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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.

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.

Hypereosinophilic syndromes (HSS) are a rare group of heterogeneous disorders characterized by prominent and persistent eosinophilia and organ dysfunction. Secondary causes of eosinophilia must be excluded. Recent advances in molecular biology and cytogenetics have permitted the characterization of different subsets of hypereosinophilic syndrome. We describe a patient with the lymphocytic variant.A 46-year old male Philippine patient presented skin lesions, fever, red eyes, enlarged lymph nodes and marked eosinophilia.Lymphocytic phenotyping by flow cytometry analysis was performed on peripheral blood and an aberrant population of T lymphocytes CD3-CD4+ producing interleukin 5 was found. TCR gene rearrangement using PCR amplification confirmed T cell clonality.The lymphocytic variant of the hypereosinophilic syndrome is a primitive lymphocytic disorder characterized by a non-malignant T cell population expansion producing eosinophilopoietic cytokines, with an indolent clinical course but that can transform into a peripheral T lymphoma. We report the first case of such a variant published in our country and review the characteristics of this variety.

Objective To investigate the contribution of T lymphocytes and monocytes to cytokine production in systemic lupus erythematosus (SLE). Methods Forty-five SLE patients and 19 healthy volunteers were included. Serum levels of tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), interleukin (IL)-6, and IL10 were quantified by ELISA. The cytokine production capacities of peripheral blood mononuclear cells were assessed by culturing in vitro with PMA+Ionomycin or LPS. The intracellular cytokine expression was measured by flow cytometry in T lymphocytes and monocytes, respectively. The influence of the disease activity (measured as the SLE-disease activity index; SLEDAI) and the treatment the patients were receiving was evaluated. Results Serum IL10, IL6, and TNFα levels were increased in patients (P ≤ 0.01), and a higher spontaneous (without stimuli) intracellular expression of IL10 in CD4+ and CD8+ T lymphocytes (P < 0.05) and of IL6 in monocytes (P = 0.01) were found. After stimulation, patients presented a higher percentage of CD4+ and CD8+ T lymphocytes producing IL4 and IL10 (P ≤ 0.01), and of monocytes producing IL6 (P = 0.04) and IL10 (P = 0.008). The SLEDAI score was positively correlated with the percentage of CD4+IL10+ and CD8+IL10+ T lymphocytes (P < 0.01), and inversely correlated with CD8+TNFα+ (P= 0.02), CD4+IFNγ+ (P = 0.04) and CD8+ IFNγ+ (P = 0.002) T lymphocytes. Patients receiving high dose prednisone produced higher IL10, but they also were the patients with a more active disease. Conclusion Monocytes and T lymphocytes (CD4+ and CD8+) contribute to an overproduction of IL6 and IL10 in SLE; this correlates with the disease activity but is independent of the treatment the patients are receiving. © 2009 Clinical Cytometry Society

The great variety of pathological entities related to the presence of circulating HSP-70 suggests a nonspecific cellular damage. As the present study shows, positive results decrease with respect to the time elapsed after the injection of the ototoxic agent. HSP-70 appears as an early and transient marker that could permit early detection of inner ear damage.The aim of this study was to determine the presence of HSP-70 at different time points by means of Western blot immunoassay in the sera of rats treated with cisplatin.Thirty-six Wistar rats were intraperitoneally injected with cisplatin at a dose of 5 mg/kg and blood samples were collected at 7 and 90 days. Determination of HSP-70 was made by means of a modified Western blot immunoassay kit originally used for human HSP-70 antigen detection. A control group of 18 animals was used for comparison.Western blot was positive in 77.8% of the animals in the 7 days group, decreasing to a 44.4% in the 90 days group. In the control group, Western blot was positive in 5.5%.

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 …

Recent evidence indicates that the slowly expanding population of CD5(+) B cells that characterizes B-cell chronic lymphocytic leukemia (B-CLL) could be related to defects in the response to cytokine produced by T cells that regulate apoptosis. We studied the intracellular expressions of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in T-helper 1 cells (Th1 response) of B-CLL.Peripheral blood mononuclear cells from 21 healthy individuals and purified T cells from 21 early-stage and 15 late-stage B-CLL patients were activated with phorbol myristate acetate and ionomycin. The Th1 cytoplasmic cytokines were evaluated in CD4(+) and CD8(+) T cells by flow cytometry.The percentages of CD4(+) and CD8(+) T cells positive for IL-2 were significantly lower in B-CLL patients than in healthy individuals (P = 0.030 and 0.049, respectively). No significant differences in TNF-alpha or IFN-gamma intracellular expressions were found between patients and healthy individuals. TNF-alpha- and IFN-gamma-expressing CD8 T cells were disease stage dependent, being significantly higher in late-stage patients (P0.001 for both cytokines).Our present observations suggested that Th1 cytokines may be of major importance in the pathogenesis of B-CLL.

The CD154 gene contains a dinucleotide repeat (CA)n in the 3' untranslated region. Allelic distribution in Spanish populations from two areas with different genetic background, the Canary Islands and Peninsula, are described. Seven alleles with different allelic distribution between the two groups, were found. This represents a highly polymorphic marker, useful for genetic studies on a critical molecule in immunity.

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.