expression in cells derived from CSF of multiple sclerosis patients.

Cytokines are produced by numerous cell types in the peripheral blood and central nervous system (CNS), and have been implicated in the immunopathogenesis of multiple sclerosis (MS). We examined the relationship between cytokine gene expression of cerebrospinal fluid (CSF) derived cells from MS patients and disease activity as measured by contrast enhanced MRI. There was a significant correlation between the number of CSF cells and the number of contrast enhancing MRI lesions. Cytokine gene expression of TNF-alpha, IFN-gamma, and IL-10 was routinely seen, but IL-4 expression was absent except in two clinically quiescent patients. Trends were observed toward decreased TNF-alpha expression, but increased IL-10 expression, after treatment with IFN-beta1b.

Antisense Elements (Genetics)↗
PubMed1997

Induction of tumor necrosis factor alpha in human neuronal cells by extracellular human T-cell lymphotropic virus type 1 Tax.

To examine the role of human T-lymphotropic virus type 1 (HTLV-1) Tax1 in the development of neurological disease, we studied the effects of extracellular Tax1 on gene expression in NT2-N cells, postmitotic cells that share morphologic, phenotypic, and functional features with mature human primary neurons. Treatment with soluble HTLV-1 Tax1 resulted in the induction of tumor necrosis factor alpha (TNF-alpha) gene expression, as detected by reverse-transcribed PCR and by enzyme-linked immunosorbent assay. TNF-alpha induction was completely blocked by clearance with anti-Tax1 monoclonal antibodies. Furthermore, cells treated with either a mock bacterial extract or with lipopolysaccharide produced no detectable TNF-alpha. Synthesis of TNF-alpha in response to soluble Tax1 occurred in a dose-dependent fashion between 0.25 and 75 nM and peaked within 6 h of treatment. Interestingly, culturing NT2-N cells in the presence of soluble Tax1 for as little as 5 min was sufficient to result in TNF-alpha production, indicating that the induction of TNF-alpha in NT2-N does not require Tax1 to be continually present in the culture medium. Treatment of the undifferentiated parental embryonal carcinoma cell line NT2 with soluble Tax1 did not result in TNF-alpha synthesis, suggesting that differentiation-dependent, neuron-specific factors may be required. These results provide the first experimental evidence that neuronal cells are sensitive to HTLV-1 Tax1 as an extracellular cytokine, with a potential role in the pathology of HTLV-1-associated/tropical spastic paraparesis.

Cell Differentiation↗
PubMed1994

Cytokine-gene expression in measles-infected adult human glial cells.

The expression of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF) alpha transcripts in cultured human glial cells was examined using reverse transcription followed by polymerase chain reaction (PCR) amplification and Southern blot quantitation. Microglial cultures derived from brain biopsy specimens from three different individuals expressed transcripts for the three cytokines under basal culture conditions. This expression was enhanced in response to measles virus (MV) infection (IL-1 beta, 2.2-8.8-fold; IL-6, 2.5-8.4-fold; TNF alpha, 2.2-3.2-fold). Neither IL-1 beta nor TNF alpha transcripts were detectable in undissociated brain tissue from two individuals, suggesting that the basal expression of these cytokines in culture may have been induced by tissue dissociation or by the culture conditions. Oligodendrocytes did not express cytokine transcripts under basal culture conditions, and IL-1 beta and IL-6 but not TNF alpha transcripts could be induced by MV. Similarly, meningeal fibroblasts expressed IL-1 beta and IL-6 but not TNF alpha in response to MV-infection, suggesting that the production of TNF alpha is more cell type-restricted than either IL-1 beta or IL-6. The results indicate that adult human microglia can participate in the inflammatory response to MV infection in the CNS by producing cytokines that contribute to inflammation and demyelination. In addition, besides their role in myelination, oligodendrocytes can potentially influence immunoreactivity in the CNS by producing IL-1 beta and IL-6.

Adolescent↗
PubMed1994

Molecular characterization of HLA class II genes in celiac disease patients of Latin American Caucasian origin.

In the present study, the polymorphic domain of HLA class II genes present in a pediatric population of Argentinian celiac disease patients was analyzed by hybridization to sequence-specific oligonucleotides and DNA sequencing. Sixteen out of 16 DR5/7 heterozygous patients bore the DQA1*0501 and DQB1*0201 alleles implicated in the DQ2 risk specificity. The second exon of DQA1, DQB1 and DRB1 genes from 2 DR5/7 patients was characterized by DNA sequencing. The following alleles were found in both patients: DRB1*1101 and DRB1*0701; DQB1*0301 and DQB1*0201; DQA1*0501 and DQA1*0201. Previous serological analysis in this population had shown the presence of DQ2 in 95% of the patients (40% in controls) and a negative association with DQ1 haplotypes, suggesting the presence of other "permissive" or neutral alleles. The following HLA-DQB1 alleles, besides DQB1*0201, were identified in 31 CD patients: DQB1*0301, 0302, 0401 and 0402. All these alleles share a common pattern of residues between positions 84 and 90, and distinct from that present in DQ1-related alleles.

