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J I Bell

Publications and source records attributed to J I Bell.

At least 55 records · Page 3Linked to original sources
PubMed1996

Production and crystallization of MHC class I B allele single peptide complexes.

Major histocompatibility complex class I B alleles, HLA B8, B53 and B3501 have been cloned, expressed, refolded and crystallized in specific complexes with a number of different 8-mer and 9-mer peptides. For some of these crystallization was initiated by cross-seeding between different B allele complexes. All crystallize in the space group P212121, with similar unit cell dimensions of approximately 52 A X 81 A X 112 A, contain one complex per asymmetric unit and diffract to approximately 2.0 A resolution.

Alleles↗
PubMed1996

A functionally significant allelic polymorphism in a T cell receptor V beta gene segment.

The effect of an allelic polymorphism in the BV1S1 gene segment on recognition of major histocompatibility complex (MHC)-peptide complexes by a specific T cell receptor (TCR) was studied using RBL 2H3 cells transfected with TCR-CD3 zeta chimeric receptors. An HLA-A2-restricted human immunodeficiency virus (HIV) pol-specific cytotoxic T lymphocyte (CTL) clone utilizing the BV1S1A2 gene in combination with AV2S1A2 was identified and the extracellular domains of the TCR were fused to CD3 zeta. In degranulation assays RBL 2H3 transfectants expressing this receptor maintained the specificity of the parental CTL clone. The allelic variant BV1S1A1N1 containing a glutamine for histidine substitution at position 48 in the loop of the second complementarity-determining region was generated by site-directed mutagenesis. Transfection of this molecule as a CD3 zeta chimera together with the original AV2S1A2 CD3 zeta molecule resulted in cell surface expression of both chains but a loss of recognition of HLA-A2 HIV pol peptide-pulsed targets. The effect of this polymorphism on MHC-peptide recognition supports current models of TCR MHC-peptide interaction and provides evidence for a functional role for polymorphism in the TCRV genes.

Alleles↗
PubMed1996

An altered position of the alpha 2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501.

The crystal structure of the human major histocompatibility complex class I B allele HLA B*3501 complexed with the 8-mer peptide epitope HIV1 Nef 75-82 (VPLRPMTY) has been determined at 2.0 angstrom resolution. Comparison with the crystal structure of the closely related allele HLA B*5301 reveals the structural basis for the tyrosine specificity of the B*3501 F pocket. The structure also reveals a novel conformation of the 8-mer peptide within the binding groove. The positions of the peptide N and C termini are nonstandard, but the classic pattern of hydrogen bonding to nonpolymorphic MHC class I residues is maintained, at the N terminus by addition of a water molecule, and at the C terminus by a substantial shift in the alpha 2 helix.

Alleles↗
PubMed1996

Bound water structure and polymorphic amino acids act together to allow the binding of different peptides to MHC class I HLA-B53.

The structure of the human MHC class I molecule HLA-B53 complexed to two nonameric peptide epitopes (from the malaria parasite P. falciparum and the HIV2 gag protein) has been determined by X-ray crystallography at 2.3 angstrom resolution. The structures reveal the architecture of a Pro-specific B pocket common to many HLA-B alleles. Relative to other alleles, the B53 peptide-binding groove is widened by a significant (up to 1.25 angstrom) shift in the position of the alpha 1 helix. Within this groove, bound water molecules, acting in concert with the side chains of polymorphic residues, provide the functional malleability of the MHC, which enables the high affinity/low specificity binding of multiple peptide epitopes.

Amino Acid Sequence↗
PubMed1996

Two stage genome-wide search in inflammatory bowel disease provides evidence for susceptibility loci on chromosomes 3, 7 and 12.

Crohn's disease (CD) and ulcerative colitis (UC), the chronic inflammatory bowel diseases (CIBD), are common causes of gastro-intestinal disease in the Western world, with a combined prevalence of 100-200/100,000 (ref. 1). Epidemiological studies, particularly concordance rates in twin pairs and siblings, strongly implicate genetic susceptibility in the pathogenesis of CIBD. In fact, the relative contribution of genetic factors to the pathogenesis of CD may be greater than in schizophrenia, asthma or hypertension, and at least equivalent to that in insulin-dependent diabetes. Systematic screening of the entire human genome now provides a strategy for the identification of susceptibility genes in complex polygenic disorders. We undertook a two-stage genome search for susceptibility genes in inflammatory bowel disease involving 186 affected sibling pairs from 160 nuclear families. We provide strong evidence for the presence of susceptibility loci for both CD and UC on chromosome 3, 7 and 12. We obtained the highest lod score (5.47; P = 2.66 x 10(-7) with the marker D12S83 and lod scores of 3.08 and 2.69 for D7S669 and D3S1573, respectively. Our data suggest that CD and UC are closely related, but distinct, polygenic disorders that share some, but not all, susceptibility genes.

