Abstract: To dissect the phenotypic and functional features of mucosal T lymphocytes in patients with gastric adenocarcinoma, we have used the Herpesvirus saimiri transformation procedure to achieve T-cell lines from gastric origin. Once achieved, cell function was assessed by in vitro stimulation with mitogens. CD2-specific monoclonal antibodies (alpha-CD2), alone or in combination with interleukin (IL)-2, rendered fewer counts in patients (34 408+/-3965 and 52 157+/-6473 c.p.m., respectively) than in controls (67 471+/-11 755 c.p.m., P<0.01 and 77 864+/-12 545 c.p.m., P<0.05, respectively). Likewise, CD3-based responses were defective in cancer cell lines: alpha-CD3 (54 794+/-9269 vs 86 104+/-10 341 c.p.m., P<0.01), alpha-CD3+IL-2 (57 789+/-8590 vs 88855+/-8516 c.p.m., P<0.01) and alpha-CD3+alpha-CD2 (52 130+/-7559 vs 120 852+/-16 552 c.p.m., P<0.01). Finally, IL-2 failed to adequately stimulate patient cell lines (39 310+/-4023 vs 60 945+/-9463 c.p.m., P<0.05). These results suggest that mucosal T lymphocytes in cancer patients are inherently impaired in their proliferative ability. This may be crucial in the control of tumour growth.
Abstract: We wished to analyse the frequency of Crohn's disease-linked CARD15 polymorphisms (P268S, R702W, G908R and 1007fs) in a group of Spanish patients with idiopathic uveitis. To this aim, DNA samples were obtained from 111 unrelated patients. P268S, R702W and G908R polymorphisms were detected using TaqMan Genotyping kits (Applied Biosystems), and the 1007fs variation by direct DNA sequencing. Control group consisted of 105 healthy subjects. None of the polymorphisms studied revealed a significant increase in the groups of patients, when compared to the control group. Thus, P268S is found in 50% of patients (gene frequency 0.284) vs 44% of control individuals (gene frequency 0.245); R702W in 7% of patients (0.036) vs 7% (0.033); G908R in 2% of patients (0.009) vs 4% (0.019) and, finally, 1007 fs in 2% of uveitis patients (0.008) vs 4% (0.021). Moreover, DNA sequencing has allowed us to define two new intronic polymorphisms in phase, in the 5' and 3' boundaries of the exon 11 (GenBank accession number #DQ 869189). Altogether, our results suggest that the Crohn's disease-linked CARD15 polymorphisms do not seem to predispose to idiopathic uveitis in the Spanish population.
Abstract: Defective CD3zeta chain expression has been reported in T lymphocytes of patients with inflammatory diseases, such as systemic lupus erythematosus or osteoarthritis, and with cancer. In lupus, the absent CD3zeta chain is replaced by the FcRgamma chain, rendering the T cells hyper responsive. However, there are no data on T lymphocytes from patients with cancer. In this study, the presence of the FcRgamma chain and its associated kinase, Syk, was analysed in patients with gastric adenocarcinoma and healthy subjects. Western blot and immunoprecipitation experiments were carried out with total cell or lipid raft extracts from fresh peripheral blood mononuclear cells or T lymphocytes, and Herpesvirus saimiri-derived T-cell lines (of blood or tissue origin). Our results revealed that the absent CD3zeta chain in cancer T lymphocytes was not replaced by FcRgamma either in fresh T cells or T-cell lines, in contrast to lupus T cells. This altered expression of signalling molecules in T lymphocytes of cancer patients, would explain their low proliferative capacity. Our T-cell lines represent tools to unveil the signalling abnormalities of cancer T lymphocytes.
Abstract: 25 patients with resectable gastric adenocarcinoma, subdivided according to the absence or presence of residual neoplasic disease (RND- or RND+, respectively), were studied. Cytofluorometric analysis and proliferative responses to mitogens was performed in peripheral blood mononuclear cells of patients. When compared to healthy subjects, the percentage of CD3-expressing cells was significantly reduced in both groups of patients studied (p < 0.0001 in all instances). However, when CD45 is considered instead of (CD3, its expression is found to be significantly reduced only in the RND+ patients (72% +/- 11), when compared with the control group (96 +/- 1%, p < 0.0001). Likewise, cells from these patients significantly less proliferated when stimulated with monoclonal antibodies to CD3 than control cells (18,920 +/- 6,019 cpm vs. 42,697 +/- 1,798 cpm, p = 0.0036); a difference not found if RND- patients (33,619 +/- 11,733 cpm) were considered. We propose that the low expression of CD45 and the poor response to CD3 are markers that are able to identify the subgroup of patients in whom the disease will tend to progress more rapidly.We also suggest the use of such markers as additional criteria for the classification of patients with gastric adenocarcinoma or to identify patients who require more aggressive therapeutic strategies.
Abstract: A rare case of type I diabetes is studied in an Amerindian (Mapuche) family from Chile, analyzing glutamic acid decarboxylase, islet-cell autoantibodies and human leukocyte antigen (HLA) genes. The affected sib is the only one that has one specific HLA haplotype combination that differs from the other sibs only in the HLA class I genes. It is concluded that HLA diabetes susceptibility factors may be placed outside the class II region or even that susceptibility factors do not exist in the HLA region in this Amerindian family.
Abstract: PURPOSE: To describe a case of meningococcemia with anterior uveitis. METHODS: Observational case report. RESULTS: A 38-year-old woman developed meningococcal septicemia caused by Neisseria meningitidis type B. During her admission, she had pain in her left eye, inflammatory cells, and a fibrinous exudate in the anterior chamber and multiple posterior synechiae, all in the context of an anterior uveitis. She was treated with topical steroids and mydriatics with resolution of ocular inflammation. CONCLUSIONS: This case illustrates the possible association between anterior uveitis and a meningococcal septicemia, and the need for careful ophthalmologic examination when a red eye develops in this clinical context.