Alleles↗
PubMed1994

Induction of HLA class I and class II expression in human T-lymphotropic virus type I-infected neuroblastoma cells.

Human T-lymphotropic virus type I (HTLV-I) is associated with a neurologic disease, HTLV-I-associated myelopathy-tropical spastic paraparesis, in which both pathological and immunological changes are observed within the central nervous system. The pathogenesis of infection in HTLV-I-associated myopathy-tropical spastic paraparesis is not well understood with respect to the cell tropism of HTLV-I and its relationship to the destruction of neural elements. In this study, neuroblastoma cells were infected with HTLV-I by coculturing with HUT-102 cells to demonstrate that cells of neuronal origin are susceptible to this retroviral infection. HTLV-I infection of the neuroblastoma cells was confirmed by verifying the presence of HTLV-I gp46 surface antigens by flow cytometry and by verifying the presence of HTLV-I pX RNA by Northern (RNA) blotting and in situ hybridization techniques. To determine whether HTLV-I infection could potentially lead to changes in cell surface recognition by the immune system, the infected neuroblastoma cells were analyzed for altered HLA expression. The HTLV-I-infected, cocultured neuroblastoma cells were shown, through cell surface antigen expression and RNA transcripts, to express HLA classes I and II. In contrast, cocultured neuroblastoma cells that did not become infected with HTLV-I expressed only HLA class I. HLA class I expression was enhanced by the cytokines tumor necrosis factor alpha and gamma interferon and in the presence of HUT-102 supernatant. In this system, expression of HLA class I and II molecules appeared to be regulated by different mechanisms. HLA class I expression was probably induced by cytokines present in the HUT-102 supernatant and was not dependent on HTLV-I infection. HLA class II expression required HTLV-I infection of the cells. The observation of HTLV-I infection leading to HLA induction in these neuroblastoma cells provides a possible mechanism for immunologic recognition of infected neuronal cells.

Cell Communication↗
PubMed1993

Direct evidence that interferon-beta mediates enhanced HLA-class I expression in measles virus-infected cells.

Viral infection results in enhancement of HLA-class I expression in a number of cell types, including glial cells, which normally do not express these molecules. This enhancement may occur through a direct interaction between a viral component and the HLA-class I gene or indirectly through virus-induced soluble factors produced by infected cells. These include cytokines such as IFN-gamma, IFN-alpha/beta, and TNF-alpha, known to enhance class I expression. Measles virus (MV) infection of a human glioma cell line (U-105 MG) and of primary human umbilical vein endothelial cells enhances the expression of HLA-class I molecules on these cells. The enhancement of HLA-class I is dependent on infectious virus, as antibody-neutralized MV has no effect on class I expression. In this study, we demonstrate the presence of an HLA-class I-enhancing factor in supernatants from MV-infected cells. The supernatant class I-enhancing factor is not IFN-gamma, IFN-alpha, or TNF-alpha because MV-infected cells did not produce measurable levels of these cytokines as detected by immunoassay or polymerase chain reaction. In contrast, IFN-beta is produced by the infected cells and the supernatant class I-enhancing factor could be entirely neutralized by antibodies to IFN-beta, but not antibodies to IFN-alpha, TNF-alpha, or non-immune sera. Furthermore, preincubation of cells with neutralizing antibodies to IFN-beta prior to infection blocked MV enhancement of HLA-class I completely in the U-105 MG cells and by as much as 74% in the umbilical vein endothelial cells. The results of these experiments provide direct evidence that enhanced HLA-class I expression in MV-infected cells is mediated primarily by IFN-beta.

Cells, Cultured↗
PubMed1991

Class II MHC antigen expression by cultured human cerebral vascular endothelial cells.

Cerebral vascular endothelial cells (EC) isolated from human brain do not constitutively express class II MHC antigens. However, incubation in the presence of human interferon-gamma (IFN gamma) resulted in the expression of both HLA-DR and -DP antigens. FACS analysis revealed that approximately 40% of the EC population expressed HLA-DR antigen. Quantitation by ELISA demonstrated that maximum expression was observed with 100 U/ml IFN gamma for 4 days. Treatment with IFN gamma also increased class II mRNA levels in all EC cultures tested.

