Biomedical subjects

S Rojo

Publications and source records attributed to S Rojo.

16 recordsLinked to original sources

PubMed1992

Peptide-presenting similarities among functionally distant HLA-B27 subtypes revealed by alloreactive T lymphocytes of unusual specificity.

Functional dissection of HLA-B27 subtypes using alloreactive or B27-restricted CTL has shown that the structurally related B*2704 and B*2706 are the most distant subtypes relative to the prototype B*2705. In particular, previous studies have failed to find anti-B*2705 CTL cross-reacting with B*2704 or B*2706. Such failure can be accounted for by the drastic effect on T cell recognition of the change at residue 152 in both subtypes relative to B*2705, as established with site-directed mutants. B*2704 and B*2706 are also related in ethnic distribution, as they are restricted to Orientals, jointly being the predominant HLA-B27 subtypes in this population. As far as it is known, there are no differences relative to B*2705 in their linkage to ankylosing spondylitis. In our study, 5 of 13 examined anti-B*2705 limiting dilution CTL lines from a particular HLA-B27- individual were shown to crossreact with B*2704, B*2706 or both. The monoclonal nature of this cross-reaction was established by cold target competition analysis. This result demonstrates that the apparent differences in T cell antigenicity among anti-B27 subtypes are strongly influenced by the responder individual, as the spectrum of clonal specificities in anti-B27 responses may show significant differences among unrelated responders. Fine specificity differences among the cross-reactive CTL allowed unambiguous functional distinction between B*2704 and B*2706. The molecular basis of such cross-reactivity was examined by correlating CTL reaction patterns with the structure of both subtypes, which differ only by two residues located in the beta-pleated sheet bottom of the peptide binding site, and with site-directed mutants mimicking HLA-B27 subtype polymorphism. The results suggest that: 1) distinct peptides are involved in the allospecific epitopes recognized by the various crossreactive CTL, and 2) B*2704, B*2706, and B*2705 differ in their peptide-presenting specificity, but can present some identical or structurally similar peptides.

Clone Cells↗

PubMed1992

[Herpes simplex virus infection in pregnancy: epidemiologic, diagnostic and therapeutic data. An unusual case of HSV-1 in monozygotic twins].

The Authors report a case of two pre-term monocorial twins affected by HSV-1 infection (Herpes Simplex Virus type 1), with generalized sepsis and involvement of the central nervous system (CNS), born by mother with primary infection who presented a typical vesicular eruption a week before delivery. As the HSV-1 was a disseminated type and the CNS was involved in both the twins, the diagnosis was based on clinical and laboratory findings (specific IgM and IgG) and on the use of Delpech-Lichtblau antibody liquoral index, a significant results both in the first and the second twin. Intensive care and early specific treatment with Acyclovir allowed a slow but progressive improvement of the twins' clinical picture. The antibody liquoral index may therapy of the viral sepsis cases involving the CNS, more than the cultural or antigen isolation of the treated virus.

Acyclovir↗

PubMed1991

Conservation and alteration of HLA-B27-specific T cell epitopes on mouse cells. Implications for peptide-mediated alloreactivity.

Alloreactive CTL responses generate a great variety of clonal specificities. Such diversity may be related to recognition of multiple peptides constitutively bound to any given MHC alloantigen. Among human alloreactive CTL, only a fraction of the clones lyse mouse P815 cells expressing class I HLA proteins. In this study the fine specificity of HLA-B27 allorecognition on human or mouse cells by five human HLA-B27-specific CTL clones was comparatively analyzed. This was done to examine what degree of variation in epitope structure is compatible with recognition of HLA Ag on mouse cells. Nine site-specific HLA-B27 mutants were expressed on both human and mouse cells, after DNA-mediated gene transfer, to construct two analogous series of target cells. The reaction patterns of four of the five CTL clones with these cell panels were compatible with conservation of their corresponding epitopes upon expression of HLA-B27 on mouse cells. The reaction pattern of the fifth clone was different with either cell panel, indicating that its epitope was structurally altered on mouse cells. It also suggested a selectively increased expression of the determinant on these cells. The results suggest that most of the epitopes recognized by allospecific CTL clones reacting across species are either independent of any bound peptide or involve identical peptides from both cell types. However, some of these clones recognize alloantigen-bound peptides that are somewhat different in structure depending on the cell type, and may be expressed at the mouse cell surface in greater amounts. Such peptides could arise from related proteins in both species, and be polymorphic as a result of phylogenetic divergence.

