Search PubMed⌕ Search

Biomedical subjects

D Jaraquemada

Publications and source records attributed to D Jaraquemada.

At least 37 records · Page 2Linked to original sources
PubMed1994

Pancreas in recent onset insulin-dependent diabetes mellitus. Changes in HLA, adhesion molecules and autoantigens, restricted T cell receptor V beta usage, and cytokine profile.

Insulin-dependent diabetes mellitus (IDDM), in which only the pancreatic beta cells are destroyed by the autoimmune response, is the paradigm of organ-specific autoimmunity. As a result of a combination of factors, the number of immunohistologic/cellular/molecular studies of pancreas in IDDM is very limited. We report here studies conducted in the pancreata of two IDDM patients: one newly diagnosed (case 1) and one long standing (case 2). In case 1, we demonstrated the presence of morphologically normal viable beta cells without evidence of viral infection. In both cases the expression of the autoantigens defined by islet cell Abs and by glutamic acid decarboxylase was markedly reduced in the islet cells whereas expression of hsp60, another putative autoantigen, was normal. Over-expression of HLA class I was detected in 58% of the islets in pancreatic sections and in cultured beta cells in case 1 and also in 30% of islets in case 2 but it was not restricted to any insular cell type. In case 1, there was "inappropriate" HLA class II expression in islets cells but it was a rare finding and not beta cell specific. The analysis of the correlation between class I overexpression, residual insulin, and insulitis suggests that the first event is the increase of HLA class I expression. Of adhesion molecules, ICAM-1, VLA, VCAM, and LFA-3 were normal and only ICAM-1 was moderately overexpressed in and around the islets of case 1 insulitis, as was detected by immunofluorescence which showed that 18% of the islets of case 1 had CD8+ lymphocytes as the predominant population. Reverse transcription-PCR demonstrated moderate V beta skewing and the profile of cytokines expected in CTLs: IL-2, IL-4, IL-10, and IFN-gamma negative, perforin positive. In addition, IFN-alpha, IFN-beta, and IL-6 transcripts were detected in the case 1 pancreas, consistent with the existence of a silent viral infection. Overall, the results indicated that, differently from spontaneous animal models of diabetes, in the pancreas of IDDM patients there are no elements of the inductive phase of the autoimmune response.

Acute Disease↗
PubMed1994

Different requirements of ICAM-1/LFA-1 adhesion in allorecognition and self-restricted antigen recognition by class II-specific T cell clones.

We have analyzed the influence of non-antigen-specific interactions between ICAM-1 and LFA-1 in target recognition by allospecific and antigen-specific T cells at the clonal level, using human and mouse fibroblasts transfected with HLA-DR1 or DR2 with or without co-expression of ICAM-1, as antigen-presenting cells. The results show a great heterogeneity in the requirements for ICAM-1/LFA-1 interactions for antigen-specific and alloreactive T cell responses and this requirement may depend on the avidity of any particular interaction. The data also show that for most alloreactive clones, ICAM-1/LFA-1 adhesion is not sufficient to facilitate efficient T cell recognition of its target molecule. HLA class II recognition by a large proportion of the DR1- and DR2-specific alloreactive clones studied was different for class II molecules expressed on murine or human fibroblasts compared to human lymphoid cells, and was independent of ICAM-1 expression on the stimulator cells. The inability of some T cell clones to recognize HLA-class II expressed on non-lymphoid cells suggests the absence of specific epitopes and could be due to the lack of the relevant peptides, either because they are derived from species-specific proteins or to differences in processing of endogenous antigen in the transfected stimulator cells.

Animals↗
PubMed1993

Gamma delta lymphocytes in endocrine autoimmunity: evidence of expansion in Graves' disease but not in type 1 diabetes.