Abstract: Low expression of the CD3zeta chain has been reported in patients with cancer and it has been suggested that tumor-derived factors are involved in its downregulation. The expression of CD3zeta chain was measured in T-cell lines from patients with gastric adenocarcinoma and healthy volunteers and grown in vitro for several months and, hence, in the absence of any tumor-derived factors. T-cell lines of mucosal origin were obtained by Herpesvirus saimiri transformation from gastric cancer patients. The expression of CD3zeta and CD3epsilon was measured by flow cytometry and Western-blot analysis. Calcium mobilization and apoptosis rate were also measured. The levels of CD3zeta, but not CD3epsilon, chain on the cell surface were significantly reduced in T-cell lines derived from patients with gastric cancer when cultured in the absence of IL-2. Western-blot analysis of total cell extracts or lipid raft fractions confirmed this finding. Calcium mobilization, a measure of signal transduction, was reduced in T cell lines from patients with gastric cancer. We conclude that T cells from patients with cancer express lower levels of CD3zeta. This downregulation is not caused by a direct effect of tumor-derived factors but, rather, it appears to be inherent to the patient cells. The low CD3zeta expression would render T lymphocytes unable to control the growth of tumor cells.
Abstract: BACKGROUND: T lymphocytes play a crucial role in the pathogenesis of inflammatory bowel disease. Achieving stable T-cell lines, rather than continuous bleeding of patients, is desirable in order to dissect their implication in the disease. METHODS: Long-lasting T-cell lines from patients with Crohn disease and ulcerative colitis and from healthy volunteers have been obtained by transformation of T lymphocytes using the lymphotropic Herpesvirus saimiri. Lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. RESULTS: Fresh cells revealed only minor differences between patients and controls, with regard to phenotype and proliferative capacity. In contrast, the use of T-cell lines showed that cells from Crohn disease patients, but not ulcerative colitis patients, over-responded to several membrane or cytoplasmic stimuli when compared to control T-cell lines. Thus, higher responses were found when stimulated with alphaCD3 and IL2, alphaCD3 and alphaCD28, IL2 alone, phorbol esters (PMA) and alphaCD3 and, finally, PMA and alphaCD2 (P < 0.05 in all instances). Further, lines from patients with Crohn disease responded more vigorously to alphaCD3 and alphaCD28 or alphaCD3 and PMA when compared to ulcerative colitis (P < 0.05 in both instances). CONCLUSIONS: The data obtained with these lines suggest that T cells from patients with Crohn disease differ in vivo in their proliferative capacity, as compared with those from ulcerative colitis patients, a finding that may reflect the clear Th-1 phenotype found in the former and absent in the latter.
Abstract: OBJECTIVES: To detect the presence of antibodies against bovine serum albumin in a cohort of Spanish patients with type 1 insulin-dependent diabetes. METHODS: Antibodies were measured using an in-house enzyme-linked immunosorbent assay test in 80 patients with type 1 diabetes, subdivided according to the presence or absence in their serum of celiac disease-related antibodies. For comparison, 30 patients with celiac disease (nondiabetic), 13 patients with autoimmune thyroiditis, and 45 healthy volunteers were used. RESULTS: Thirty-one percent of patients with diabetes yielded a positive result, with a mean value of 26.1 +/- 21.8 arbitrary units (AU). If the group was split into those with celiac disease-related antibodies and those lacking them, the percentages were 53% and 25%, respectively, with a mean value of 39.6 +/- 28.4 AU and 22.4 +/- 18.3 AU (P = 0.003), respectively. Seventy-three percent of celiac patients showed bovine serum albumin antibodies with a mean level of 38.8 +/- 27.7 AU, comparable to that of patients with diabetes with celiac antibodies, but higher than the group lacking them (P = 0.001). Although 46% of patients with autoimmune thyroiditis had positive results, the level detected (22.1 +/- 8.7 AU) was significantly lower than that recorded in patients with type 1 diabetes who had celiac disease antibodies (P = 0.04) and celiac patients (P = 0.04). Healthy volunteers showed no antibodies against bovine serum albumin. CONCLUSIONS: These data suggest that bovine serum albumin antibodies appears in patients with a compromised epithelial permeability, and they reflect a general defect in the process of immunologic tolerance associated with a predisposition to autoimmunity, rather than immunity specific to beta cells.
Abstract: We have taken advantage of a recently described technique of transformation and immortalization of T lymphocytes using the lymphotropic Herpesvirus saimiri, to achieve long-lasting T-cell lines from gastric cancer patients and healthy volunteers. Blood samples were drawn and T lymphocytes were transformed. Once sustained growth was observed, lines were subjected to phenotypic and functional analyses, and the results compared with freshly isolated peripheral blood mononuclear cells. Cytofluorometric analysis revealed that CD3 and CD45 were found at lower proportion in primary cells from patients than from control individuals (54% vs 75%, p<0.001, 90% vs 96%, p<0.05, respectively), and in HVS-derived T-cell lines (90% vs 98%, p<0.05, 97% vs 100%, p<0.05, respectively). Proliferative analyses showed that primary isolated cells were unable to respond adequately to CD3-, CD2-, and PHA-mediated stimulation, as compared to controls. Similarly, T-cell lines from patients proliferated to a lesser extent when CD3- and CD2-mediated stimuli were considered, especially when simultaneous stimulation via CD3 and CD2 molecules was carried out (47,824 counts per minute [cpm] vs 121,478 cpm, p<0.05). Altogether these results show that the defects reported in T cells from patients with cancer are not exclusively due to tumour-derived factors, since the alterations persist in long-lasting, HVS-transformed, T-cell lines, suggesting that this model seems a suitable one to disclose them.
Abstract: The frequency of reticulin (ARA), endomysium (EmA), and gut epithelial cell (GECA) autoantibodies, and gliadin antibodies (AGA), was investigated in 86 Spanish diabetic patients by indirect immunofluorescence (IFI) and ELISA, along with their HLA phenotype. Four patients (5%) showed ARA-IgG (R1 pattern), eight (9%) showed AGA-IgG, and eight (9%) showed AGA-IgA. No EmA or GECA-positive patients were found. In diabetic patients, HLA-DR7 is increased in ARA-IgG+ vs. ARA-IgG- (though not significantly), and HLA-DR6 and HLA-DQ1 are significantly increased in the AGA-IgG+ group vs. the AGA-IgG- group. Comparison with a non-diabetic coeliac group showed that HLA-DR4 and HLA-DQ3 are significantly increased in the AGA-IgA+ group, whereas HLA-DQ2 shows a significant decrease in the AGA-IgG+ and AGA-IgA+ patients. Finally, when compared to the healthy group, HLA-DR7 frequency is decreased in the ARA-IgG- group, while HLA-DQ3 is significantly increased and HLA-DR6 and HLA-DQ1 significantly decreased in the AGA-IgG- group.Altogether, these data suggest that the genetic background leading to the appearance of coeliac-specific autoantibodies in Spanish diabetic patients differ depending on the autoantibody produced and is also different to the genetic background leading to diabetes in Spain.
