Immobilization of primary cultures of insulin-releasing human pancreatic cells

Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type 1 diabetes, however, it is severely limited by the shortage of organ donors. Ex vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 diabetes. It has recently been shown that, even in the absence of fibrotic over-growth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells. Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing. Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.

Dose-dependent effect of Resveratrol on bladder cancer cells: Chemoprevention and oxidative stress

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of Th1/Th2 cytokines and nitric oxide in murine systemic infection induced by Sporothrix schenckii

In an attempt to elucidate the effects of Sporothrix schenckii infection on the immune response, our laboratory has developed a murine model of disseminated sporotrichosis. Helper T cells can be further subdivided into Th1 and Th2 phenotypes. The differentiation of two subsets of T lymphocytes is driven by IL-12 and IL-4 cytokines, respectively. Th1 cells produce IFN-gamma that activate macrophages and promote cell-mediated immunity. In addition, we found low levels of iNOS and NO production in the initial (1st and 2nd weeks) and final (9th and 10th weeks) periods of the infection, in contrast with the period of week 4 to 7 of elevated values. The determination of IFN-gamma and IL-12 are in agreement with NO/iNOS detection, showing the presence of cellular immune response throughout the infectious process. However, the production of IL-4 shows an increase in levels after the 5th and 6th weeks suggesting a participation of Th2 response in this period as well. Regarding these results, the study demonstrated that in experimental sporotrichosis infection the cellular immune response participated throughout the period analyzed as a nitric oxide dependent mechanism. In contrast, the presence of Th2 response began in the 5th week, suggesting the participation of humoral immune response in advanced stages of sporotrichosis.

Low protein diet confers resistance to the inhibitory effects of interleukin1 beta on insulin secretion in pancreatic islets

High protein content in the diet during childhood and adolescence has been associated to the onset insulin-dependent diabetes mellitus. We investigated the effect of interleukin-1 beta (IL-I beta) on insulin secretion, glucose metabolism, and nitrite formation by islets isolated from rats fed with normal protein (NP, 17%) or low protein (LP, 6%) after weaning. Pretreatment of islets with IL-1 beta for 1 h or 34 h inhibited the insulin secretion induced by glucose in both groups, but it was less marked in LP than in NP group. Islets from LP rats exhibited a decreased IL-1 beta -induced nitric oxide (NO) production, lower inhibition of D-[(UC)-C-14]-glucose oxidation to (CO2)-C-14, and less pronounced effect of IL-1 beta on alpha -ketoisocaproic acid-induced insulin secretion than NP islets. However, when the islets were stimulated by high concentrations of K+ the inhibitory effect of IL-1 beta on insulin secretion was not different between groups. In conclusion, protein restriction protects beta -cells of the deleterious effect of IL-1 beta, apparently, by decreasing NO production. The lower NO generation in islets from protein deprived rats may be due to increased free fatty acids oxidation and consequent alteration in Ca2+ homeostasis. (C) 2001 Elsevier B.V. All rights reserved.

Insulin and glucose sensitivity, insulin secretion and beta-cell distribution in endocrine pancreas of the fruit bat Artibeus lituratus

The fruit bat Artibeus lituratus absorbs large amounts of glucose in short periods of time and maintains normoglycemia even after a prolonged starvation period. Based on these data, we aimed to investigate various aspects related with glucose homeostasis analyzing: blood glucose and insulin levels, intraperitoneal glucose and insulin tolerance tests (ipGTT and ipITT), glucose-stimulated insulin secretion (2.8, 5.6 or 8.3 mmol/L glucose) in pancreas fragments, cellular distribution of beta cells, and the amount of pAkt/Akt in the pectoral muscle and liver. Blood glucose levels were higher in fed bats (6.88 +/- 0.5 mmol/L) than fasted bats (4.0 +/- 0.8 mmol/L), whereas insulin levels were similar in both conditions. The values of the area-under-the curve obtained from ipGTT were significantly higher when bats received 2 (5.5-fold) or 3 g/kg glucose (7.5-fold) b.w compared to control (saline). These bats also exhibited a significant decrease of blood glucose values after insulin administration during the iplTT. Insulin secretion from fragments of pancreas under physiological concentrations of glucose (5.6 or 8.3 mmol/L) was similar but higher than in 2.8 mmol/L glucose 1.8- and 2.0-fold, respectively. These bats showed a marked beta-cell distribution along the pancreas, and the pancreatic beta cells are not exclusively located at the central part of the islet. The insulin-induced Akt phosphorylation was more pronounced in the pectoral muscle, compared to liver. The high sensitivity to glucose and insulin, the proper insulin response to glucose, and the presence of an apparent large beta-cell population could represent benefits for the management of high influx of glucose from a carbohydrate-rich meal, which permits appropriate glucose utilization. (C) 2010 Elsevier B.V. All rights reserved.