Endocrine autoimmune disorders are mediated by T cell-dependent responses to organ-specific antigens, but the mechanisms initiating the process remain unknown. Lymphocytes which use the gamma delta heterodimer as T cell receptor (TCR) for antigen constitute a distinct subset of T cells whose function remains elusive. In order to investigate their possible involvement in endocrine autoimmunity we have determined the proportion of gamma delta T cells in the peripheral blood of 23 patients with type 1 (insulin-dependent) diabetes mellitus (type-1 DM) and 30 patients with autoimmune thyrotoxicosis (Graves' disease). T lymphocyte TCR expression was assessed by fluorescence-activated flow cytometry on peripheral blood mononuclear cells using MoAbs UCHT1 (CD3), TCR delta 1 (gamma delta TCR), WT31 and beta F1 (alpha beta TCR) and both the percentage of T cells expressing gamma delta and the ratio gamma delta/alpha beta were calculated. In the diabetic patients gamma delta cells were not significantly different from the control group (7.7 +/- 54% versus 8.0 +/- 5.5% of T cells, P NS). There was no relation between the proportion of gamma delta lymphocytes and the presence of islet cell antibodies (ICA) in the sera. The Graves' patients showed a tendency towards a higher proportion of gamma delta T lymphocytes than the controls (gamma delta/alpha beta ratios: 0.095 +/- 0.047 versus 0.063 +/- 0.022, P = 0.03). In 14 Graves' patients the number of gamma delta were measured in paired samples of peripheral and intrathyroidal lymphocytes, demonstrating an expansion of gamma delta within the thyroid glands (0.21 +/- 0.3 versus 0.095 +/- 0.047, P = 0.032). Immunohistochemical studies showed that gamma delta cells were scattered among the predominant alpha beta lymphocytes infiltrating the thyroid gland and that they account for 10% of intraepithelial lymphocytes. No relation was found between the increase of gamma delta lymphocytes and any clinical features.

Adolescent↗
PubMed1992

Processing pathways for presentation of cytosolic antigen to MHC class II-restricted T cells.

Antigens presented to CD4+ T cells derive primarily from exogenous proteins that are processed into peptides capable of binding to class II major histocompatibility complex (MHC) molecules in an endocytic compartment. In contrast, antigens presented to CD8+ T cells derive mostly from proteins processed in the cytosol, and peptide loading onto class I MHC molecules in an early exocytic compartment is dependent on a transporter for antigen presentation encoded in the class II MHC region. Endogenous cytosolic antigen can also be presented by class II molecules. Here we show that, unlike class I-restricted recognition of antigen, HLA-DR1-restricted recognition of cytosolic antigen occurs in mutant cells without a transporter for antigen presentation. In contrast, DR1-restricted recognition of a short cytosolic peptide is dependent on such a transporter. Thus helper T-cell epitopes can be generated from cytosolic antigens by several mechanisms, one of which is distinct from the classical class I pathway.

Amino Acid Sequence↗
PubMed1991

A myelin basic protein peptide is recognized by cytotoxic T cells in the context of four HLA-DR types associated with multiple sclerosis.

We have examined previously the peptide specificity of the T cell response to myelin basic protein (MBP) in patients with multiple sclerosis (MS) and healthy controls, and demonstrated that an epitope spanning amino acids 87-106 was frequently recognized. Because this region is encephalitogenic in some experimental animals, it has been postulated that the response to the epitope may have relevance to MS. In this study, the fine specificity of this response is studied using four well-characterized, monospecific T cell lines from three MS patients and an identical twin of a patient. Each of the lines recognized a peptide with the same core sequence, amino acids 89-99, although the responses were affected to various degrees by truncations at the COOH- or NH2 terminal ends of the 87-106 epitope. Importantly, the epitope was recognized in conjunction with four different HLA-DR molecules. Also, the T cell receptor beta chain usage was heterogeneous, and each line expressed a different VDJ sequence. The four HLA-DR molecules restricting the response to this epitope have been shown to be overrepresented in MS populations in various geographic areas, suggesting that the response to this region of the MBP molecule may be relevant to the pathogenesis of MS. These findings may have important implications in designing therapeutic strategies for the disease.

Amino Acid Sequence↗
PubMed1991

On the relative immunogenicity of DR alloantigens: T cell recognition of HLA-DR2a and HLA-DR2b.