Abstract: Caco-2 is a colonic tumour cell line which, when cultured, spontaneously exhibits enterocyte-like characteristics. Given the difficulties in maintaining long-lasting cultures of enterocytes, this cell line may be a suitable in vitro model to carry out experiments trying to delineate the involvement of enterocytes in local immune responses, and their role in pathology. It seems then reasonable to obtain a detailed immune analysis of Caco-2, and compare it with available data on enterocytes. Cytofluorometry revealed several leukocyte markers on Caco-2, present also on human enterocytes. These markers include surface proteases (CD10, CD13 and CD26), antigen-presenting cell markers (CD13, CD14, CD35 and CD63), integrins (CD18 and CD61), epithelial/endothelial markers (CD21, CD31, CD47 and CD59) and finally, CD25 and CD28. In contrast to enterocytes, HLA-class 11 molecules are not found on Caco-2, whether resting or gamma-IFN-stimulated. Moreover, culture experiments with allogeneic lymphocytes revealed that Caco-2 cells were unable to induce their proliferation. Cytokine analysis showed an increased RANTES synthesis and IL-2 transcription upon stimulation with IL-1beta. Finally, amongst RANTES receptors, CCR1 is found on Caco-2 cells, whereas CCR3 and CCR5 are not.
Abstract: Cultured Caco-2 cells were stimulated with Lens culinaris, Phaseolus vulgarisandVicia fabalectins. The production of IL-1, IL-6, IL-8 and MCP-1 was measured by ELISA and RT-PCR. IL-8 production appeared to be specifically triggered upon stimulation with all three lectins used, since none of the other cytokines tested were produced. The IL-8 secreted may induce the extravasation of activated neutrophils and generate tissue damage. A similar mechanism may be implicated in the lesions observed after infection by some enteric pathogens, with lectin-like domains on their membrane. Finally, this model may be suitable one to study the regulation of IL-8 production.
Abstract: The presence of autoantibodies and autoimmune diseases was tested in all available members of five families with at least one member affected with X-linked chronic granulomatous disease. Patients and carriers relatives possess autoantibodies more frequently than non-carriers relatives (95% vs 10%, p < 1.0 x 10(-5), Fisher test). Further, a survey of the literature revealed that in X-linked immunodeficiencies with X-chromosome random inactivation, clear features of autoimmunity are observed, not found in those with non-random inactivation. It appears then as if random inactivation of the X-chromosome in these pathologies, may favor the expression of an autoimmune phenotype in patients and carriers.
Abstract: The HLA-B locus is the most polymorphic of the class I genes encoded within the human major histocompatibility complex. This polymorphism is mainly located in exons 2 and 3, which code for the molecule's alpha1 and alpha2 domains and includes the antigenic peptide binding site. However, information about adjacent non-coding regions (introns 1 and 2) has not been extensively reported but could be very important in establishing an understanding of the evolutionary mechanisms involved in the polymorphism generation of HLA-B and the Mhc loci. In the present work, introns 1 and 2 of 14 HLA-B alleles are studied and their significance is discussed; 10 have been sequenced in our own laboratory and the other 4 have been previously reported by others. Different serological families share the complete intron 1 sequence; at this region, 12 out of 14 HLA-B alleles could be included in four groups with the same intron 1 sequence: a) B*0702, B*4201, B*4801; b) B*27052, B*4002, B*4011; c) B*40012, B*4101, including B*4501, B*5001 (these latter two alleles have specific characteristics in both introns 1 and 2, which may reflect a common evolutionary pathway); and d) B*44031, B*44032. The other alleles, B*1402, and B*1801, do not have identical intron 1 sequences compared to any of the described groups, but share many similarities with them. The B*1801 evolutionary pathway seems to be very specific since it branches separately from other alleles both in intron 1 and intron 2 dendrograms. On the other hand, HLA-B allelic group distribution and similarities according to intron 1 sequences were not confirmed when using intron 2, especially in the cases of B*4002, B*4101 and B*4801. This would suggest that both point mutations fixed by genetic drift and gene conversion events are involved in HLA-B diversification. The latter events could be supported by the strong homology between intron 1 and, to a lesser extent, intron 2, and also the CG content within them. Finally, the precise knowledge of these non-coding regions could be important for developing DNA base typing strategies for the HLA-B alleles.
Abstract: HLA class I and class II expression was analyzed weekly by cytofluorometry on spermatozoa samples from four donors during a 15-wk trial. On the same day that semen samples were studied, and to analyze whether this expression was hormone-controlled, serum levels of testosterone, LH, FSH, inhibin B, activin, and pro-alphaC on the one hand, and seminal plasma levels of inhibin B, activin, and alpha-inhibin on the other, were also measured. Inhibin B and related peptides were quantitated using a novel two-site assay with monoclonal antibodies to the alpha and beta subunits of inhibin. Our results showed that HLA class I and class II molecules were expressed on the spermatozoa's surface, following a cyclic pattern, and that there was a simultaneous and coordinated expression of both types of molecules (r = 0.801, P < 0.0001). Furthermore, when the expression of these molecules was plotted against the different hormone levels, serum inhibin B showed a clear inverse correlation with HLA class I (r = -0.612, P < 0.0001) and class II (r = -0.534, P < 0.0001). This finding reveals unexpected functions of inhibin B, which may be relevant in the fertilization process and on male fertility control.
