Toll-like receptor 4 (TLR4) expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis

Background: Toll-like receptor 4 (TLR4) is widely recognized as an essential element in the triggering of innate immunity, binding pathogen-associated molecules such as Lipopolysaccharide (LPS), and in initiating a cascade of pro-inflammatory events. Evidence for TLR4 expression in non-immune cells, including pancreatic beta-cells, has been shown, but, the functional role of TLR4 in the physiology of human pancreatic beta-cells is still to be clearly established. We investigated whether TLR4 is present in beta-cells purified from freshly isolated human islets and confirmed the results using MIN6 mouse insulinoma cells, by analyzing the effects of TLR4 expression on cell viability and insulin homeostasis. Results: CD11b positive macrophages were practically absent from isolated human islets obtained from nondiabetic brain-dead donors, and TLR4 mRNA and cell surface expression were restricted to beta-cells. A significant loss of cell viability was observed in these beta-cells indicating a possible relationship with TLR4 expression. Monitoring gene expression in beta-cells exposed for 48h to the prototypical TLR4 ligand LPS showed a concentration-dependent increase in TLR4 and CD14 transcripts and decreased insulin content and secretion. TLR4-positive MIN6 cells were also LPS-responsive, increasing TLR4 and CD14 mRNA levels and decreasing cell viability and insulin content. Conclusions: Taken together, our data indicate a novel function for TLR4 as a molecule capable of altering homeostasis of pancreatic beta-cells.

Leptin and the Immune Response An Active Player or an Innocent Bystander?

Leptin is involved in the control of energy storage by the body. Low serum leptin levels, as seen in starvation, are associated with impaired inflammatory T cell responses that can be reversed by exogenous leptin. Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia and recurrent infections. Several defects in T cell function have also been described, and allergy, autoimmune disease, and lymphomas or other malignancies can be present. Previous studies in Brazilian CVID patients have shown that, in contrast with mononuclear cells from healthy controls, CVID cells cultured with phytohemagglutinin and added leptin increased the proliferative response and decreased activation-induced apoptosis. Interleukin (IL)-2 and especially IL-4 production also increased significantly, although the effects of exposure to leptin were not observed uniformly in CVID patients. The majority, however, responded in some degree, and some exhibited completely restored values of the four parameters. These remarkable results indicate leptin could be used to improve immune function in these patients. On the other hand, we found no specific correlation between serum leptin levels and the number of infectious events over a 24-month period, presence of autoimmunity, allergies, or cancer in these patients. The results suggest that the absolute value of serum leptin does not determine the clinical behavior of patients or responses to leptin in vitro. Of note is the divergence between serum leptin, response to leptin in vitro, and the presence of autoimmunity, indicating the need to identify the cellular and molecular players involved in the regulation of the immune response by leptin in CVID.

Prevalência de sobrepeso e obesidade e fatores associados em adolescentes na região centro-oeste do estado de São Paulo (SP, Brasil)

Objetivou-se avaliar a relação entre o Índice de Massa Corporal (IMC) e o índice CPOD em 207 adolescentes de 12 anos, de 8 escolas públicas e particulares da região centro-oeste do estado de São Paulo. A amostra foi constituída por 380 adolescentes aos 12 anos, de ambos os gêneros, sendo examinados 207. Utilizou-se o índice CPOD, IMC para peso, medida de estatura, e aplicou-se questionário sobre hábitos alimentares, características antropométricas e atividade física. Quanto ao peso corpóreo, 55,93% apresentaram normal (G4), 35,59% de baixo peso (G3), 8,47% de pré-obesos (G2), nas escolas particulares. Nas públicas, 52,03% apresentaram normal, baixo peso 41,22%, pré-obesos 4,73% e obesos (G1) 2,03%; não houve diferença significativa (p=0,45). Verificou-se que o CPOD nas escolas públicas foi 2,16 e nas particulares, 0,23 (p<0,05), sendo que 39,2% das crianças estavam livres de cárie nas municipais e nas particulares, 88,1%. Não houve correlação do maior IMC com o incremento de CPOD. Houve correlação negativa entre condições socioeconômicas e índice de cárie dentária. Concluiu-se que os grupos pré-obesos e obesos, embora houvesse maior frequência de ingestão de alimentos, não apresentaram correlação com o incremento de cárie dentária, mas as condições socioeconômicas foram determinantes para essa ocorrência.

