Study of association of CTLA4 gene variants to non-anterior uveitis.

This study was undertaken to investigate the possible genetic association of functional CTLA4 polymorphisms with susceptibility to non-anterior uveitis. Four hundred and seventeen patients with endogenous non-anterior uveitis and 1517 healthy controls of Spanish Caucasian origin were genotyped for the CTLA4 polymorphisms rs733618, rs5742909 and rs231775, using predesigned TaqMan(©) allele discrimination assays. PLINK software was used for the statistical analyses. No significant associations between the CTLA4 polymorphisms and susceptibility to global non-anterior uveitis were found. It was also the case when the potential association of these genetic variants with the anatomical localization of the disease, such as intermediate, posterior or panuveitis, was assessed. Our results do not support a relevant role of these CTLA4 polymorphisms in the non-anterior uveitis genetic predisposition.

New CD3zeta allele showing a 9-bp deletion in the 3'-UT region of the gene.

We report here a non-previously described 9-bp deletion in the 3'-UT region of the CD3zeta gene, located in between two AREs.

Analysis of Crohn's disease-related CARD15 polymorphisms in Spanish patients with idiopathic uveitis.

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.

Anterior uveitis and meningococcemia: a case report.

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.

Informe sobre el ejercicio de estandarización de inmunofluorescencia indirecta (IFI) para la detección de anticuerpos antinucleares (ANA). Taller de autoinmunidad, Santander 2004 / Report on the indirect immunofluorescence (IFI) standardisation assay for the detection of antinuclear antibodies (ANA). Workshop on autoimmunity, Santander, May 2004

Para reducir las diferencias que se observan a la hora de titular por diferentes laboratorios un mismo suero ANA+, sugerimos el uso de unidades estándar. Con esta finalidad, en el presente Taller de Autoinmunidad, se distribuyeron cinco alícuotas de un mismo suero control, diluido seriadamente con PBS. A los laboratorios participantes se les pidió que diluyeran cada alícuota hasta su título final. Con estos datos se construyó una recta patrón para cada laboratorio. Además, se enviaron cuatro muestras problema. Estas muestras consistían en duplicados y diluciones de un mismo suero, con la finalidad de determinar si los laboratorios participantes eran capaces de replicar los duplicados y ordenar correctamente las diluciones. Los títulos remitidos para cada suero en diluciones se transformaron en unidades usando la curva patrón previamente obtenida por cada laboratorio. Los resultados obtenidos muestran que: 1) algunos laboratorios participantes no fueron capaces de replicar los duplicados o de ordenar adecuadamente las diluciones; 2) el uso de unidades en vez de diluciones disminuye la dispersión de los resultados obtenidos por los laboratorios participantes, para un suero dado. Por tanto, proponemos el uso de unidades estándar en lugar de diluciones cuando se informen títulos de ANA (AU)

El sistema inmunitario de las mucosas: una breve revisión / Mucosal immune system: a brief review

La mayoría de los antígenos que entran en contacto con el sistema inmune durante la vida de un ser vivo lo hacen a través de la superficie de la mucosa de los tractos respiratorio, gastrointestinal y urogenital. Ocupan una superficie de 400 m2 y forman el área de mayor tamaño en contacto directo con el ambiente externo. Las mucosas separan el ambiente externo del ambiente interno, estéril, y representan una primera línea de defensa. Esta barrera está en contacto tanto con patógenos que han desarrollado mecanismos eficaces para la colonización de epitelios e invasión de mucosas, como con antígenos inocuos, tales como comida, o la flora bacteriana comensal. En el primer caso se necesita una respuesta inmune eficaz y robusta, mientras que en el segundo se requiere una respuesta caracterizada por ignorancia o supresión activa. En estas condiciones, las mucosas han desarrollado un complejo sistema inmune, con características anatómicas y funcionales particulares, capaz de generar rigurosas respuestas frente a antígenos patogénicos, mientras mantiene una situación de ignorancia o supresión activa frente a antígenos no patogénicos (AU)

Higher proliferative capacity of T lymphocytes from patients with Crohn disease than from ulcerative colitis is disclosed by use of Herpesvirus saimiri-transformed T-cell lines.

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.

Intrinsic defects explain altered proliferative responses of T lymphocytes and HVS-derived T-cell lines in gastric adenocarcinoma.