Chromosome Mapping↗
PubMed1996

Large clonal expansions of CD8+ T cells in acute infectious mononucleosis.

Primary infection with Epstein-Barr virus often results in the clinical syndrome of acute infectious mononucleosis (glandular fever). This illness is characterized by a striking lymphocytosis, the nature of which has been controversial. We show that large monoclonal or oligoclonal populations of CD8+ T cells account for a significant proportion of the lymphocytosis and provide molecular evidence that these populations have been driven by antigen. The results suggest that the selective and massive expansion of a few dominant clones of CD8+ T cells is an important feature of the primary response to this virus.

Acute Disease↗
PubMed1996

Analysis of peptide-binding motifs for two disease associated HLA-DR13 alleles using an M13 phage display library.

Major histocompatibility complex (MHC) molecules bind peptides bearing an appropriate 'sequence motif' for MHC binding. The use of phage display libraries exploits the ability of MHC class II molecules to exchange peptides in solution and thus select out peptide sequences with high-affinity binding from a large array of random peptides. We have analysed the peptide binding motifs of HLA-DRB1*1301 and *1302 using affinity purified HLA-DR13 molecules to purify sequentially HLA-DR13-binding peptides from a large random library of M13 phage containing nonamer inserts in the pIII coat protein. These DR13 alleles differ only at position 86 of the HLA-DR beta chain, where they contain valine and glycine residues respectively. These alleles were chosen because of their association with protection from severe malaria and chronic hepatitis B virus infection in West Africa. Analysis of the phage bound to these DR molecules suggests binding motifs. We compare the results derived from the use of the phage display library with results obtained from analysis of eluted peptides and peptide-binding studies. This analysis shows that although there is a common theme to motifs derived using different methods, there are also subtle variations between them.

Alleles↗
PubMed1995

CD8highCD57+ T lymphocytes in normal, healthy individuals are oligoclonal and respond to human cytomegalovirus.

CD8+CD57+ lymphocytes from normal peripheral blood are divided into T cells expressing high levels of CD8 and NK cells expressing low levels of surface CD8. Increased numbers of CD8highCD57+ T cells correlate with previous exposure to human cytomegalovirus (HCMV) infection, but no virus-specific function or function for CD8highCD57+ cells has been recorded. We have studied the TCR repertoire and responses of the CD8highCD57+ population induced by virus-infected fibroblasts in healthy individuals. Using three-color flow cytometry of PBL, we detected restricted TCRBV usage in the CD8highCD57+ subset of 11/15 subjects, compared with 1/15 in the CD8+, CD57- subset. The results of anchored PCR and sequencing also showed oligoclonality of TCR; 40-70% of CD8highCD57+ lymphocytes (10-20% of total CD8+ T cells) were derived from single clones. Such expansions were stable with time and detected in one subject over a 2-yr period. Functionally, CD8highCD57+ lymphocytes proliferated strongly to HCMV-, but not HSV-, VZV-, or influenza-infected fibroblasts in an MHC-unrestricted manner in vitro, including preferential augmentation of particular in vivo oligoclonally expanded subpopulations. HCMV-specific MHC-restricted CTL were detected, but limiting dilution analysis showed that these were a minority (< 10%) and not the oligoclonal subsets. In contrast, depletion of oligoclonally expanded CD8highCD57+ subpopulations, resulted in the increase of HCMV-specific CTL, suggesting functional heterogeneity in these cells.

Adult↗
PubMed1995

Identification and characterization of three members of the human SR family of pre-mRNA splicing factors.

SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C-terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta-thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.

Alternative Splicing↗
PubMed1995

Naturally processed peptides from two disease-resistance-associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DR beta chain.

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR beta chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly-->Val change at position 86. These data suggest a molecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection.

Alleles↗
PubMed1995

Persistent high frequency of human immunodeficiency virus-specific cytotoxic T cells in peripheral blood of infected donors.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTLs) are thought to play a major role in the immune response to HIV infection. The HIV-specific CTL response is much stronger than previously documented in an infectious disease, yet estimates of CTL frequency derived from limiting-dilution analysis (LDA) are relatively low and comparable to other viral infections. Here we show that individual CTL clones specific for peptides from HIV gag and pol gene products are present at high levels in the peripheral blood of three infected patients and that individual CTL clones may represent between 0.2% and 1% of T cells. Previous LDA in one donor had shown a frequency of CTL precursors of 1/8000, suggesting that LDA may underestimate CTL effector frequency. In some donors individual CTL clones persisted in vivo for at least 5 years. In contrast, in one patient there was a switch in CTL usage suggesting that different populations of CTLs can be recruited during infection. These data imply strong stimulation of CTLs, potentially leading some clones to exhaustion.

Amino Acid Sequence↗
PubMed1995

Increased expression of CD44v6 and CD44v3 in ulcerative colitis but not colonic Crohn's disease.