The HLA-DR2 haplotype encodes two highly polymorphic DR molecules, DR2a and DR2b. Because little is known regarding the relative immunogenicity of different HLA-DR molecules, we have studied the T-cell recognition of DR2a and DR2b molecules from the DRw15, Dw2 haplotype. A series of DR2-specific alloreactive T-cell clones were analyzed with murine L-cell transfectants expressing either the DR2a or the DR2b molecules as stimulator cells in proliferation assays. Somewhat surprisingly, both DR2a and DR2b were capable of stimulating DR2-specific T-cell clones with equal magnitude and similar frequency. In addition, DR2a and DR2b are functionally distinct, that is, no clone was identified which was stimulated by both DR2a and DR2b molecules.

Amino Acid Sequence↗
PubMed1991

Efficient cDNA expression vectors for stable and transient expression of HLA-DR in transfected fibroblast and lymphoid cells.

cDNA expression vectors with several useful features were constructed. First, the long terminal repeat of Rous sarcoma virus was used as a promoter to obtain high levels of expression in various cells of human and mouse origin. Second, cis-linked expression units that confer resistance either to mycophenolic acid or the neomycin analog G418 were inserted to facitate the isolation of transfected cells expressing the cDNA of interest. Third, by replicating in simian COS cells, these vectors can be used for efficient transient expression. cDNA fragments encoding the DR alpha or DR beta chains of human class II major histocompatibility complex antigens were inserted into these vectors and high levels of cell surface HLA-DR antigen were obtained after cotransfection into mouse and human fibroblasts. These vectors were also successfully used to correct the inability of a class II-negative B cell line, derived from a patient with a congenital immunodeficiency, to present peptide antigen to DR-restricted T cells.

Animals↗
PubMed1990

HLA-DR2a is the dominant restriction molecule for the cytotoxic T cell response to myelin basic protein in DR2Dw2 individuals.

The HLA-DR2 restriction of the T cell response to myelin basic protein (MBP) was studied using murine L cells transfected with DRalpha and either DR2a or DR2b beta-chain cDNA. DR2a and DR2b represent the two isotypic DRbeta chains expressed in DR2Dw2 haplotypes. Eleven MBP-specific cytolytic T cell lines derived from patients with multiple sclerosis were isolated. Two of these cell lines recognized MBP-pulsed DR2-expressing L cell transfectants and four of them could only recognize the L cells if the adhesion molecule ICAM-1 was expressed in addition to HLA-DR2. Five of the six lines were restricted by HLA-DR2a; one line recognized Ag in conjunction with DR2b, but only if ICAM-1 was coexpressed. The remaining five lines did not lyse MBP-pulsed L cells. The ability of the DR2b molecules on transfected cells to stimulate T cells was confirmed with DR2b-allospecific T cell clones. Although five MBP-specific lines were restricted by DR2a, they recognized different parts of the MBP molecule, as demonstrated by the presentation of shorter peptides. Thus, our results suggest that DR2a is a dominant restriction molecule in MBP-specific responses by DR2+ MS patients. The results also indicate that the reported heterogeneity in MBP epitopes recognized by DR2-restricted T cells, may not be due to the use of different restriction elements but rather to the binding of different MBP peptides to DR2a molecules.

Animals↗
PubMed1990

An endogenous processing pathway in vaccinia virus-infected cells for presentation of cytoplasmic antigens to class II-restricted T cells.

The recognition of virus-infected cells by class I MHC-restricted cytotoxic T cells requires endogenous processing of antigen for presentation. It is still unclear whether endogenous processing of antigen can be utilized by class II MHC molecules for presentation. To test this possibility, a human B cell line expressing HLA-A2 and HLA-DR1 was infected with a recombinant vaccinia virus expressing the Influenza A virus M1 matrix protein (VAC-M1) and was assayed for lysis by different M1-specific cytolytic T cell lines, restricted by either HLA-A2 or by HLA-DR1. Class II-restricted lysis of VAC-M1-infected cells did occur. This lysis required de novo M1 synthesis and was not due to exogenous antigen. Several properties of the endogenous processing pathway for class II-restricted presentation were different from those of the pathway utilized by class I molecules. First, class II-mediated recognition of VAC-M1 infected cells was less efficient, requiring higher doses of virus and longer infection times, than the class I-mediated recognition. Second, chloroquine completely blocked presentation of endogenous M1 to class II-restricted T cells but had no effect on the class I-restricted presentation. Third, the class II-restricted presentation of M1 was only mildly affected by Brefeldin A, a drug that prevents transport from the endoplasmic reticulum to the Golgi, whereas the class I-restricted presentation of M1 was completely abrogated by this drug. These data demonstrate the existence of an endogenous processing pathway for the presentation of cytosolic antigen by class II molecules and show that this pathway is distinct from the one used for presentation by class I molecules.