Abstract: OBJECTIVE: To study the influence MHC class II and TAP2 alleles exert on systemic lupus erythematosus (SLE) susceptibility and on the clinical and serological manifestations of the disease, in a cohort of Spanish patients. METHODS: HLA-DR serological typing and HLA-DQA, DQB, and TAP2 DNA sequence specific oligotyping, were carried out in 85 unrelated Spanish SLE patients and 186 healthy controls. Autoantibodies detection was carried out by indirect immunofluorescence and counter immunoelectrophoresis. RESULTS: Total SLE group: the frequency of HLA-DR3 and HLA-DQA1*0501 is significantly increased in this group (pc < 0.005, delta = 0.34 and pc < 0.005, delta = 0.45, respectively) although the highest delta value (delta = 0.87) is obtained when the TAP2*01 alleles are considered. No DQB allele shows significant deviation from the control group. Renal damage: it mainly occurs in HLA-DR3 patients (pc < 0.0005 and delta = 0.72). HLA-DQA1*0501 (p < 0.05, delta = 0.57 and DQB1*0201 (pc NS, delta = 0.56) are weaker susceptibility factors. Ro+ (but not LA) group: this autoantibody response is associated with TAP2*01 alleles in homozygosity (p < 0.05, delta = 0.81). R0/La+ group: it has a different genetic background as HLA-DQA1*0501 (delta = 1) and HLA-DQB1*0201 (delta = 1) are the main susceptibility factors. CONCLUSIONS: A differential association between HLA-DR, DQA1, and DQB1 alleles and SLE or its clinical and serological manifestations are found. Furthermore, the associations are different to the ones reported in other ethnic groups. Finally, TAP2*01 group of alleles are associated with the highest susceptibility to SLE (higher than HLA-DR3) and may influence Ro (but not La) autoantibodies production, whereas HLA-DQA1*0501 and DQB1*0201 mediates concomitant Ro and La productions.
Abstract: DRB genes have been studied for the first time in green monkeys (Cercopithecus aethiops). Eleven new DRB alleles (exon 2, exon 3) have been obtained and sequenced from cDNA. A limited number of lineages have been identified: DRB1*03 (4 alleles), DRB1*07 (3 alleles), DRB5 (1 allele), DRB*w6 (1 allele), and DRB*w7 (2 alleles). The existence of Ceae-DRB1 duplications is supported by the finding of 3 DRB1 alleles in 3 different individuals. Ceae-DRB1*0701 may be non-functional because it bears serine at position 82, which hinders molecule surface expression in mice; the allele is only found in Ceae-DRB duplicated haplotypes. Base changes in cDNA Ceae-DRB alleles are consistent with the generation of polymorphism by point mutations or short segment exchanges between alleles. The eleven green monkey DRB alleles meet the requirements for functionality as antigen-presenting molecules (perhaps, excluding DRB1*0701), since: 1) they have been isolated from cDNA and do not present deletions, insertions or stop codons: 2) structural motifs necessary for a correct folding of the molecule, for the formation of DR/DR dimers and for CD4 interactions are conserved, and 3) the number of non-synonymous substitutions is higher than the number of synonymous substitutions in the peptide binding region (PBR), while the contrary holds true for the non-PBR region.
Abstract: Sequencing studies of HLA class II molecules have focused almost exclusively on exon 2. In this study the complete cDNA sequence of the DRB1*09012 allele is reported for the first time. This sequence was previously only partially published. In the DR9 antigen, two synonymous allelic variants (DRB1*09011 and 09012) were officially recognized, though it was later found that the first one contained an error and both sequences were, thus, identical.
Abstract: Mucosal intestinal lymphocytes form the first immune-cell line of defense in the intestine. Several methodologies, most of them cumbersome and time consuming, have been used to obtain T-cell clones to unveil their physiological role. In the present work we take advantage of the recently described technique of transformation of T lymphocytes using Herpesvirus saimiri to show that it is possible to immortalize intestinal T-cell lines derived from healthy and diseased colonic samples and thence easily obtain in vitro intestinal T-cell lines as a model for physiopathological studies. Intestinal samples were obtained by colonoscopy and digested with dispase and collagenase. Mucosal lymphocytes (assessed by the expression of the CD3 and CD103 markers) were isolated using a Percoll gradient centrifugation and transformed with Herpesvirus saimiri. Sustained growth was observed 3 months later, showing that the cells were successfully transformed, a finding further confirmed by PCR. All cell lines were CD8+TcRalphabeta+ and HLA-DR+. CD25 was expressed on 1% of Crohn's disease-derived cells and on 25% of cells derived from patients with ulcerative colitis. CD80 expression was found on 80-90% of the cells. These immortal cell lines of intestinal origin may be useful in future experiments aimed at elucidating the role of mucosal lymphocytes in health and disease.
Abstract: Sequencing studies of HLA class II molecules have focused almost exclusively on its exon 2. The complete cDNA sequence of the DRB*1302 allele is reported here for the first time. A conservative polymorphism was detected outside the exon 2 at residue 206; these changes can only be recorded if complete cDNA sequences are studied, and may be of value in drawing evolutive inferences.
Abstract: Epithelial cells of the intestine seem to act as antigen-presenting cells to surrounding lymphoid tissue and may be crucial to maintain the pool of peripheral T lymphocytes. The scope of this study was to carry out an immunophenotypic and ultramicroscopic analysis of purified human enterocytes to elucidate their role as antigen-presenting cells, in the immune responses in the gut-associated lymphoid tissue. A method has been developed to obtain purified and viable human enterocyte populations, later labeled with relevant monoclonal antibodies directed to leukocyte antigens and subjected to cytofluorometric analysis. Phenotypic analysis revealed the presence of markers common to "classical" antigen-presenting cells (CD14, CD35, CD39, CD43, CD63 and CD64), reinforcing the idea that enterocytes may act as such. Moreover, several integrins (CD11b, CD11c, CD18, CD41a, CD61 and CD29) were also found. CD25 (IL-2 receptor alpha chain) and CD28, characteristic of T cells, were detected on the surface of these cells; this latter finding rises the possibility that enterocytes could be activated by IL-2 and/or via CD28 through binding to its ligands CD80 or CD86. Finally, the presence of CD21, CD32, CD35 and CD64 that may bind immune complexes via Fc or C3, suggests their participation in the metabolism of immune complexes. Furthermore, the finding of a Birbeck's-like granule in the cytoplasm of the cells, shows that enterocytes contain an ultramicroscopic feature previously thought to be characteristic of Langerhans' cells, an antigen-presenting cell. The phenotype detected on the surface of enterocytes, along with their ultramicroscopic characteristics, suggests that they may play an important role in the immune responses elicited in the gut, presenting antigens to surrounding lymphoid cells, and establishing cognate interactions with them.