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.

Informe sobre el ejercicio de estandarización de inmunofluorescencia indirecta (IFI) para la detección de anticuerpos antinucleares (ANA). Taller de autoinmunidad. Madrid, Junio 2001 / Report on the indirect immunofluorescence (IFI) standardisation assay for the detection of antinuclear antibodies (ANA). Workshop on autoimmunity. Madrid, june 2001

Para reducir las diferencias que se observan a la hora de titular por diferentes laboratorios un mismo suero ANA+, sugerimos el uso de unidades estándar. Con esta finalidad, en el presente Taller de Autoinmunidad, se distribuyeron cinco alícuotas de un mismo suero control, diluido de forma seriada con suero humano normal. A los laboratorios participantes se les pidió que diluyeran cada alícuota hasta su título final. Con estos datos se construyó una recta patrón para cada laboratorio. Además, se enviaron ocho muestras problema. Estas muestras consistían en duplicados y diluciones de distintos sueros, con la finalidad de determinar si los laboratorios participantes eran capaces de replicar los duplicados y ordenar correctamente las diluciones. Los títulos remitidos para cada suero en diluciones se transformaron en unidades usando la curva patrón previamente obtenida por cada laboratorio. Los resultados obtenidos muestran que: a) algunos laboratorios participantes no fueron capaces de replicar los duplicados o de ordenar adecuadamente las diluciones; y b) el uso de unidades en vez de diluciones disminuye la dispersión de los resultados obtenidos por los laboratorios participantes, para un suero dado. (AU)

Informe sobre el ejercicio de estandarización de inmunofluorescencia indirecta para la detección de anticuerpos antinucleares / Report on the indirect immunofluorescence standardisation assay for the detection of antinuclear antibodies. Workshop on autoimmunity

Para reducir las diferencias que se observan a la hora de titular por diferentes laboratorios un mismo suero ANA+, sugerimos el uso de unidades estándar. Con esta finalidad, en el presente Taller de autoinmunidad, se distribuyeron cuatro alícuotas de un mismo suero control, diluido serialmente con suero humano normal. A los laboratorios participantes se les pidió que diluyeran cada alícuota hasta su título final. Con estos datos se construyó una recta patrón para cada laboratorio. Además, se enviaron cuatro muestras problema. Estas muestras consistían en duplicados y diluciones de un mismo suero, con la finalidad de determinar si los laboratorios participantes eran capaces de replicar los duplicados y ordenar correctamente las diluciones. Los títulos remitidos para cada suero en diluciones se transformaron en unidades usando la curva patrón previamente obtenida por cada laboratorio. Los resultados obtenidos muestran que: a) algunos laboratorios participantes no fueron capaces de replicar los duplicados o de ordenar adecuadamente las diluciones; b) el uso de unidades en vez de diluciones disminuye la dispersión de los resultados obtenidos por los laboratorios participantes, para un suero dado (AU)

Coeliac- and enteropathy-associated autoantibodies in Spanish insulin-dependent diabetes mellitus patients and their relation to HLA antigens.

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.

Cell surface phenotype and cytokine secretion in Caco-2 cell cultures: increased RANTES production and IL-2 transcription upon stimulation with IL-1beta.

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.

Lens culinaris, Phaseolus vulgaris and vicia faba lectins specifically trigger IL-8 production by the human colon carcinoma cell line CACO-2.

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.

Cyclic expression of HLA class I and II molecules on the surface of purified human spermatozoa and their control by serum inhibin B levels.

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.

Evolutionary relationships between HLA-B alleles as indicated by an analysis of intron sequences.

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.

Higher incidence of autoantibodies in X-linked chronic granulomatous disease carriers: random X-chromosome inactivation may be related to autoimmunity.

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.

Successful in vitro immortalization of human intestinal mucosal lymphocytes with Herpesvirus saimiri.

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.

Complete cDNA sequence of the HLA-DRB1*09012 allele.

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.

Differential contribution of HLA-DR, DQ, and TAP2 alleles to systemic lupus erythematosus susceptibility in Spanish patients: role of TAP2*01 alleles in Ro autoantibody production.

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.

Polymorphism of Mhc-DRB alleles in Cercopithecus aethiops (green monkey): generation and functionality.

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.