Animals↗

PubMed1991

Peptide-mediated allo-recognition of HLA-B27 by cytotoxic T lymphocytes.

The molecular basis of the recognition of class-I HLA antigens by allo-reactive cytotoxic T lymphocytes (CTL) remains obscure. This article reviews our work in which HLA-B27-specific allo-reactive CTL clones were obtained and their fine specificity was analyzed with a panel of structurally defined HLA-B27 natural variants and site-directed mutants expressed on human and mouse cells. The results have implications for the involvement of endogenous peptides in determining the clonal diversity of HLA-B27 allogeneic responses and the fine specificity of T-cell recognition when HLA-B27 is expressed on different cell types.

Animals↗

PubMed1990

Structure and diversity of HLA-B27-specific T cell epitopes. Analysis with site-directed mutants mimicking HLA-B27 subtype polymorphism.

HLA-B27 subtype polymorphism is amenable to differential recognition by CTL. Site-directed mutagenesis was used to construct a series of HLA-B27 mutants reproducing most of the changes occurring in the natural subtypes. The reactivity of 21 anti-HLA-B27 CTL clones was examined with these mutants to address three issues concerning the alloreactive response against HLA-B27: 1) diversity of clonotypic specificities, 2) structural features of the epitopes recognized by these clones, and 3) role of individual positions in the differential recognition of HLA-B27 subtypes. Virtually all CTL clones displayed unique reaction patterns with the mutants, indicating a corresponding diversity of epitopes. However, these share some molecular features, such as certain amino acid residues and related locations. Individual mutations induced complex effects on multiple B27-specific CTL epitopes, revealing some of their very precise stereochemical constrains. An important feature of HLA-B27 subtype polymorphism is that every individual change was relevant, altering recognition by many CTL clones. Although the specific set affected by each mutation was partially different, the global number of clones affected by most changes was very similar. This suggests that the antigenic profile of any given subtype is not dominated by one particular change but is uniquely defined by its corresponding set of changes. An exception was the change at position 152, which totally abrogated recognition by all 20 anti-B*2705 CTL clones. This effect decisively influences the profound differences in T cell recognition between B*2705 and the two subtypes, B*2704 and B*2706, carrying this change. The results are compatible with the idea that HLA-B27 allorecognition may involve multiple peptides bound to the alloantigen on the cell surface.

Amino Acid Sequence↗

PubMed1990

Structure and immune recognition of HLA-B27 antigens: implications for disease association.

HLA-B27 is strongly associated with susceptibility to ankylosing spondylitis and other spondyloarthropathies. Structural analysis of this antigen has revealed the existence of multiple variants, or subtypes, in human populations. The structural microheterogeneity of these subtypes deeply affects allospecific T cell recognition and most of it occurs at an spatial cluster within the peptide binding groove of the molecule. Many polymorphic residues whose combination is unique to HLA-B27 but is conserved among subtypes are clustered in a spatially separated site of the groove from that where most subtype polymorphism occurs. Site-directed mutagenesis and DNA-mediated gene transfer has been used to show that the positions that are polymorphic among subtypes are highly relevant for modulating T cell recognition, so that immunologically silent changes do not occur. These studies have also revealed an extremely high clonotypic diversity in the alloreactive response against HLA-B27. The structural basis for this diversity has been examined by sequencing the clonotypic T cell receptors. The analysis shows a certain bias in V beta gene segment usage, as well as other recurrent structural motives, among T cell receptor beta chains from HLA-B27-specific cytotoxic T cell clones.

Amino Acid Sequence↗

PubMed1988

Avidity dictates the lytic capacity of human cytolytic T lymphocyte clones with similar fine specificity against murine cells expressing HLA-B27 antigen.