Anti-Bacterial Agents↗
PubMed1990

Fine specificity and HLA restriction of myelin basic protein-specific cytotoxic T cell lines from multiple sclerosis patients and healthy individuals.

Myelin basic protein (MBP) is a candidate Ag for the autoimmune process believed to be involved in the pathogenesis of multiple sclerosis (MS). To investigate the fine specificity and HLA restriction of human MBP-specific CTL, long term T cell lines (TCL) were established from 22 MS patients and 16 healthy individuals by repeated antigenic restimulation. By using this approach, MBP-specific cytotoxic TCL were generated from 81% of the lines from MS patients and 69% of those from controls. TCL from both groups expressed the CD3+, CD4+, CD8- phenotype and secreted substantial amounts of IFN-gamma. By using large enzymatic and small synthetic peptides of MBP, TCL were primarily specific for the C-terminal part of the molecule and to a lesser extent for the N-terminal portion. Two regions of the molecule, MBP peptide 87-106 and MBP peptide 154-172, were recognized by the majority of the polyspecific lines and by four and three of 14 monospecific TCL, respectively. These highly immunogenic regions are of interest because they include sequences encephalitogenic in other species. The HLA restriction of each line was determined by using antibody blocking as well as various target cells including EBV-transformed B cells, homozygous typing cells, and fibroblasts transfected with cDNA for DR-alpha and DR-beta genes. All TCL were restricted by HLA-DR Ag. Several HLA-DR molecules restricted multiple cathepsin D-derived and synthetic MBP peptides, including the regions of peptides 87-106 and 154-172 which, respectively, were recognized in conjunction with four and three HLA-DR types. Three of these HLA-DR types are overrepresented in MS patients in different geographic regions. Together, these findings suggest that the MBP-specific cytotoxic T cell response, although not sufficient for disease, may be important for the pathogenesis of MS.

Amino Acid Sequence↗
PubMed1990

Structural requirements for pairing of alpha and beta chains in HLA-DR and HLA-DP molecules.

To test for the assembly of human MHC class II molecules having an alpha chain from one isotype (HLA-DR, -DQ, or -DP) and the beta chain of another (mixed isotypic pairs), murine fibroblasts were transfected with expressible cDNAs encoding the different class II alpha and beta chains. A rapid and efficient transient transfection system was developed using a polyoma virus-based vector. Typically, 30-50% of cells transfected using this system expressed high levels of class II molecules on their surface, but only with matched isotypic pairs. Biochemical analysis of cells transfected with matched or mixed isotypic pairs of the DR and DP molecules revealed that only matched chains could pair efficiently inside the cell. Thus, the lack of expression of the two mixed isotypic pairs is due to inefficient primary assembly of the class II molecule and not to a processing or transport defect. To define what region of the beta chains controlled their assembly with alpha chains, a series of chimeric cDNA molecules containing both DR and DP beta chain sequences were constructed. Expression of these chimeric beta chains with DR and DP alpha chains was determined by cytofluorimetry and biochemical analysis. Both alpha chains paired with beta chains in which only the beta 1 domain was isotypically matched. In contrast, the pattern of expression of chimeras made at other points within the beta 1 domain was different for DR and DP. These data show that different areas of primary sequence are important for the assembly of different human class II isotypes, and suggest that HLA-DR and -DP molecules have different secondary or tertiary structures in their NH2-terminal domains.

