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The purpose of this study was to examine whether several allelic variants in the polymorphic interleukin (IL)-10 promoter region were related with an increased risk of developing systemic lupus erythematosus (SLE) in Spanish patients from Canary Islands. Microsatellites (MS) at positions -4000 and -1200 (IL10R and IL10G, respectively) and single nucleotide polymorphisms (SNPs) (MS) at positions -1082G/A, -819C/T and -592C/A of the IL-10 promoter were analysed in patients with SLE and healthy controls from Canary Islands (Spain). We found that SNPs but not MS were associated with SLE. The GCC haplotype frequency was significantly higher in SLE patients (0.43) than in healthy donors (0.33) [P = 0.02; OR = 1.50 (95% CI = 1.06-2.14)], whereas the ACC haplotype was less represented in patients (0.28 vs. 0.37) [P = 0.02; OR = 0.64 (95% CI = 0.44-0.92)]. To assess the functional role of genotypes, serum IL-10 levels from patients and controls were quantified by ELISA. Also, the lipopolysaccharide-induced IL-10 secretion by monocytes from healthy controls was evaluated in vitro. Serum IL-10 levels were higher in patients [median (interquartile range) = 2.8 pg/mL (1.8-4.2)] than in controls [0.9 pg/mL (0-3.5)] (P = 0.02), but no association was observed between serum IL-10 levels or lipopolysaccharide-induced IL-10 secretion and the IL-10 promoter haplotypes. These data suggest that the IL-10 promoter haplotype that produces higher levels of cytokine is associated with SLE in patients from Canary Islands.

Objective: To investigate the association of the (CA)n dinucleotide repeat in the 3' untranslated region (3'UTR) of the CD154 gene with systemic lupus erythematosus (SLE), and its functional role in protein expression. Methods: The allelic and genotypic distributions of the polymorphism were compared in 80 patients with SLE and 80 controls. A complete clinical and analytical database was recorded in each patient in order to correlate the clinical manifestations in SLE with different alleles. To investigate the functional role of the polymorphism, the CD154 protein expression on activated lymphocytes from healthy homozygous controls was evaluated by flow cytometry. Results: The 24 CA allele was the most represented in controls (p = 0.029), whereas the alleles containing >24 CA repeats were found in patients (p = 0.0043). Furthermore, when only homozygous women were considered, most controls carried two 24 CA alleles (p = 0.041), whereas most patients carried two alleles containing >24 CA repeats (p = 0.032). Also, patients carrying at least one 24 CA allele had less neurological involvement (p = 0.034), and carriers of at least one allele with fewer than 24 CA repeats presented more livedo reticularis (p = 0.006) and anti-Sm (p = 0.01) and anti-RNP (p = 0.038) autoantibodies. CD154 maximum expression in activated lymphocytes from all controls was reached after 54 hours, but it was more prolonged in controls carrying two alleles with >24 CA repeats (p = 0.0068). Conclusion: The CD154 3'UTR microsatellite is associated with SLE, and the most represented alleles in patients were accompanied by a more prolonged protein expression in activated lymphocytes from controls.

Genetic variants of endosomal toll like receptors (TLR) have been associated with many infectious, autoimmune and inflammatory diseases, but few studies have been reported in the Spanish population. The aim of this study was to describe the allelic and genotypic distributions of some common nucleotide substitutions of endosomal TLRs in healthy Spanish women and to compare them with those already published in other population groups. Nine substitutions were analysed in 150 DNA samples from 150 Spanish, non-related healthy females: TLR3 rs3775291 and rs5743305; TLR7 rs179008 and rs5743781; TLR8 rs3764880 and TLR9 rs187084, rs5743836, rs352139 and rs352140. Genotyping was carried out by real time PCR and melting curve analysis in a LightCycler 480. A systematic review was performed in order to compare the genotypic distributions in our cohort with those previously published in other population groups. The comparative study was performed with the two tailed Fisher's test or the Yates continuity correction for the Chi-square test when appropriate. No homozygotes for rs5743781 in TLR7 were found, and rs352139 and rs352140 of TLR9 were in strong linkage disequilibrium. Genotype distributions in endosomal TLR are similar to other Spanish series previously reported. As expected, most differences were found when comparing our distributions with Asiatics, but differences were also found with other Caucasian populations. Since there are significant variations in genotypic distributions of TLRs in both interracial groups and within the same ethnic group, to carry out studies of disease susceptibility in more restricted groups is mandatory.