The role of the avidity of human CTL in the recognition and lysis of murine P815 cells expressing HLA-B27.1 Ag has been examined. Seven B27-specific alloreactive CTL clones were tested for their ability to lyse a B27.1+-P815 transfectant clone 1-7E, obtained after cotransfection of P815-HTR cells with HLA-B27.1 and human beta 2-microglobulin genes. The expression level of HLA-B27.1 on 1-7E cells was comparable to that on a human lymphoblastoid cell line, as determined by flow cytometry. Of the seven CTL clones used, CTL 1, 26, and 29 displayed the same fine specificity as established with a panel of target cells expressing six structurally different HLA-B27 variants. However, CTL 1 and 29 were of higher avidity than CTL 26, in that the lysis of human target cells by only this latter clone was inhibited by an anti-CD8 mAb. Based on the same criteria, CTL 2, 15, and 48 possessed the same or very similar fine specificity, but CTL 48 was of higher avidity than CTL 2 or 15. The seventh clone, CTL 40, was of a different fine specificity and its lysis of human target cells was also inhibited by the same anti-CD8 mAb. Only those clones whose lysis of human targets could not be inhibited by anti-CD8 antibody were able to lyse the 1-7E murine transfectants. These results indicate that, for human CTL clones with identical or very similar fine specificity, only those of higher avidity are able to lyse P815 murine cells expressing the HLA-B27 antigen. The lysis of HLA-B27.1+-murine transfectants by relevant clones was inhibited by anti-CD8 antibody. This result strongly suggests that the relative contribution of CD8 in stabilizing the interaction between human CTL and HLA-B27+-murine target cells is more significant than with human target cells.

Animals↗

PubMed1988

Clonal heterogeneity of HLA-B27 cellular allorecognition. Delineation of immunodominant sites.

The fine specificity of nine cytolytic T lymphocyte (CTL) clones obtained after stimulation of HLA-B27- responder lymphocytes with B27.1+ lymphoblastoid cell lines has been analyzed. These clones defined three different reaction patterns when tested against a panel of target cells including those expressing all known HLA-B27 subtypes: (a) specific recognition of HLA-B27.1, B27.2 and B27d, (b) selective reactivity with B27.1, B27d and HLA-B40 and (c) selective recognition of B27.1, B27.2, B27d, B27f and B40. Representative clones within each group were analyzed in detail. Differences in lytic ability of the various susceptible targets within each group were established by cold target inhibition analyses and by blocking experiments with anti-CD3 and anti-CD8 monoclonal antibodies. When correlated with the known structure of the HLA-B27 subtypes, these results demonstrate the critical relevance of amino acid changes within residues 77-81 and at position 152 in modulating allospecific CTL recognition of HLA-B27.1 and suggest that these residues could be involved in the structure of immunodominant regions of this antigen. The observed cross-reactions with HLA-B40, differing from B27.1 in 16 amino acid residues, suggest that the simultaneous occurrence of multiple amino acid changes could have mutually compensatory effects, so that a cross-reactive epitope might result from various combinations of polymorphic residues.

Antibodies, Monoclonal↗

PubMed1988

Modulation on immunogenicity by HLA-B27 subtype polymorphism.

Cells from the same HLA-B27- individual, PA, were stimulated in vitro in primary mixed lymphocyte culture, with either B*2705+ or B*2704+ lymphoblastoid cell lines, in independent experiments. Cytolytic T lymphocytes (CTL) were cloned at limiting dilution and the clones obtained were screened for anti-B27 alloreactivity. Most of the CTL clones generated against the B*2705+ stimulator cells were directed against the B*2705 antigen. In contrast, no anti-B27 CTL clones were found among those derived against the B*2704+ stimulator cells. This was not due to a poor cytotoxic response against these cells because a large proportion of the T cell clones derived from this stimulation were cytotoxic. B2704 differs from B*2705 by only two amino acid changes at positions 77 and 152. Previous studies (Aparacio, P. et al., Eur. J. Immunol. 1988.18: 203) have shown that none of the anti-B*2705 CTL clones derived from donor PA and amenable to detailed characterization cross-reacted with B*2704, suggesting that most of this cytotoxic response was directed against an immunodominant determinant contributed for by residues 77 and/or 152 from B*2705. The present results further suggest that the changes at these positions in B*2704 alter this determinant in such a way that B*2704 becomes less immunogenic for the particular individual PA. Furthermore, a similar poor anti-B*2704 CTL response was obtained from a second B27- responder individual, AE, stimulated with another B2704+ cell line. The single anti-B*2704 CTL clone, 64.8P, isolated from this second individual, displayed an unusual reaction pattern in that it cross-reacted with all B27 subtypes with changes only at or close to positions 77 and 152, including B*2705. Significantly, the only HLA-B27 subtype that was not recognized by CTL 64.8P was B*2703, which differs from B*2705 only at residue 59. This residue is located in the three-dimensional structure at the opposite end from residues 77 and 152 at the surface of the antigen-binding groove of the class I molecule. Thus, the area around residues 77 and 152 is not an essential part of the epitope recognized by CTL 64.8P.