Animals↗
PubMed1990

Recombinant CD4-Pseudomonas exotoxin hybrid protein displays HIV-specific cytotoxicity without affecting MHC class II-dependent functions.

The present study describes several in vitro activities of CD4(178)-PE40, a recombinant protein containing a portion of human CD4 linked to active regions of Pseudomonas aeruginosa exotoxin A. Using assays for cell viability, we demonstrate that the hybrid toxin displays highly selective cytotoxicity for HIV-infected T lymphocytes. In a latently infected human T-cell line which is inducible for HIV expression, toxin sensitivity is observed only upon virus induction. At concentrations which readily kill HIV-infected T cells, CD4(178)-PE40 has no observable cytotoxic effects on uninfected human cell lines expressing surface major histocompatibility complex (MHC) Class II molecules, and does not interfere with cellular responses known to be dependent on functional association between CD4 and MHC Class II molecules.

ADP Ribose Transferases↗
PubMed1989

HLA-D region heterogeneity in a Nigerian population.

HLA class II antigens were studied in a panel of 130 Nigerians. Complex patterns of associations were seen between HLA-Dw, -DR and -DQ specificities, differing widely from those reported for other populations. A number of Dw types were associated with the same DR antigen: Dw'1N' and Dw'BERN' with DR1, Dw2 and Dw'2N' with DR2, Dw5 and Dw'5N' (Dw5 + Dw'F5') with DRw11. It was also observed that a Dw type associated with more than one DR antigen: HLA-Dw3 was assigned to individuals who were DR3 negative and similarly Dw10, Dw13 and Dw14 to individuals negative for DR4. HLA-DRw8 and Dw8 were completely dissociated in Nigerians, and Dw8 did not show a preferential DR association. These results demonstrate that DR and DQ identity between HTC stimulator and responder cell is not necessarily a prerequisite for Dw to be assigned. Preliminary studies show that subtypes of HLA-Dw1 and Dw8 detected by HTC typing correlate with restriction fragment length polymorphisms (RFLPs) detected with a combination of Bgl II enzyme and DRA/DRB cDNA probes. HLA-DP antigen frequencies differed between Nigerians and British Caucasoids. The most common DP antigen in Nigerians was DPw1, compared with DPw4 in Caucasoids. HLA-DPw6 appeared to be absent or rare in both Nigerians and British Caucasoids. Only five out of 68 Nigerians tested were assigned two DP specificities. The association between HLA-DR3 and DPw1 reported in Caucasoid panels was absent in Nigerians.

Black People↗
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

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

Alloreactive cytolytic T cell clones with dual recognition of HLA-B27 and HLA-DR2 antigens. Selective involvement of CD8 in their class I--directed cytotoxicity.

HLA-B27- responder cells were stimulated in vitro with HLA-B27.1+ lymphoblastoid cell lines, and alloreactive CTL clones were obtained by limiting dilution. Three of these clones specifically lysed B27.1+ targets. In addition, they also lysed homozygous DR2 targets with various degrees of efficiency, depending on the Dw specificity of the target cell. All three clones possessed a homogeneous CD3+,CD8+,CD4- phenotype and were also homogeneous upon subcloning. Cold-target inhibition analyses showed mutual inhibition of B27.1 target lysis by DR2 targets and vice versa. Lysis of B27.1 targets was selectively inhibited by anti-class I mAbs. In contrast, lysis of DR2 targets was inhibited only by anti-class II and anti-DR monomorphic antibodies, but not by anti-class I, anti-DQw1, or anti-DP antibodies. The results indicate that these clones display dual recognition for HLA-B27.1 and for HLA-DR2 and suggest that HLA-B27.1 may share at least one epitope that is closely related to some stimulatory Dw determinants present on the HLA-DR2 antigens. Lysis of both B27+ and DR+ targets was inhibited by an anti-CD3 mAb. In contrast, an anti-CD8 antibody selectively inhibited the B27- but not the DR2-directed killing by these clones. The data support a stabilizing role of CD8 through its binding to the same class I (but not class II) molecule on the target cell bound by the T cell antigen receptor.

Antibodies, Monoclonal↗