Despite a fludarabine-based treatment is the first choice of therapy in chronic lymphocytic leukemia (CLL), not all patients achieve a partial or complete response and some of them develop autoimmune manifestations. The aim of this study was to evaluate the influence of CD154 on these adverse effects because CD154 is involved in both B-cell survival and autoimmunity.Peripheral blood mononuclear cells (PBMC) from 36 patients with CLL were cultured in vitro with fludarabine or 2-chlorodeoxyadenosine for 24, 48, and 72 hours.Seven patients (19.4%) presented CD154 expression in PBMC cultured with purine analogues in vitro for 24 and/or 48 hours, while no expression was found when cultured in media alone. These seven patients showed a decreased apoptotic rate in vitro after purine analogues compared with those patients who did not express CD154 (p = 0.01 for fludarabine; p0.001 for 2-chlorodeoxyadenosine). CD154 expression was found to have prognostic value for response to fludarabine in vivo and was associated with the development of autoimmune manifestations (odds ratio = 25; 95% confidence interval = 3.5-166.7; p0.001).Our preliminary results suggest that CD154 expression in CLL patients, which may be induced by purine analogues, is associated with resistance to fludarabine and with development of autoimmune manifestations.

A program has been developed for the control of Vapour Pressure Osmometry (VPO) measurements. The output signal of a Vapour Pressure Osmometer is read by an A/D converter card installed in one of the expansion slots of a PC microcomputer. The stability of the measurements is checked by analysing the first derivative of the smoothed signals, which is calculated in real time. Sets of repeated measurements are carried out under the supervision of a computer program as a check for their reproducibility. When the set is ended the program calculates the average and its standard deviation.The program is particularly valuable for evaluating VPO measurements for solution equilibria studies. This interfacing strategy may be applied to any kind of technique in which the time stability of the signals is the basis for defining measurement stability. The automated VPO has other advantages, including low cost and time saving.

Background: Not all the patients with sarcoidosis need pharmacological therapy, and the decision to start therapy is based mainly on clinical conditions. The aim of this study was to evaluate the prognostic value of the leukocyte and lymphocyte subpopulations in the bronchoalveolar lavage fluid from these patients. Methods: Thirty-three nonsmoking patients with sarcoidosis were included and classified based on the presence of Lofgren's syndrome (n = 11), the radiological stage (12 at Stage I, 17 at Stage II, and 4 at Stage III), and their follow-up. Differential leukocyte subsets and the lymphocyte subpopulations were determined by flow cytometry. Results: The percentage of neutrophils was lower in patients with Lofgren's syndrome (P = 0.038) and in patients at Stage I (P = 0.002). Patients with a poor outcome had a higher percentage of neutrophils (P = 0.004) and NK cells (P = 0.023) than those with a stable disease. Finally, a higher percentage of NK cells was found in those patients who needed a steroid treatment (P = 0.012). Conclusions: Increased percentages of neutrophils and NK cells in the bronchoalveolar lavage fluid from patients with sarcoidosis are associated with a poor outcome and a higher probability to need steroids treatment. The percentage of neutrophils was also lower in patients with Lofgren's syndrome. © 2006 International Society for Analytical Cytology

Current evidence indicates that regulatory T cells (Tregs) actively suppress autoreactive lymphocytes that escape central tolerance.1,2 Tregs are characterised by a high intensity CD25 constitutive expression3 on the surface of CD4 T lymphocytes, which distinguishes them from the non-regulatory CD4+CD25+ T lymphocytes.4 Because of their important role in the maintenance of tolerance, it has been suggested that Tregs are decreased in patients with systemic lupus erythematosus (SLE).5 We have quantified Tregs in patients with SLE and controls and evaluated their association with disease activity and treatment. Thirty three patients with SLE (26 women, 7 men) and 14 healthy volunteers matched for age and sex (11 women, 3 men) with a mean age of 39.9 and 36 years, respectively, were studied. All patients fulfilled at least four of the American College of Rheumatology 1982 revised criteria for the classification of SLE.6 Disease activity at the time of blood sampling was assessed using the SLE Disease Activity Index (SLEDAI).7 …