Antibodies, Monoclonal↗

PubMed1987

Structural analysis of an HLA-B27 functional variant, B27d, detected in American blacks.

The structure of a new functional variant B27d has been established by comparative peptide mapping and radiochemical sequencing. This analysis completes the structural characterization of the six known histocompatibility leukocyte antigen (HLA)-B27 subtypes. The only detected amino acid change between the main HLA-B27.1 subtype and B27d is that of Tyr59 to His59. Position 59 has not been previously found to vary among class I HLA or H-2 antigens. Such substitution accounts for the reported isoelectric focusing pattern of this variant. HLA-B27d is the only B27 variant found to differ from other subtypes by a single amino acid replacement. The nature of the change is compatible with its origin by a point mutation from HLA-B27.1. Because B27d was found only in American blacks and in no other ethnic groups, it is suggested that this variant originated as a result of a mutation of the B27.1 gene that occurred within the black population.

Amino Acid Sequence↗

PubMed1987

Fine specificity of HLA-B27 cellular allorecognition. HLA-B27f is a functional variant distinguishable by cytolytic T cell clones.

Three HLA-B27 allospecific cytolytic T lymphocyte (CTL) clones were isolated by limiting dilution of HLA-B27-negative responder cells stimulated with HLA-B27.1-positive lymphoblastoid cells. These clones displayed three distinct reaction patterns when tested for their lytic ability against target cells expressing various structurally defined HLA-B27 subtypes. One of the clones was specific for HLA-B27.1; a second CTL clone reacted only with B27.1 and, less efficiently, with B27.2; the third clone recognized both B27.1 and B27f targets but not cells expressing any other B27 subtype. These results indicate that HLA-B27f is a functional variant amenable to differential recognition by alloreactive CTL. A correlation of the structure of the HLA-B27 subtypes with the reactivity of these clones revealed that multiple B27-specific alloreactive CTL are activated against epitopes of the HLA-B27.1 molecule sharing common structural features. This illustrates the complexity and fine specificity of the allogeneic CTL response against class I HLA antigens and suggests that their immunodominant regions are those which are capable of eliciting a diverse polyclonal response against each of these regions, rather than inducing the selective expansion of a single T cell clone.

Amino Acid Sequence↗

PubMed1987

Structural analysis of an HLA-B27 population variant, B27f. Multiple patterns of amino acid changes within a single polypeptide segment generate polymorphism in HLA-B27.

The structure of a new HLA-B27 variant, B27f, distinguishable from other HLA-B27 subtypes by isoelectric focusing and serologic criteria, has been established by comparative peptide mapping and radiochemical sequence analysis. HLA-B27f differs from the major B27.1 subtype in three clustered amino acid replacements: Asp74, Asp77, and Leu81 in B27.1 are changed to Tyr74, Asn77, and Ala81, respectively in B27f. This pattern of differences is analogous to that of HLA-B27.2 in that this subtype also differs from B27.1 in multiple clustered substitutions within the same segment. Thus, polymorphism within the HLA-B27 system is being achieved by introducing different sets of amino acid changes within a particular short segment of the alpha 1 domain. The most likely mechanism for the introduction of multiple changes within this segment is a nonreciprocal recombination event, such as gene conversion. The structural analogies and ethnic distribution of B27f and B27.2 as compared with those of B27.3, and B27.4 support a dynamic model of HLA-B27 evolution in which polymorphism has been created after the separation of the major ethnic groups. In this model, a Caucasian branch would be characterized by subtypes differing from B27.1 in a few changes within the alpha 1 domain, which were probably generated by single genetic steps. An Oriental branch would include those subtypes which differ from B27.1 by changes in both alpha 1 and alpha 2, involving multiple genetic steps for their generation.

Amino Acid Sequence↗

PubMed1986

HLA-B27 antigenicity: antibodies against the chemically synthesized 63-84 peptide from HLA-B27.1 display alloantigenic specificity and discriminate among HLA-B27 subtypes.

