In Vitro Effect of the Cell-Free Supernatant of the Lactobacillus casei Strain IMAU60214 against the Different Pathogenic Properties of Diarrheagenic Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) and enterohemorrhagic E. coli (EHEC) are E. coli pathotypes associated with unmanageable diarrhea in children and adults. An alternative to the treatment of infections caused by these microorganisms is the use of the bacteria of the Lactobacillus genus; however, the beneficial effects on the intestinal mucosa are specific to the strain and species. The interest of this study consisted of analyzing the coaggregation properties of Lactobacillus casei IMAU60214, as well as the effect of cell-free supernatant (CSF) on growth and anti-cytotoxic activity in a cell model of the human intestinal epithelium for an agar diffusion assay (HT-29) and the inhibition of biofilm formation on plates of DEC strains of the EAEC and EHEC pathotypes. The results showed that L. casei IMAU60214 exhibits time-dependent coaggregation (35-40%) against EAEC and EHEC that is similar to the control E. coli ATCC 25922. The CSF showed antimicrobial activity (20-80%) against EAEC and EHEC depending on the concentration. In addition, the formation and dispersion of biofilms of the same strains decrease, and the proteolytic pre-treatment with catalase and/or proteinase K (1 mg/mL) of CSF reduces the antimicrobial effect. When evaluating the effect in HT-29 cells pre-treated with CFS on the toxic activity induced by the EAEC and EHEC strains, a reduction of between 30 and 40% was observed. The results show that L. casei IMAU60214 and its CSF have properties that interfere with some properties associated with the virulence of the EAEC and EHEC strains that cause intestinal infection, which supports their use for the control and prevention of infections caused by these bacteria.

Probiotic Properties and Immunomodulatory Activity of Lactobacillus Strains Isolated from Dairy Products.

Lactobacilli species are an effective biotherapeutic alternative against bacterial infections and intestinal inflammatory disorders. However, it is important to evaluate their beneficial properties, before considering them as probiotics for medical use. In this study we evaluated some probiotic properties of Lactobacillus rhamnosus GG, Lactobacillus rhamnosus KLSD, Lactobacillus helveticus IMAU70129, and Lactobacillus casei IMAU60214 previously isolated from dairy products and as control Lactobacillus casei Shirota. Experimental evaluations revealed that all strains expressed hydrophobicity (25-40%), auto-aggregation (55-60%), NaCl tolerance (1-4%), adhesion to Caco-2 cells (25-33%), partial inhibition on adherence of Escherichia coli ATCC 35218, Salmonella Typhimurium ATCC 14028, and Staphylococcus aureus ATCC 23219. Cell-free supernatants (CFS) of Lactobacilli also inhibit growth of these pathogens. In immunomodulatory properties a reduction of interleukin-8 (IL-8) and nitric oxide (NO) release was observed in assays with Caco-2 cells stimulated with interleukin-1ß (1 ng/mL), or lipopolysaccharide (0.1 µg/mL). On the other hand, the damage induced to Caco-2 cells with sodium dodecyl sulfate (SDS) was attenuated when the cultured cells were pretreated with L. rhamnosus KLDS, L. helveticus IMAU70129 and L. casei IMAU60214. These Lactobacilli possess probiotic properties determined by both an antagonistic activity on pathogenic bacteria and reduction in the inflammatory response of cells treated with SDS, a pro-inflammatory stimulant.

Association of Genetic Polymorphisms and Serum Levels of IL-6 and IL-8 with the Prognosis in Children with Neuroblastoma.