Immune mechanisms, possibly involving cell-surface molecules such as CD44, have been invoked to explain the pathogenesis of inflammatory bowel disease. We used monoclonal antibodies against epitopes encoded within the variable region of CD44 to investigate CD44 isoform expression in colon, small intestine, and liver in patients with various intestinal disorders and in controls. Biopsy samples from patients with ulcerative colitis showed significantly increased epithelial expression of CD44 isoforms containing the v6 and v3 epitopes, detected with antibodies 2F10 and 3G5, respectively. CD44v6 was detected on colonic crypt epithelial cells in 23 of 25 ulcerative colitis samples compared with 3 of 18 colonic Crohn's disease samples (p = 3.0 x 10(-6); odds ratio 57.5 [95% CI 6.83-702]) and 3 of 52 controls (22 normal colon, 10 infective colitis, 2 radiation colitis, and 18 colonic Crohn's disease; p < 1 x 10(-8); odds ratio 199 [25.5-2294]). No significant expression of CD44v6, CD44v3, or CD44v8/9 was found in samples of normal proximal colon from 4 patients with distal ulcerative colitis, whereas samples from the affected area showed staining for CD44v6 and CD44v3. No expression of CD44 variants was found in 15 samples of normal small intestine, 11 small-bowel pouchitis, 8 coeliac disease, 3 small-bowel Crohn's disease, 6 normal liver, 6 primary biliary cirrhosis, or 9 primary sclerosing cholangitis. The high intensity of CD44v6 and v3 epitope expression on crypt epithelial cells in ulcerative colitis suggests that CD44 isoforms may have an important role in ulcerative colitis. Their detection could have diagnostic potential in differentiating ulcerative colitis from other forms of colonic inflammation including Crohn's disease.

Antibodies, Monoclonal↗
PubMed1995

Human HLA-A0201-restricted cytotoxic T lymphocyte recognition of influenza A is dominated by T cells bearing the V beta 17 gene segment.

The major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response is important in the clearance of viral infections in humans. After influenza A infection, a peptide from the matrix protein, M58-66, is presented in the context of the MHC allele HLA-A0201 and the resulting CTL response is detectable in most HLA-A0201 subjects. An initial study suggested that M58-66-specific CTL clones show conserved T cell receptor (TCR) alpha and beta gene segments. We have addressed the significance of this observation by determining the expression of V beta 17 during the development of M58-66-specific CTL lines in 21 unrelated HLA-A0201 subjects, and analyzing TCR usage by M58-66-specific CTL clones. TCR V beta 17 was the dominant V beta segment used and CD8 V beta 17 expansion correlated with M58-66-specific lysis. Limiting dilution analysis from five subjects showed the M58-66 CTL precursor frequency to vary between 1/54,000 and less than 1/250,000, and that up to 85% of the matrix peptide (M58-66)-specific CTL used the V beta 17 gene segment. The M58-66 specific CTL response was dependent on previous viral exposure and specific V beta 17 expansion, as it was not found in cord blood, despite a readily expandable V beta 17+ CD8+ T cell subpopulation. Sequence analysis of 38 M58-66-specific V beta 17 transcripts from 13 subjects revealed extensive conservation in the CDR3 region including conservation of an arginine-serine motif. To test the dependence of this CTL response on the V beta 17 gene segment, peripheral blood lymphocytes were depleted of CD8+ TCR V beta 17+ cells, before the generation of M58-66-specific CTL. In most cases such depletion blocked or severely reduced the generation of the M58-66-specific response, and under limiting dilution conditions could abolish M58-66-specific CTL precursors. These studies reveal the dependence of this natural human immune response on a particular TCR gene segment.

Alleles↗
PubMed1995

Selection of T cell receptor variable gene-encoded amino acids on the third binding site loop: a factor influencing variable chain selection in a T cell response.

The 3' end of the T cell receptor V beta 7.1 gene contains the five nucleotides CAAGA between the broadly conserved consensus sequence of nucleotides TGC/T GCC AGC AGC (which encode cysteine, alanine, serine and serine at positions 92-95 of the beta chain) and the heptamer that signals rearrangement. These nucleotides are frequently preserved during gene rearrangement, resulting in the common presence of glutamine at position 96 and of aspartate or glutamate at position 97 of the V beta 7.1 chain CDR3 loop in peripheral blood lymphocytes. There is selection of V beta 7.1 and of the V beta 7.1 gene-encoded glutamate at position 97 of the beta chain CDR3 loop in the cytotoxic T lymphocyte response to the HLA B2705-restricted influenza A nucleoprotein epitope SRYWAIRTR. Our results indicate that selection of V beta 7.1 gene-encoded amino acid residues on CDR3 loops may be one factor driving selection of V beta 7.1 in this response.

B-Lymphocytes↗