Assessment of interleukin-10 promoter variant (-1082A/G) and cytokine production in patients with hemolytic uremic syndrome.

Introduction: Hemolytic uremic syndrome (HUS) is a condition that results in acute kidney failure mainly in children, which is caused by Shiga toxin-producing Escherichia coli and inflammatory response. Although anti-inflammatory mechanisms are triggered, studies on the implication in HUS are scarce. Interleukin-10 (IL-10) regulates inflammation in vivo, and the interindividual differences in its expression are related to genetic variants. Notably, the single nucleotide polymorphism (SNP) rs1800896 -1082 (A/G), located in the IL-10 promoter, regulates cytokine expression. Methods: Plasma and peripheral blood mononuclear cells (PBMC) were collected from healthy children and HUS patients exhibiting hemolytic anemia, thrombocytopenia, and kidney damage. Monocytes identified as CD14+ cells were analyzed within PBMC by flow cytometry. IL-10 levels were quantified by ELISA, and SNP -1082 (A/G) was analyzed by allele-specific PCR. Results: Circulating IL-10 levels were increased in HUS patients, but PBMC from these patients exhibited a lower capacity to secrete this cytokine compared with those from healthy children. Interestingly, there was a negative association between the circulating levels of IL-10 and inflammatory cytokine IL-8. We observed that circulating IL-10 levels were threefold higher in HUS patients with -1082G allele in comparison to AA genotype. Moreover, there was relative enrichment of GG/AG genotypes in HUS patients with severe kidney failure. Discussion: Our results suggest a possible contribution of SNP -1082 (A/G) to the severity of kidney failure in HUS patients that should be further evaluated in a larger cohort.

Nasal immunization with H7 flagellin protects mice against hemolytic uremic syndrome secondary to Escherichia coli O157:H7 gastrointestinal infection.

Introduction: Shiga-toxin (Stx) producing Escherichia coli (STEC) O157:H7 is the most frequent serotype associated with hemolytic uremic syndrome (HUS) after gastrointestinal infections. Protection against HUS secondary to STEC infections has been experimentally assayed through the generation of different vaccine formulations. With focus on patients, the strategies have been mainly oriented to inhibit production of Stx or its neutralization. However, few approaches have been intended to block gastrointestinal phase of this disease, which is considered the first step in the pathogenic cascade of HUS. The aim of this work was to assay H7 flagellin as a mucosal vaccine candidate to prevent the systemic complications secondary to E. coli O157:H7 infections. Materials and methods: The cellular and humoral immune response after H7 nasal immunization in mice were studied by the analysis of systemic and intestinal specific antibody production, as well as cytokine production and lymphocyte proliferation against H7 flagellin ex vivo. Results: Immunized mice developed a strong and specific anti-H7 IgG and IgA response, at systemic and mucosal level, as well as a cellular Th1/Th2/Th17 response. H7 induced activation of bone marrow derived dendritic cells in vitro and a significant delayed-type hypersensitivity (DTH) response in immunized mice. Most relevant, immunized mice were completely protected against the challenge with an E. coli O157:H7 virulent strain in vivo, and surviving mice presented high titres of anti-H7 and Stx antibodies. Discussion: These results suggest that immunization avoids HUS outcome and allows to elicit a specific immune response against other virulence factors.

Alendronate inhibits triglyceride accumulation and oxidative stress in adipocytes and the inflammatory response of macrophages which are associated with adipose tissue dysfunction.

Alendronate, a bisphosphonate used to prevent osteoporosis, stimulates osteogenesis but impairs adipogenesis. Different clinical trials suggest that the incidence of diabetes may be lower in patients treated with alendronate. Taking into account the importance of adipocytes and macrophages of adipose tissue in insulin resistance and type 2 diabetes, it is necessary to evaluate the effect of alendronate in both cell types. In this paper, we investigated the effect of alendronate on the differentiation to adipocytes of 3T3-L1 fibroblasts, the cell line most used to study adipogenesis, and also its effect on lipid content and oxidative stress in mature adipocytes as well as on the inflammatory response of macrophages. We found that alendronate inhibits differentiation of 3T3-L1 fibroblasts to adipocytes in keeping with reports in other cell lines. On the other hand, treatment of 3T3-L1 adipocytes with alendronate was able to decrease triglyceride content and to prevent H2O2-induced lipid peroxidation which was evaluated as an indicator of oxidative stress. In addition, it was found that activation of RAW 264.7 macrophages to a pro-inflammatory M1 type is inhibited by this bisphosphonate. These results suggest that alendronate may contribute to prevent adipocyte excessive enlargement and the induction of oxidative stress in 3T3-L1 adipocytes as well as the activation of macrophages to a pro-inflammatory M1 type, which are events associated with adipose tissue dysfunction and insulin resistance. In this study, we unraveled the underlying mechanisms of events that were previously observed in clinical trials.

Effect of lead on proliferation, oxidative stress and genotoxic damage of 3T3-L1 fibroblasts.

Lead (Pb) is an environmental and industrial contaminant that still represents a public health problem. In this paper, we investigated the effect of Pb on proliferation, lipid peroxidation and the number of micronucleated cells in exponentially growing 3T3-L1 fibroblasts, a cell line previously used to evaluate different environmental contaminants. We found that Pb (10 µM or higher) was able to inhibit proliferation of exponentially growing cells after 24-h treatment, which was evaluated by the MTT assay and cell counting in Neubauer chamber, but cell survival was not affected according to the trypan blue exclusion assay. On the other hand, Pb was able to increase lipid peroxidation and the number of micronucleated cells, which are indicative of oxidative stress and genotoxic damage respectively. We also found that removal of Pb after 24-h treatment allowed cells to recover proliferation. Our results indicate that Pb was able to induce oxidative stress and genotoxicity in this cell line under standardized conditions, which supports the involvement of Pb in similar effects observed in human exposed to this heavy metal. In addition, Pb inhibits proliferation of exponentially growing fibroblasts but cells resume proliferation after removal of this metal, which suggests that it is important to move away Pb-exposed individuals from the source of contamination.