Abstract: HLA-DR and DQ alleles have been detected by DNA typing in Ashkenazi and non-Ashkenazi Jews from Israel. Allele frequencies, characteristic DR/DQ linkage disequilibria, population distances and their corresponding dendrogram by using the Neighbor-Joining method were used to study relatedness between Jewish and other Mediterranean and non Mediterranean populations. Closest relatedness is observed between Ashkenazi and non-Ashkenazi Jews, and, in decreasing order, also with Algerians, Spaniards (including Spanish-Basques), French and Italians. Also, particular characteristic Central European alleles are observed in Ashkenazi Jews and Mediterranean/African alleles in non-Ashkenazi Jews. This is consistent with historical data, Jews being an ancient Mediterranean population, who have had a certain degree of admixture with their 2000-3000 years old neighbors in spite of cultural and religious traditions which have preserved identity outside Israel.
Abstract: A diploid expression of class I and class II human leukocyte antigens (HLA) has been found in purified spermatozoa by using double fluorescence labeling cytofluorometry and relevant monoclonal antibodies; this expression has been confirmed for the first time by the analysis of specific HLA mRNA and metabolic 35S labeling followed by immunoprecipitation, which demonstrates an active ongoing translation of HLA proteins in germinal cells. Long-living mRNA coming from diploid germinal cells may be translated to HLA molecules in spermatozoa. This translation is controlled (or at least inversely correlated) by a testicular hormone (inhibin) in a cyclic fashion. Remarkably, serum levels of inhibin, synthesized by Leydig and Sertoli cells, follow a 12- to 13-day cycle, with a peak level at Day 6; this is probably controlled by FSH (not cyclic in males) and other testicular and/or unknown hormones. Peak levels of inhibin concur with the lower density and percentage of spermatozoa expressing both HLA class I and II molecules (close to 3% by cytofluorometry); lowest levels of inhibin coincide with the highest numbers (35-40%) of spermatozoa positive for both HLA molecules and a higher surface density. These observations could put to an end a disconcerting and long-lasting controversy on the expression/non-expression of HLA antigens on spermatozoa. The possibility that HLA-bearing spermatozoa are more capacitated for fertilization than those that do not bear HLA, and the implications of our results on male fertility control are also discussed.
Abstract: We investigate whether a heat-shock protein gene (HSP70-2) is involved in determining susceptibility to systemic lupus erythematosus (SLE) in a Spanish population. A HSP70-2 PstI polymorphism was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction-amplified genomic DNA in 90 SLE patients and 117 controls. The PstI site containing allele (B) was significantly increased in SLE patients compared to healthy controls. This was due to a significant increase in the BB homozygous genotype in patients, particularly in those with diffuse proliferative nephritis. Neither allelic nor genotypic differences were detected when compared by the presence or absence of DR3. The HSP70-2 B allele seems tightly linked to the human leucocyte antigen (HLA) haplotypes carrying susceptibility to SLE in our population. An independent role for this gene cannot be confirmed due to its linkage with HLA DR3.
Abstract: TAP2 genes are placed within the HLA complex, have limited genetic variability and encode two main groups of peptide transporter proteins, the so-called TAP2*01 alleles, with a short ATP-binding domain, and the TAP2*0201 allele with a long domain. These transporters carry antigenic peptides from cytoplasm across the endoplasmic reticulum membrane to release them into nascent HLA class I molecules, which will then travel towards the plasma membrane. The shorter TAP2*01 alleles are present in 99% of diabetics and 90% of controls; these alleles may add slight, although significant and independent, susceptibility to diabetes, particularly in subjects carrying non-Asp 57 at beta DQ. Moreover, this increased susceptibility is not due to linkage disequilibrium with other HLA markers (i.e.: DR4), which does not exist in our Spanish population.
Abstract: Three new allelic forms of the HLA-G DNA sequence (HLA-G*II, HLA-G*III, and HLA-G*IV) have been identified. With the HLA-G*I sequence (previously designated HLA 6.0) as a reference, HLA-G*II shows a silent (G-->A) mutation at the third base of codon 57, HLA-G*III bears a non-synonymous (A-->T), but conservative, (Thr-->Ser) substitution at the first base of codon 31, and HLA-G*IV shows two silent substitutions: (A-->T) at the third base of codon 107 and (G-->A) at the third base of codon 57. A rapid method of singling out each allele on genomic DNA has been developed by using polymerase chain reaction amplification followed by restriction endonuclease treatment. Also, more or less strong linkage disequilibria has been found between most HLA-A alleles and either HLA-G*I or *II, both being the most prevalent alleles in the population, with a genotypic frequency of 0.55 and 0.38, respectively; HLA-G*III is very rare and HLA-G*IV has a genotypic frequency of 0.07. An evolutive classification of HLA-A alleles results according to their association with either HLA-G*I or HLA-G*II, which does not correlate with the classical serological cross-reacting groups classification. The finding of a strong and selective A/G linkage disequilibria with most HLA-A alleles, together with the existence of less frequent random A/G associations, may suggest that there exist in different haplotypes true and varied A/G genetic distances (and not a recombinational hotspot). It may be inferred from preliminary data that in primates HLA-A/G haplotypes bearing G*II may have appeared later than those bearing G*I.
Abstract: OBJECTIVE--To find the degree of HLA antigen sharing in couples with infertility of unknown etiology and compare it with that in couples with infertility of known etiology as well as couples with normal fertility. SUBJECTS--Eleven couples with infertility of unknown etiology, 26 with infertility of known etiology, and 31 fertile couples were tested for HLA class I (A, B, C), class II (DR, MLC), and class III (Bf) antigens and GLO alleles. HLA lymphocytotoxic antibodies and anti-sperm antibodies in both partners were also searched. RESULTS--Gene frequencies and the number of HLA antigens shared between the two members of the couple were similar in all groups. When "intra-couple" MLC reactivity was measured, no quantitative differences were found in the infertile group with unknown etiology, as compared to the group of known etiology: spouses in couples of either group usually reacted in both ways as expected for unrelated individuals" lymphocytes. Lymphocytotoxic antibody frequency did not differ between the nonfertile groups, and anti-sperm antibodies, found in ten patients in the group of known etiology, were not associated with any HLA antigen. In spite of that, GLO and all the HLA antigen classes described (I, II and III) were analyzed. CONCLUSIONS--Although the results are negative, these and others' (negative) results concerning couples with infertility of unknown etiology do not disprove that HLA has a role in fertilization, because HLA-related factors still unknown may exist. Exhaustive HLA available marker typing in international cooperative efforts may be needed to reach a significant number of carefully selected couples fully HLA typed to elucidate this problem.