There is evidence that high circulating levels of IL-6 and IL-8 are markers of a poor prognosis in various types of cancer, including NB. The participation of these cytokines in the tumor microenvironment has been described to promote progression and metastasis. Our objective was to evaluate the prognostic role of genetic polymorphisms and serum levels of IL-6 and IL-8 in a cohort of Mexican pediatric patients with NB. The detection of the SNPs rs1800795 IL-6 and rs4073 and rs2227306 IL-8 was carried out by PCR-RFLP and the levels of cytokines were determined by the ELISA method. We found elevated circulating levels of IL-8 and IL-6 in NB patients compared to the control group. The genotype frequencies of the rs1800795 IL-6 and rs4073 IL-8 variants were different between the patients with NB and the control group. Likewise, the survival analysis showed that the GG genotypes of rs1800795 IL-6 (p = 0.014) and AA genotypes of rs4073 IL-8 (p = 0.002), as well as high levels of IL-6 (p = 0.009) and IL-8 (p = 0.046), were associated with lower overall survival. We confirmed the impact on an adverse prognosis in a multivariate model. This study suggests that the SNPs rs1800795 IL-6 and rs4073 IL-8 and their serum levels could be promising biomarkers of a poor prognosis, associated with overall survival, metastasis, and a high risk in Mexican children with NB.

Role of Genetic Polymorphisms of Deoxycytidine Kinase and Cytidine Deaminase to Predict Risk of Death in Children with Acute Myeloid Leukemia.

Cytarabine is one of the most effective antineoplastic agents among those used for the treatment of acute myeloid leukemia. However, some patients develop resistance and/or severe side effects to the drug, which may interfere with the efficacy of the treatment. The polymorphisms of some Ara-C metabolizing enzymes seem to affect outcome and toxicity in AML patients receiving cytarabine. We conducted this study in a cohort of Mexican pediatric patients with AML to investigate whether the polymorphisms of the deoxycytidine kinase and cytidine deaminase enzymes are implicated in clinical response and toxicity. Bone marrow and/or peripheral blood samples obtained at diagnosis from 27 previously untreated pediatric patients with de novo AML were processed for genotyping and in vitro chemosensitivity assay, and we analyzed the impact of genotypes and in vitro sensitivity on disease outcome and toxicity. In the multivariate Cox regression analysis, we found that age at diagnosis, wild-type genotype of the CDA A79C polymorphism, and wild-type genotype of the dCK C360G polymorphism were the most significant prognostic factors for predicting the risk of death.

[Analysis of genetic polymorphisms of thiopurine S-methyltransferase (TPMT) in Mexican pediatric patients with cancer]. / Análisis de los polimorfismos génicos de Tiopurina S-Metiltransferasa (TPMT) en pacientes pediátricos mexicanos con cáncer.

BACKGROUND: Thiopurine S-Methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs, such as 6-mercaptopurine, 6-thioguanine and azathioprine, leading to their inactivation. Individuals who carry TPMT allele variants are more likely to experience life-threatening toxicity when these drugs are given at a standard dose. Wildtype phenotype TPMT*1 exhibits high level of catalytic activity, while all variants manifest with a decreased enzymatic activity. Ethnic-related differences in the distribution of TPMT variant alleles have been found. In Mexico, limited information is available; so far only two studies have been published and clear differences exist between them. MATERIAL AND METHODS: Allelic variants and genotypes of the TPMT gene were determined in 240 Mexican children with leukemia and solid tumors using DNA extracted from peripheral blood. Polymorphisms G460A and A719G were identified by PCR-RFLP and G238C by the specific-allele PCR assay. The enzyme variants were detected by allelic discrimination. RESULTS: Homozygous wild-type genotype TPMT*1/TPMT*1 was found in 173 patients (72.1%); 67 cases (27.9%) were heterozygous: 18 with genotype TPMT*1/TPMT*3B (7.5%), 17 TPMT*1/TPMT*3C (7.1%), 16 TPMT*1/TMPT*2 (6.7%), 14 TPMT*1/TPMT*3A (5.8%), and 2 (0.8%) were homozygous for two variants: TPMT*2/ TPMT*3B in both. The allele frequencies were TPMT*1 in 411 (85.62%), TPMT*3B in 20 (4.1%), TPMT*2 in 18 (3.75%), TPMT*3C in 17 (3.55%) and TPMT*3A in 14 (2.9%). CONCLUSIONS: A high frequency and diversity of variant TPMT genotypes was found in this series with predominance of the TPMT*3B allele.