A chemically synthesized peptide with an amino acid sequence identical to that of the segment spanning residue 63-84 of the major HLA-B27.1 subtype antigen has been obtained. Specific antibodies were raised in rabbits against this peptide, coupled to keyhole limpet hemocyanin carrier. These antibodies lysed lymphoblastoid cell lines expressing HLA-B27.1 in a complement-mediated cytotoxicity assay. They lysed neither B27-negative target cells, nor B27-positive cells expressing other B27 subtype antigens. Complement-mediated lysis of B27.1-positive targets was inhibited by free peptide and by peptide coupled to an unrelated carrier. In addition, the lytic action of the rabbit antiserum was blocked by a monoclonal antibody with no complement-activating capacity that under the conditions of the assay, was specific for HLA-B27. These results indicate that rabbit antibodies against the 63-84 peptide recognize the native HLA-B27.1 antigen; this antiserum is allospecific in character; and it discriminates among B27 subtypes. Thus the data provide direct evidence on the contribution of the hypervariable region spanning residues 63-84 to the alloantigenic specificity of HLA-B27.

Antibody Specificity↗

PubMed1985

Structural analysis of an HLA-B27 functional variant: identification of residues that contribute to the specificity of recognition by cytolytic T lymphocytes.

The structure of a variant HLA-B27 antigen, B27.2, that is distinguished from the HLA-B27.1 and HLA-B27.3 subgroups by specific cytolytic T lymphocytes has been established by comparative peptide mapping and sequence analysis. There are only three amino acid substitutions between B27.1 and B27.2: aspartate-77, threonine-80, and leucine-81 in HLA-B27.1 are changed to asparagine-77, isoleucine-80, and alanine-81 in HLA-B27.2. These changes account for their single charge difference detectable by isoelectric focusing. The three clustered substitutions of HLA-B27.2 are identical to the corresponding residues in HLA-A24, so that both molecules become identical in their amino acid sequence between residues 72 and 96. This suggests that gene conversion may have occurred during the diversification of the HLA-B27 antigens. HLA-B27.2 has no changes in the alpha 2 domain and is similar in its pattern of substitutions to the murine bm11 mutant. It is suggested that residues 77-81 are of major significance in determining the specificity of cellular recognition of class I HLA antigens. This study, together with the previous analyses of HLA-B27.1 and HLA-B27.3, completes the structural characterization of the three major HLA-B27 functional subtypes and establishes the molecular basis of their functional and serological differences.

Amino Acid Sequence↗

PubMed1985

Delineation of functional sites in HLA-B27 antigens. Molecular analysis of HLA-B27 variant Wewak I defined by cytolytic T lymphocytes.

An HLA-B27 positive, Epstein Barr virus-transformed cell line Wewak I was not lysed by Epstein Barr virus-specific cytolytic T lymphocytes restricted by HLA-B27. This line is weakly reactive with a B27-specific monoclonal antibody, M2, which recognizes a majority, but not all, of the HLA-B27-positive cells. To establish the molecular basis for this lack of recognition, the structure of the variant HLA-B27 antigen was compared with the known structure of HLA-B27 from the LG-2 cell line, which is representative of a major subtype of this antigen. Both molecules were almost indistinguishable by isoelectric focusing. However, comparative peptide mapping and sequencing of the difference peptides revealed two amino acid changes: At position 77, Asp in donor LG-2 had changed to Ser in the variant, and at position 152, Val in LG-2 had changed to Glu in the variant. The nature of these substitutions was consistent with the extreme similarity of the isoelectric focusing pattern. An evaluation of these findings in the context of studies on other HLA variants and H-2Kb mutants suggests that both positions 77 and 152 contribute to the determinants recognized by B27-specific cytolytic T lymphocytes. The change at position 152 adds to previous evidence suggesting that the segment 149-156 is critical for cellular recognition. In addition, it is proposed on the basis of structural comparisons that residue 77 may also participate in the epitope recognized by the B27M2 antibody.

Amino Acid Sequence↗

PubMed1983

Physiological and biochemical properties and population studies in the M lactate dehydrogenase polymorphism of Discoglossus pictus (Amphibia, Anura).

1. M LDH polymorphism has been previously described in Discoglossus pictus. 2. Gene frequency at the LdM locus varied significantly in two isolated areas which differed markedly in water oxygen tension. 3. Sex distribution, relative activity of the LDH isozymes, percentage of total H and M subunits and enzyme kinetics did not differ among the distinct LDH phenotypes. 4. Under decreasing oxygen conditions, mean time to death, LT50 and oxygen consumption were similar in tadpoles of the three LDH phenotypes (fast, intermediate and slow). 5. Our results are compatible with a neutral role for this LDH polymorphism.

Animals↗