Abstract: The particular histocompatability antigen (HLA) gene(s) that may confer systemic lupus erythematosus (SLE) susceptibility remains unknown. In the present study, 58 unrelated patients and 69 controls have been analyzed for their class I and class II serologic antigens, class II (DR and DQ) DNA restriction fragment length polymorphism, their deduced DQA1 and B1 exon 2 nucleotide sequences and their corresponding amino acid residues. By using the etiologic fraction (delta) as an almost absolute measure of the strongest linkage disequilibrium of an HLA marker to the putative SLE susceptibility locus, it has been found that the strength of association of the HLA marker may be quantified as follows: DQA1*0501 (associated to DR3) or DQB1*0201 (associated to DR3) > non Asp 57 beta DQ/Arg 52 alpha DQ > DR3 > non Asp 57 beta DQ. Thus, molecular HLA DQ markers tend to be more accurate as susceptibility markers than the classical serologic markers (DR3). However, dominant or recessive non Asp 57 beta DQ susceptibility theories, as previously postulated for insulin-dependent diabetes mellitus, do not hold in our SLE nephritic population; indeed, three patients bear neither Arg 52 alpha DQ nor Asp 57 beta DQ suscepibility factors. On the other hand, nonsusceptibility factors are included in our population in the A30B18CF130-DR3DQ2(Dw25) haplotype and not in A1B8CS01-DR3DQ2(Dw24); this distinctive association has also been recorded in type I diabetes mellitus and may reflect the existence of common pathogenic HLA-linked factors for both diseases only in the A30B18CF10DR3DQ2(Dw25) haplotype. Finally, the observed increase of deleted C4 genes (and not 'null' C4 proteins) in nephritic patients shows that C4 genes are disease markers, but probably without a pathogenic role.
Abstract: The HLA-DRB6 gene (also called DRB sigma/V1) has been found only in about 26% of human HLA haplotypes, i.e.; DR1, DRw10, and DR2-bearing ones (Corell et al. 1991). In contrast, exon-2 DRB6 sequences have been obtained from all tested primates: nine chimpanzees (Pan troglodytes), three gorillas (Gorilla gorilla) and three orangutans (Pongo pygmaeus); other apes which had already been sequenced (one gorilla and one chimpanzee) also had the DRB6 gene. Thus, all apes tested from three different species, some of them evolutionary separated by at least 14-16 million years, bear the DRB6 gene. In addition, more than one gene copy per haplotype has been found in one chimpanzee; this, together with the apparent loss of this gene in some of the human DR haplotypes, may indicate that the DR genome has undergone evolutionary changes more recently and more actively than class I or III genes. In addition, ten different and presumably allelic DRB6 exon-2 sequences have been obtained, and some of them coming from different species are more similar to each other than the one from the same species; this finding goes in favor of the trans-species theory of major histocompatibility complex polymorphism generation. Also, data are presented supporting that DRB6 may be one of the eldest genes of the DRB family, thus one of the first to diverge from the ancestral DRB gene.
Abstract: The question of HLA susceptibility to Type 1 (insulin-dependent) diabetes mellitus remains unresolved. In the present study, 127 diabetic patients and 177 unrelated control subjects have been analysed for their class I and class II serological antigens, class II (DR, DQ) DNA restriction fragment length polymorphisms and DQA1 and B1 exon-2 nucleotide sequences and their corresponding amino acid residues. By using the aetiologic fraction (delta) as an almost absolute measure of the strongest linkage disequilibrium of an HLA marker to the putative Type 1 diabetes susceptibility locus, it has been found that the strength of association of the HLA markers may be quantified as follows: DR4 less than DR3 less than DR3 or DR4 less than non-Aspartate 57 beta DQ and Arginine 52 alpha DQ less than Arginine 52 alpha DQ. Thus, molecular HLA-DQ markers appear to be more accurate as susceptibility markers than the classic serologically defined ones (DR3 and DR4); however, any effect of DQ markers disappears when non-DR3/DR4 individuals are considered, suggesting that DR factors (or others in between DQ and DR) are also important. In addition, a dominant non-Aspartate 57 beta DQ susceptibility theory does not hold (but a recessive one does) in our diabetic population (probably due to the high frequency of the protective DR7-non-Aspartate 57 beta DQ haplotypes); Arginine 52 alpha DQ is the best single HLA marker found in our population, both as a recessive or as a dominant one. Also there are 13 patients in our sample who bear neither Arginine 52 alpha DQ nor non-Aspartate 57 beta DQ susceptibility factors.(ABSTRACT TRUNCATED AT 250 WORDS)
Abstract: The recent description of a selective human CD3 gamma deficiency and other T-cell receptor (TCR)/CD3 structural and functional defects, together with previous biochemical data on the structure and interactions of the TCR/CD3 complex, may aid in elucidating the physiology of this multi-subunit membrane ensemble. CD3 gamma seemed to be required for the commitment and thymic maturation of an important fraction of T lymphocytes to the CD8 (but not CD4) lineage, perhaps by participating with the CD8 co-receptor in the instructive signal delivered through the alpha beta TCR during intrathymic positive selection by HLA class I molecules. The homologous CD3 delta component would, in contrast, be necessary for the selection of CD4 lymphocytes by HLA class II molecules. The interaction of CD4 and CD8 with the TCR/CD3 complex during antigen recognition may thus be asymmetrical, taking place through CD3 delta and gamma, respectively. Also, the existence of in vivo functional TCR/CD3 hemireceptors (lacking either CD3 gamma or CD3 delta) is suggested, and defects in their relative amount on the T-cell surface may disrupt unresponsiveness to self antigens and generate autoimmunity.
Abstract: C3 nephritic factor (NEF) has been found in 3 siblings presenting different (or none) degrees of renal disease. Other relatives, including their dead father, suffered from a renal illness. In 2 of the siblings, NEF activity was restricted to IgG1 and IgG3 subclasses. Familial NEF incidence and a shared C3 allotype and a common HLA haplotype including BfS alleles for the 3 NEF-positive siblings suggest that at least in our cases genetical factors may be involved in NEF generation.