Nd:YAG laser irradiation effect on apical intracanal dentin - a microleakage and SEM evaluation

The purpose of this in vitro study was to evaluate the effect of neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation on intracanal dentin surface by SEM analysis and its interference in the apical seal of filled canals. After endodontic treatment procedures, 34 maxillary human incisors were randomly assigned to 2 groups. In the negative control group (n=17), no additional treatment was performed and teeth were filled with vertically condensed gutta-percha; in the laser-treated group (n=17), the root canals were irradiated with Nd:YAG laser (1.5 W, 100 mJ, 15 Hz) before filling as described for the control group. Two specimens of each group were prepared for SEM analysis to evaluate the presence and extent of morphological changes and removal of debris; the other specimens were immersed in 0.5% methylene blue dye (pH 7.2) for 24 h for evaluation of the linear dye leakage at the apical third. SEM analysis of the laser-treated group showed dentin fusion and resolidification without smear layer or debris. The Student’s t-test showed that the laser-treated group had significantly less leakage in apical third than the control group. Within the limitations of this study, it may be concluded that the morphological changes on the apical intraradicular dentin surface caused by Nd:YAG laser resulted in less linear dye apical leakage.

Protocols for obtainment and isolation of two mesenchymal stem cell sources in sheep

PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.

Avaliação de metais traços e íons majoritários em águas de chuva na cidade de São Paulo

Rainfall samples collected in the downtown area of São Paulo city, during 2003, exhibited average concentrations of cadmium, lead and copper of 1.33, 8.52 and 49.5 nmol L-1, respectively. Among the major ions, NH4+ was the predominant species followed by NO3-, SO4(2-) and Ca2+, with volume weighed mean (VWM) concentrations of 37.1, 20.1, 11.9 and 10.8 µmol L-1, respectively. All the determined species showed high inter-events variability, including free H+ ions whose VWM concentration was 4.03 µmol L-1, corresponding to a pH value of 5.39.

A hybrid heuristic for the multi-plant capacitated lot sizing problem with setup carry-over

This paper addresses the capacitated lot sizing problem (CLSP) with a single stage composed of multiple plants, items and periods with setup carry-over among the periods. The CLSP is well studied and many heuristics have been proposed to solve it. Nevertheless, few researches explored the multi-plant capacitated lot sizing problem (MPCLSP), which means that few solution methods were proposed to solve it. Furthermore, to our knowledge, no study of the MPCLSP with setup carry-over was found in the literature. This paper presents a mathematical model and a GRASP (Greedy Randomized Adaptive Search Procedure) with path relinking to the MPCLSP with setup carry-over. This solution method is an extension and adaptation of a previously adopted methodology without the setup carry-over. Computational tests showed that the improvement of the setup carry-over is significant in terms of the solution value with a low increase in computational time.

Adaptação transcultural e consistência interna do Early Trauma Inventory (ETI)

As experiências traumáticas precoces são um fator de risco preditivo de problemas psicopatológicos futuros. O Early Trauma Inventory (ETI) é um instrumento que avalia em indivíduos adultos experiências traumáticas ocorridas antes dos 18 anos de idade. Tal instrumento foi traduzido, transculturalmente adaptado e sua consistência interna foi avaliada. Vítimas de violência que preencheram os critérios de inclusão e exclusão foram submetidas a uma entrevista diagnóstica (SCID-I) e ao ETI. Foram incluídos 91 pacientes com o transtorno do estresse pós-traumático (TEPT). O alfa de Cronbach nos diferentes domínios variou de 0,595-0,793, e o escore total foi de 0,878. A maior parte dos itens nos vários domínios, com exceção do abuso emocional, apresentou índices de correlação interitem entre 0,51-0,99. A versão adaptada foi útil tanto na clínica quanto na pesquisa. Apresentou boa consistência interna e na correlação interitem. O ETI é um instrumento válido, com boa consistência para se avaliar a presença de história de traumas precoces em indivíduos adultos.