Blotting, Northern↗
PubMed1991

Interleukin-1 beta decreases HLA class II expression on a glioblastoma multiforme cell line.

Antigens encoded within the major histocompatibility complex (MHC) are not normally expressed in the central nervous system (CNS), but can be induced by treatment with interferon-gamma (IFN-gamma). Other cytokines released during an inflammatory process can potentially influence MHC expression as well. One cytokine of interest is interleukin-1 (IL-1), an immunoregulatory polypeptide that is produced by macrophages and also by cells in the CNS. In this study, the effect of IL-1 beta on MHC expression in a human glioblastoma multiforme cell line, U-105 MG, has been examined. Treatment of U-105 MG with 10 U IL-1 beta/ml for a period of 5 days resulted in a decrease in constitutive cell surface HLA class II expression and limited the induction of class II by IFN-gamma. This effect was also observed on steady-state levels of class II RNA and could be neutralized with antibodies to IL-1 beta. All class II transcripts examined (HLA-DR, -DQ, and -DP alpha and beta) were affected. Class I expression was only marginally changed by IL-1 beta treatment. A minimal concentration of 1 U IL-1 beta/ml was required to reduce class II expression and a kinetics experiment indicated that U-105 MG must be treated for at least 4 days with IL-1 beta for a decrease in class II expression to be observed. This study suggests that IL-1 may play a role in limiting immunoreactivity in the CNS by limiting class II induction.

Dose-Response Relationship, Drug↗
PubMed1991

HLA-DR and -DQ allelic sequences in multiple sclerosis patients are identical to those found in the general population.

The HLA-DR2/Dw2 haplotype is associated with multiple sclerosis (MS) in the North American Caucasian population. HLA-DRB, -DQA, and -DQB N-terminal domain sequences derived from amplified cDNA in a series of North American Caucasian MS patients were examined to determine if unique or rare class II alleles could be found. In addition, class II allelic sequences were analyzed from clinically discordant, HLA-genoidentical siblings from a multiplex MS family. All alleles observed, whether from HLA-DR2/Dw2 positive or negative individuals, were identical to those most commonly expressed in the general population. These data demonstrate that, if HLA class II truly confers susceptibility to MS, commonly expressed alleles are involved.

Alleles↗
PubMed1989

Differential effects of amino acid substitutions in the beta-sheet floor and alpha-2 helix of HLA-A2 on recognition by alloreactive viral peptide-specific cytotoxic T lymphocytes.

Crystallographic studies of the HLA-A2 molecule have led to the assignment of a putative peptide binding site that consists of a groove with a beta-pleated sheet floor bordered by two alpha-helices. A CTL-defined variant of HLA-A2, termed HLA-A2.2F, differs from the common A2.1 molecule by three amino acids: a Leu to Trp substitution at position 156 in the alpha-2 helix, a Val to Leu substitution at position 95 in the beta-sheet floor of the groove, and a Gln to Arg substitution at position 43 in a loop outside of the groove. Another HLA-A2 variant, termed CLA, has a single Phe to Tyr substitution at position 9 that is sterically located adjacent to position 95 in the beta-sheet floor of the groove. We have determined which of the amino acid substitutions at positions 9, 43, 95, or 156 could individually affect recognition by panels of A2.1 allospecific and A2.1-restricted influenza viral matrix peptide-specific CTL lines, using a panel of site-directed mutants and CLA. Recognition by allospecific CTL lines was generally unaffected by any one of the amino acid substitutions, but was eliminated by the double substitution at positions 95 and 156. Allorecognition by some CTL lines was eliminated by a single substitution at position 9 or 95. In contrast, recognition by A2.1-restricted matrix peptide specific CTL was totally eliminated by a single substitution at position 9 or 156. The substitution at position 43 in a loop away from the peptide binding groove had no effect on allorecognition or matrix peptide recognition. These results indicate that amino acid residues in the floor or alpha-2 helical wall of the peptide binding groove of the HLA-A2 molecule can differentially affect allorecognition and viral peptide recognition.

Amino Acid Sequence↗
PubMed1989

A single amino acid substitution in HLA-A2 can alter the selection of the cytotoxic T lymphocyte repertoire that responds to influenza virus matrix peptide 55-73.