Abstract: A novel DRB gene member has recently been identified (DRB6). It defines a new supratypic group of HLA-DR alleles comprising the DR1-, DR2-, and DRw10-carrying haplotypes. The comparison of this gene either at the nucleotide or at the protein level with other HLA-DRB genes supports its inclusion in the DRB gene family and not in DPB or DQB gene families. Moreover, the construction of an evolutionary gene-tree shows that this gene is probably the eldest member of the DRB gene family. Contradictory expression results do not clarify whether or not DRB6 is a pseudogene.
Abstract: A novel TaqI restriction fragment length polymorphism (RFLP) of 4.15 kb is reported using a DR beta probe (pRTV1). This fragment corresponds to the DRB1 locus and allows the subdivision at the DNA level of the DRB1*0301 allele (DR3 antigen), which had not previously been reported. Both splits also distinguish each of the two DR3-bearing extended haplotypes (HLA-B8,SCO1,DR3,DQw2,Dw24 and B18,F1C30,DR3,DQw2,Dw25) found associated to several autoimmune diseases as insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE) and myasthenia gravis. The fact that no polymorphism in the DRB1*0301 coding DNA sequence has been detected indicates that DRB1*0301 intronic, regulatory of neighbouring sequences might also contribute to differential disease associations (and pathogenic mechanisms) found linked to each of the two DR3-bearing haplotypes, i.e. IDDM and B8,DR3,Dw24 in North European/American Caucasoids vs IDDM and B18,DR3,Dw25 in Mediterraneans; SLE and B8,DR3,Dw24 in children vs SLE and B18,DR3,Dw25 in Spanish adults.
Abstract: Two new allelic exon-2 HLA-DRB sequences have been identified by using universal and also specific DRB primers. They may correspond to a previously unidentified DRB gene (DRB sigma) and define a new supratypic group ("DRw54") which includes DR1, DR"Br", DR2 and DRw10 bearing HLA haplotypes. This is probably the last HLA-DRB gene to be described in the standard DR haplotypes on the bases of the number of TaqI RFLPs obtained. Sequence comparison with their respective DP and DQ sequences shows that DRB sigma is unequivocally placed within the DRB family and also a constructed "neighbouring homology tree" indicates that DRB sigma gene is probably the eldest in the DRB family, thus the first to diverge from the ancestral DRB gene. An hypothetically deduced DRB sigma beta 1 protein domain was found to be quite different from the corresponding DRB1, DRB3, DRB4 and DRB5 products, since residues 40-55 would bear a longer alpha-helical conformation and would also exist a loss of both the extended conformation at residues 50-54 and the alpha-helix at residues 64-71. Thus, the putative DRB sigma protein would be remarkably different to other DRB ones. Also, a DRB sigma partial transcript (exon-2) has been obtained by PCR of cDNA by using specific DRB sigma oligonucleotides, but a specific Northern blot hybridization has not been achieved.
Abstract: A study of DR4 subtypes has been done in Spanish unrelated controls and insulin-dependent diabetics by using dot blot hybridization with specific DR4B1 exon-2 oligonucleotides and automated dideoxy DNA sequencing. Dw15-DQw8 is the predominant DR4 subtype present in our normal population (37%); this DR4 frequency characteristic singles out our population from all other Caucasoids tested so far and may also be a marker of the original Iberian paleo-North African population. Dw15-DQw8 is not significantly increased in our insulin-dependent diabetics sample and despite its relative high frequency in the control population it does not have a bearing in lowering insulin-dependent diabetes mellitus frequency of DR4-positive Spaniards. In addition, no particular DR4 split is by itself significantly increased in Spanish diabetics; this may indicate that selective diabetogenic environmental factors may be working upon DR4-positive individuals, but on genes (or gene products) other than DR or at least not upon the polymorphic sites of DRB1 exon-2 products.
Abstract: Necropsic lymphoid tissues obtained from an infant with a novel type of immunodeficiency consisting of a peripheral blood T lymphocyte antigen receptor (TCR) surface expression defect, were analyzed by immunohistochemistry for the expression of various TCR-associated epitopes. The work was aimed to characterize the biochemical basis of this kind of disorder and confirm the defect in different lymphoid tissues. Within an assessed lymphoid depletion, the patient's tissues showed a normal expression of several TCR epitopes (those associated to CD3 epsilon, CD3 delta and the clonotypic -Ti- alpha and beta chains). In contrast, the expression of the epitopes recognized by the monoclonals OKT3, WT31, and BMA031 was severely diminished. Our results therefore support that CD3 epsilon, CD3 delta, Ti alpha and Ti beta are probably not involved in this type of immunodeficiency, and strongly suggest that CD3 gamma (forming part of the epitope recognized by OKT3) may rather be the affected chain giving rise to the defective surface T cell phenotype; however, alternative interpretations are not ruled out. The disrupted TCR thus formed, containing Ti alpha beta heterodimers and CD3 epsilon and CD3 delta subunits, but lacking normal CD3 gamma, would in this scheme lack the conformational framework determinants recognized by WT31 and BMA031.
Abstract: Gut epithelial cell autoantibodies have been considered a hallmark of autoimmune enteropathy, a disorder occurring in children with protracted diarrhea of unknown etiology. Four patients (two male and two female) with such autoantibodies were studied. Immunofluorescence analysis showed two different disjunctive staining patterns: complement-fixing apical (three of four) and cytoplasmic (the remaining fourth one), which are shown to be directed against different structures. All three patients positive for complement-fixing apical gut epithelial cell autoantibodies had abnormal T-cell responses in vitro, one of them with an immunoglobulin G2 immunoglobulin deficiency and another with an immunoglobulin A deficiency. An immunoglobulin A deficiency without T-cell alterations was also diagnosed in the cytoplasmic gut epithelial cell autoantibody-positive patient. These findings suggest that different immunologic alterations (either a T-cell abnormality or immunoglobulin deficiency) may favor the appearance of gut epithelial cell autoantibodies (complement-fixing apical or cytoplasmic, respectively). Furthermore, these autoantibodies should not be considered a specific marker of autoimmune enteropathy, because they may not always be associated with such a disease: two patients with apical gut epithelial cell autoantibodies showed no signs of intestinal lesion or diarrhea.