Background IL10 is a pleiotrophic cytokine that inhibits the antigen specific activation of Th1 clones by reducing both the antigen presenting capacity and the production of pro-inflammatory cytokines by monocytes. It is also a potent activation and differentiation factor for B lymphocytes. SLE patients show increased IL10 serum levels associated with the pathogenesis of the disease. Peripheral blood mononuclear cells from SLE patients, and their relatives, constitutively produce high levels of the cytokine, suggesting an hereditary component in IL10 production. Anti-IL10 antibodies administration to SLE patients may be beneficial in the management of refractory cases. The IL10 gene promoter contains two dinucleotide repeats (microsatellites): IL10G and IL10R, close to transcription factor binding sites, probably playing an important role in the transcription regulation. Objectives To evaluate the association of the IL10G microsatellite with SLE in Spanish patients. Methods 80 unrelated SLE patients from Canary Islands (Spain) fulfilling at least four of the ACR criteria and 79 sex-matched healthy donors were included. A segment of the IL10 gene containing the microsatellite was amplified by PCR and the resulting products were electrophoresed in denaturing polyacrilamide gels. Chi-square test was used for statistical analysis. Results No particular IL10G allele was found to be significantly associated with SLE. Neverthaless, we found an elevated risk of serositis in carriers of the allele 19 (p = 0.007), a significant association of the 19/23 genotype with seizures (p = 0.018), and a reduced risk of renal disorder (p = 0.047) and thrombocytopenia (p = 0.049) in carriers of the allele 23. Conclusion Although IL10G alleles seem not to influence susceptibility to SLE in Spaniards, some of them may be related to the presence of several clinical manifestations of the disease.

Background PARP is a DNA-binding protein that catalyses the covalent addition of ADP-ribose to many nuclear proteins. It is involved in DNA repair and apoptosis, processes that have been implicated as causative factors for SLE. It has been shown that patients suffering SLE have decreased PARP mRNA levels and a low enzime activity. The PARP gene is located at 1q41-q42 and contains a microsatellite (CA)n polymorphism in the promoter region that probably affects the transcription and mRNA levels. A recent study found preferential transmission of the allele containing 12 CA repeats to affected offspring in Caucasian SLE families. Objectives To investigate the association between PARP polymorphism and SLE susceptibility in Spaniards. Methods 76 unrelated SLE patients from Canary Islands (Spain) fulfilling at least four of the ACR criteria and 79 sex-matched healthy donors were included. A segment of the PARP gene containing the microsatellite was amplified by PCR and the resulting products were electrophoresed in denaturing polyacrilamide gels. Chi-square test was used for statistical analysis. Results Allelic distributions of PARP polymorphism showed no statistical differences between SLE patients and controls. But considering particular clinical features we found several associations with specific alleles: a) homozygotes for 11rep. allele suffered more lymphopenia (p = 0.04) but less hypocomplementemia (p = 0.03), b) autoantibodies anti-Ro/La were more frequent (p = 0.02) in carriers of 15rep., c) carriers of 16rep. allele presented more malar rash (p = 0.03) and seizures (p = 0.04), and finally d) carriers of 17rep. allele showed a reduced risk of production of several autoantibodies as anti-Sm (p = 0.007) and anti-Ro/La (p = 0.04). Conclusion Although PARP alleles seem not to influence susceptibility to SLE in Spaniards, some of them may be related to the presence of several clinical and analytical manifestations of the disease.

CD40–CD154 interaction is an important mediator of inflammation and has been implicated in T helper type 1-mediated autoimmune diseases including rheumatoid arthritis (RA). Linkage studies have shown association of markers in the proximity of the CD154 gene. In the present work we investigated whether specific allele variants of the microsatellite in the 3' UTR of the CD154 gene might modulate the risk of RA. The study, in a case-control setting, included 189 patients and 150 healthy controls from the Canary Islands, Spain. The 24CAs allele was less represented in female patients than in controls (0.444 in controls versus 0.307 in patients, P = 0.006, odds ratio (OR) 0.556, 95% confidence interval (CI) 0.372 to 0.831) but not in males (0.414 versus 0.408), and only when homozygous (P = 0.012; OR 0.35, 95% CI 0.16 to 0.77). We also verified that CD154 association with RA was independent of human leukocyte antigen (HLA) phenotype. A further functional study showed that after stimulation anti-CD3, CD154 mRNA was more stable in CD4+ T lymphocytes from patients with RA bearing the 24CAs allele (mRNA half-life 208 minutes) than in patients without the 24CAs allele (109 minutes, P = 0.009). However, a lower percentage of CD154+CD4+ T lymphocytes was seen in freshly isolated peripheral blood mononuclear cells from patients carrying 24CAs alleles (mean 4.28 versus 8.12; P = 0.033), and also in CD4+ T lymphocytes stimulated with anti-CD3 (median 29.40 versus 47.60; P = 0.025). These results were concordant with the smaller amounts of CD154 mRNA isolated from stimulated T lymphocytes with 24CAs alleles. The CD154 microsatellite therefore seems to affect the expression of the gene in a complex manner that implies not only mRNA stability. These data suggest that the CD154 microsatellite contributes to the regulation of mRNA and protein expression, although further studies will be necessary to elucidate its role in disease predisposition.