Previous studies have demonstrated that certain amino acid substitutions in the alpha two domain at positions 152 and 156 in the alpha two helix of the HLA-A2 molecule can affect presentation of the influenza virus matrix peptide M1 55-73 without abolishing binding of the M1 peptide. HLA-A2.1-restricted M1 55-73 peptide-specific CTL lines obtained from almost all HLA-A2.1+ individuals fail to recognize the M1 peptide presented by site-directed mutants of HLA-A2 that have either a Val----Ala or Val----Gln substitution at position 152 or a Leu----Trp substitution at position 156. Only one HLA-A2+ individual (donor Q66, HLA-A2,-B53,-B63) has been found who is able to generate a unique repertoire of HLA-A2-restricted M1 peptide-specific CTL that can recognize peptide presented by HLA-A2 mutants with either an Ala or Gln substitution at position 152 or a Trp substitution at position 156. These Q66 M1 peptide-specific CTL could be selected by stimulation with M1 peptide-pulsed transfectants that express the mutant HLA-A2 gene with the Trp substitution at 156. To determine if the presence of the unique CTL repertoire could be attributed to a variant HLA-A2 molecule in Q66, sequences were determined from polymerase chain reaction-amplified segments of the HLA-A2 RNA. Two different HLA-A2 genes were found expressed in Q66 cells: one is identical to HLA-A2.1 and the other is identical to HLA-A2.2F (Gln----Arg at position 43, Val----Leu at position 95, and Leu----Trp at position 156). These results demonstrate that a different CTL repertoire specific for HLA-A2 plus the M1 55-73 peptide is generated in an individual that expresses both HLA-A2.1 and HLA-A2.2F compared to individuals who express HLA-A2.1 alone, and that the unique repertoire can be selected by the presence of an HLA-A2 molecule with a single amino acid substitution at position 156.

Adult↗
PubMed1989

Genetic regulation of class I major histocompatibility complex (MHC) antigen induction on astrocytes.

Neural cells, including astrocytes, normally do not express detectable levels of class I major histocompatibility complex (MHC) molecules, unlike cells of most tissues. However, upon cultivation in vitro, astrocytes begin to express class I molecules, increasing with time after plating. This spontaneous expression was examined in the present study to characterize inducible expression on astrocytes among various strains of mice. Inducible expression, either as a consequence of cultivation or standard gamma-interferon treatment, differed markedly among the strains examined. Analysis of congenic strains on a C57BL/10 (B10) background showed that expression was controlled by genes within the MHC locus. Examination of additional congeneic animals with various recombinations within the MHC showed that high or low expression of MHC molecules correlates with the presence of particular MHC class I genes. In general, H-2a and H-2d class I products are expressed much higher on astrocytes than H-2b and H-2s products. This difference in expression is not seen on spleen cells indicating tissue specificity. Moreover, levels of expression at the cell surface are reflected by the steady-state level of RNA message within astrocytes of the different strains.

Animals↗
PubMed1988

Analysis of the molecular basis of HLA-A3 recognition by cytotoxic T cells using defined mutants of the HLA-A3 molecule.

The structure-function relationships in human class I HLA molecules have been examined by the analysis of two T cell-defined subtypes of HLA-A3 (A3.1 and A3.2). These subtypes differ by two amino acid residues that are located at positions 152 (GluA3.1 vs ValA3.2) and 156 (LeuA3.1 vs GlnA3.2). By the methods of site-directed mutagenesis and DNA-mediated gene transfer, mammalian cell transfectants have been produced that express only one of the above A3.2 amino acid residues at either position 152 or position 156. Previous studies using murine transfectants have shown that A3.1- and A3.2-expressing cells can be distinguished by A3.1-restricted type A influenza virus-specific CTL and A3.2-allospecific CTL and have implied that amino acid position 152 plays a key role in this specificity. To test whether these results were a function of the virus specificity, the alloantigen, or the cell type expressing the class I molecules, we have tested the recognition of human and murine cell transfectants by A3.1-restricted, A/JAP/305/57 and B/Ann Arbor-specific CTL and by A3.1- and A3.2-allospecific CTL. The results indicate that the Glu at position 152 is critical for recognition by all of the A3.1-restricted CTL populations tested and 15 of 16 of the A3.1-allospecific CTL populations tested. The A3.1 Leu at position 156 was sufficient for recognition by only one A3.1-allospecific CTL line. Substitution of the charged Glu residue for the polar Gln at position 156 of A3.2 affected recognition of some but not all A3.2-alloreactive CTL. These data demonstrate that the structural basis for epitopes that are recognized by almost all CTL that discriminate between A3.1 and A3.2 is primarily the amino acid at position 152. The implications of these data for Ag presentation and CTL recognition are discussed.

Adult↗