Abstract: HLA-DR3 antigen included in the compound phenotype B18BfF1 (but not the one linked to the B8BfS compound phenotype) was found to be significantly increased in our SLE patients. It is remarkable that in our Southern-Mediterranean population, B18BfF1DR3 individuals (but not B8BfSDR3) are prone to SLE with renal disease, in contrast with other Northern European and Caucasoid populations. Also, patients with autoantibodies to Ro/La have a significant increase of the B8DR3 compound phenotype. Production of autoantibodies against Ro alone was associated to DR2 and production of anti-Sm/nRNP to DR3 (either B18BfF1 or B8BfS associated) only in the subgroup without renal disease. The distinctive HLA and autoimmune associations to SLE with and without renal disease suggests that both clinical forms may not share a common identical pathogenesis.
Abstract: DQA1, DQA2, DQB1, and DRB1 alleles have been determined and the DQA1 and DQB1 DNA gene sequences assigned by using restriction fragment length polymorphisms in 67 diabetic individuals and 72 controls. It has been found that: 1) DQA2 (U allele) is not a susceptibility factor, 2) non-aspartic acid homozygosity in residue 57 (Asp 57 negative) of the DQ beta chains is positively correlated with insulin-dependent diabetes mellitus (IDDM), and 3) DQ beta Asp-57-negative and DQ alpha arginine-52-positive (Arg-52-positive) individuals are increased among diabetic patients; this latter analysis shows a higher etiologic fraction (delta) value than the one obtained when considering only homozygous DQ beta Asp-57-negative individuals. However, if only non-DR3 or DR4 individuals were considered (both in DQ beta Asp-57-negative homozygous and in DQ beta Asp-57-negative/DQ alpha Arg-52-positive individuals) the correlation with disease disappears. In addition, the postulated risk DQ beta Asp 57-negative and DQ alpha Arg 52 positive is absent in six patients. These data do not discard the possibility that DR3/DR4 may contain the primary susceptibility factors. It is concluded that it is not possible to assign the susceptibility to IDDM to a specific HLA locus and that several loci within the same or the trans haplotype may be involved.
Abstract: HLA-DR3 or -DR4 segregation distortion to normal or insulin-dependent (ID) diabetic offspring of 108 Spanish families whose parents were healthy was not observed; however, DR3 or DR4 ID offspring is significantly increased in the present study, since parents were chosen after tracing ID children. These results are discrepant with those found by others in families with diabetic parents in other ethnic groups. These conflicting data could be due to sampling errors or segregation distortion. Thus, ethnic group differences in a genetic (T/t-like) or metabolic mechanism might confer advantages to DR3- or DR4-bearing gametes from ID diabetic parents, but segregation distortion might only affect certain HLA DR3 or DR4 extended haplotypes which are frequent and characteristic for certain ethnic groups (i.e. B8-DR3-BfS-C4AQOB1 and Bw62-DR3-BfS-C4A383 in most caucasians) but not for other haplotypes in other ethnic groups (Spaniards; B18-DR3-BfF1-C4A3BQO and BwX-DR4-BfX-C4AXBX).
Abstract: Exon 2 nucleotide sequence of the DRB1 gene encoding the HLA-DRw13b allele defined by DNA-RFLP (Restriction Fragment Length Polymorphism) typing, has been obtained by using five heterozygous individuals genomic DNA and a non isotopic automated "dideoxi" methodology. Its comparison with other known homologous DRB1 sequences suggests that two different mechanisms which generate HLA allele variability may have occurred in this particular exon 2: a gene conversion between DRw11 or DRw13 as acceptors and DR4-Dw15 or DRw8.1 as donors and in addition, a non-conservative point mutation at base 221. The relationship between this HLA sequence characteristics and certain diseases susceptibility is discussed.
Abstract: We report a case of a 14 1/2-year-old boy who was diagnosed of systemic lupus erythematosus in the background of an acute nephritic syndrome, 3 1/2 years after being diagnosed of idiopathic thrombocytopenic purpura. The familial history suggested the presence of other cases of SLE, which were proven with relevant clinical and laboratory studies. A genetic study for disease markers was carried out and a correlation was found with haplotypes HLA A25, B18, BW6, DRX, and DQW; C2 deficiency was ruled out. We conclude that it is of paramount importance to rule out the existence of familial SLE in front of infantile SLE, particularly in boys, and we emphasize the necessity of keeping on further searching for genetic markers of the disease.
Abstract: A polyclonal T-cell receptor complex (TCR) expression defect (as detected with monoclonal antibody WT31) has been found in two children belonging to an otherwise healthy Spanish family. One of the sibs (V, who had been vaccinated with attenuated poliomyelitis virus) showed clinical signs of immunodeficiency with an autoimmune syndrome, but the other (older) sib (D, vaccinated with attenuated rubella, measles, mumps, and poliomyelitis viruses) has been symptomless throughout life. In contrast to both sibs' normal expression of other peripheral leucocyte markers, as measured by flow cytometry (including CD1, CD2, CD4, CD8, and CD16), only about 6% of CD2+ polyclonal T cells expressed surface antigen-specific T-cell receptor (Ti/WT31), and only about 23% weakly expressed surface CD3 determinants. On the remaining CD2+ T cells in each sib the expression of Ti and CD3 was undetectable; the defect in CD3 expression is very likely secondary to the defect in Ti expression. Natural killer (NK) activity was not increased in any of the sibs, ruling out a high content of NK cells among their CD2+ lymphocytes. Functional data indicate that CD3-mediated T-cell activation with anti-CD3 monoclonals and Ti-mediated responses to allogeneic and tetanus toxoid antigens were severely depressed, whereas activation via CD2 was normal in the T lymphocytes of both sibs. Genes encoding for Ti alpha, beta, and gamma chains did not show major alterations by southern blot analysis, and polyclonal beta chain genes rearrangements were detected in both children's T-cell blasts. Family clustering suggests a genetic pathogenesis, but linkage to HLA or other blood group markers has not been found. Sib V had a concomitant autoimmune disease and died after a severe autoimmune haemolytic anaemia, indicating a relationship between the TCR and generation of autoimmune clones. However, the resistance of both individuals to infection and to vaccination with attenuated viruses, and the fact that sib D has been symptomless to date questions the relative importance of the TCR in the immune response against infection, and suggests that alternative T-cell activation pathways and non-specific defence mechanisms (external surfaces--bound and/or cellular) may suffice under certain circumstances.