Triagem sorológica e influência do conhecimento sobre a dengue em pacientes do ambulatório de especialidades do SUS

INTRODUÇÃO: A dengue é causa de preocupação em países como o Brasil. OBJETIVO: Verificar a soropositividade dos pacientes do ambulatório de especialidades do Sistema Único de Saúde (SUS) para anticorpos antidengue, relacionando os resultados com os dados sociodemográficos. METODOLOGIA: Foram respondidos 184 questionários de avaliação sociodemográfica e de conhecimento sobre a transmissão da dengue. Foi utilizado o método de imunoensaio enzimático (ELISA) para pesquisar imunoglobulina da classe M (IgM) e da classe G (IgG) contra os vírus. RESULTADOS: Quinze por cento dos pacientes apresentaram IgG contra o vírus, sem a presença de IgM. CONCLUSÃO: Os pacientes demonstraram conhecimento sobre a doença e sua prevenção, independentemente da classe econômica. A infecção assintomática deve ser avaliada, principalmente nos casos de doença febril.

Effects of ascorbic acid supplementation in ileum myenteric neurons of streptozotocin-induced diabetic rats

The exacerbation of the oxidative stress and of the polyol pathway which impair damage myenteric plexus are metabolic characteristics of diabetes. The ascorbic acid (AA) is an antioxidant and an aldose reductase inhibitor, which may act as neuroprotector. The effects of AA supplementation on the density and cellular body profile area (CP) of myenteric neurons in STZ-induced diabetes in rats were assessed. Four groups with five animals each were formed: normoglycemic (C); diabetic (D); AA-treated diabetic (DS) and AA-treated normoglycemic (CS). Dosagen of 50mg of AA were given, three times a week, for each animal (group DS and CS). Ninety days later and after euthanasia, the ileum was collected and processed for the NADPH-diaphorase technique. There were no differences (P>0.05) in the neuronal density among the groups. The CP area was lower (P<0.05) in the DS and CS groups, with a higher incidence of neurons with a CP area exceeding 200µm² for groups C and D. The AA had no influence on the neuronal density in the ileum but had a neuroprotective effect, preventing the increase in the CP area and allowing a higher number of neurons with a CP area with less than 200µm².

Performance of transport and selective media for swine Bordetella bronchiseptica recovery and it comparison to polymerase chain reaction detection

Three comparative assays were performed seeking to improve the sensitivity of the diagnosis of Bordetella bronchiseptica infection analyzing swine nasal swabs. An initial assay compared the recovery of B. bronchiseptica from swabs simultaneously inoculated with B. bronchiseptica and some interfering bacteria, immersed into three transport formulations (Amies with charcoal, trypticase soy broth and phosphate buffer according to Soerensen supplemented with 5% of bovine fetal serum) and submitted to different temperatures (10ºC and 27ºC) and periods of incubation (24, 72 and 120 hours). A subsequent assay compared three selective media (MacConkey agar, modified selective medium G20G and a ceftiofur medium) for their recovery capabilities from clinical specimens. One last assay compared the polymerase chain reaction to the three selective media. In the first assay, the recovery of B. bronchiseptica from transport systems was better at 27ºC and the three formulations had good performances at this temperature, but the collection of qualitative and quantitative analysis indicated the advantage of Amies medium for nasal swabs transportation. The second assay indicated that MacConkey agar and modified G20G had similar results and were superior to the ceftiofur medium. In the final assay, polymerase chain reaction presented superior capability of B. bronchiseptica detection to culture procedures.

Prevalence, Incidence Density, and Genotype Distribution of GB Virus C Infection in a Cohort of Recently HIV-1-Infected Subjects in Sao Paulo, Brazil

Background: The results of previous studies elsewhere have indicated that GB virus C (GBV-C) infection is frequent in patients infected with the human immunodeficiency virus type 1 (HIV-1) due to similar transmission routes of both viruses. The aim of this study was to determine the prevalence, incidence density and genotypic characteristics of GBV-C in this population. Methodology/Principal Findings: The study population included 233 patients from a cohort primarily comprised of homosexual men recently infected with HIV-1 in Sao Paulo, Brazil. The presence of GBV-C RNA was determined in plasma samples by reverse transcriptase-nested polymerase chain reaction and quantified by real-time PCR. GBV-C genotypes were determined by direct sequencing. HIV viral load, CD4+ T lymphocyte and CD8+ T lymphocyte count were also tested in all patients. The overall prevalence of GBV-C infection was 0.23 (95% CI: 0.18 to 0.29) in the study group. There was no significant difference between patients with and without GBV-C infection and Glycoprotein E2 antibody presence regarding age, sex, HIV-1 viral load, CD4+ and CD8+ T cell counts and treatment with antiretroviral drugs. An inverse correlation was observed between GBV-C and HIV-1 loads at enrollment and after one year. Also, a positive but not significant correlation was observed between GBV-C load and CD4+ T lymphocyte. Phylogenetic analysis of the GBV-C isolates revealed the presence of genotype 1 and genotype 2, these sub classified into subtype 2a and 2b. Conclusion/Significance: GBV-C infection is common in recently HIV -1 infected patients in Sao Paulo, Brazil and the predominant genotype is 2b. This study provides the first report of the GBV-C prevalence at the time of diagnosis of HIV-1 and the incidence density of GBV-C infection in one year.

Frequency and genotypic distribution of GB virus C (GBV-C) among Colombian population with Hepatitis B (HBV) or Hepatitis C (HCV) infection

Background: GB virus C (GBV-C) is an enveloped positive-sense ssRNA virus belonging to the Flaviviridae family. Studies on the genetic variability of the GBV-C reveals the existence of six genotypes: genotype 1 predominates in West Africa, genotype 2 in Europe and America, genotype 3 in Asia, genotype 4 in Southwest Asia, genotype 5 in South Africa and genotype 6 in Indonesia. The aim of this study was to determine the frequency and genotypic distribution of GBV-C in the Colombian population. Methods: Two groups were analyzed: i) 408 Colombian blood donors infected with HCV (n = 250) and HBV (n = 158) from Bogota and ii) 99 indigenous people with HBV infection from Leticia, Amazonas. A fragment of 344 bp from the 5' untranslated region (5' UTR) was amplified by nested RT PCR. Viral sequences were genotyped by phylogenetic analysis using reference sequences from each genotype obtained from GenBank (n = 160). Bayesian phylogenetic analyses were conducted using Markov chain Monte Carlo (MCMC) approach to obtain the MCC tree using BEAST v. 1.5.3. Results: Among blood donors, from 158 HBsAg positive samples, eight 5.06% (n = 8) were positive for GBV-C and from 250 anti-HCV positive samples, 3.2%(n = 8) were positive for GBV-C. Also, 7.7% (n = 7) GBV-C positive samples were found among indigenous people from Leticia. A phylogenetic analysis revealed the presence of the following GBV-C genotypes among blood donors: 2a (41.6%), 1 (33.3%), 3 (16.6%) and 2b (8.3%). All genotype 1 sequences were found in co-infection with HBV and 4/5 sequences genotype 2a were found in co-infection with HCV. All sequences from indigenous people from Leticia were classified as genotype 3. The presence of GBV-C infection was not correlated with the sex (p = 0.43), age (p = 0.38) or origin (p = 0.17). Conclusions: It was found a high frequency of GBV-C genotype 1 and 2 in blood donors. The presence of genotype 3 in indigenous population was previously reported from Santa Marta region in Colombia and in native people from Venezuela and Bolivia. This fact may be correlated to the ancient movements of Asian people to South America a long time ago.