Análise imuno-histoquímica de proteínas relacionadas às respostas Th1, Th2 e Th17 na doença periodontal

Periodontal disease is an inflammatory condition of infectious nature characterized by destruction of protecting and supporting dental tissues. It happens as a response produced by the host when attacked by microorganisms. Several factors are involved in this process. Among them, cytokines are key regulatory molecules in this immune response, playing a role either protective and/or destructive in lesion progression. Thus, this study investigated the immunohistochemical expression of IFN- , GATA-3, IL-17, IL-23, IL-6 and TGF- in gingival tissues of humans, in an attempt to gain a better understanding of the participation of Th1, Th2 and Th17 immune responses in the development of periodontal disease processes. To this end, eighty-two samples of gingival tissues were divided into three groups: Group 1 = 15 (samples of healthy gum tissue as controls), Group 2 = 36 (samples with chronic gingivitis) and Group 3 = 31 (samples with chronic periodontitis). All cases were submitted to morphological analysis from sections stained with hematoxylin and eosin and then subjected to staining by immunohistochemistry using the streptavidin-biotin method. Results showed positive labeling for all proteins. Nonetheless, we observed a greater expression of Th1 cytokines and Th17 cells in group 3. We found statistically significant difference between TGF- expression and the clinical condition of the samples (p=0.02). We conclude that Th1 and Th17 responses may act synergistically in the destructive process of periodontal tissue, overlapping the Th2 response that was also present in these tissues

Ovary peltate trichomes of Zeyheria montana (Bignoniaceae): Developmental ultrastructure and secretion in relation to function

center dot Background and Aims Nectar production in the Bignoniaceae species lacking a nectariferous functional disc is ascribed to trichomatic glands around the ovary base and/or on the inner corolla wall. Nevertheless, knowledge about the secretion and function of these glands is very incomplete. The purpose of this paper is to study, from a developmental viewpoint, the ultrastructure, histochemistry and secretory process of the peltate trichomes on the ovary of Zeyheria montana, a species in the Bignoniaceae which has a rudimentary disc.center dot Methods Samples of the gynoecium at various developmental stages were fixed and processed for light and electron microscopy. Histochemistry and cytochemistry tests were performed to examine the chemical composition of exudates. Thin layer chromatography was used to determine the presence of alkaloids and terpenes in gynoecium and fruit extracts, and in fresh nectar stored in the nectar chamber.center dot Key Results Peltate trichomes at different developmental stages appear side by side from floral budding up to pre-dispersal fruit. Large plastids with an extensive internal membrane system consisting of tubules filled with lipophilic material, abundant smooth endoplasmic reticulum, few Golgi bodies, lipophilic deposits in the smooth endoplasmic reticulum and mitochondria, and scattered cytoplasmic oil droplets are the main characteristics of mature head cells. The secretion which accumulates in the subcuticular space stains positively for hydrophilic and lipophilic substances, with lipids prevailing for fully peltate trichomes. Histochemistry and thin layer chromatography detected terpenes and alkaloids. Fehling's test to detect of sugars in the secretion was negative.center dot Conclusions the continuous presence and activity of peltate trichomes on the ovary of Z. montana from early budding through to flowering and fruiting set, and its main chemical components, alkaloids and terpenes, suggest that they serve a protective function and are not related to the floral nectar source or to improving nectar quality.

Dendroid colleters on vegetative and reproductive apices in Alibertia sessilis (Rubiaceae) differ in ultrastructure and secretion

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

OIL GLANDS IN PTERODON PUBESCENS BENTH. (LEGUMINOSAE-PAPILIONOIDEAE): DISTRIBUTION, STRUCTURE, and SECRETION MECHANISMS

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Regulation of angiotensin type 2 receptor in bovine granulosa cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

FGF10 inhibits dominant follicle growth and estradiol secretion in vivo in cattle

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Propolis effect on Th1/Th2 cytokines production by acutely stressed mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Th1/Th2 cytokine production by clove-treated mice

Although clove possesses several biological and therapeutic properties, its immunomodulatory action has not been fully investigated. The goal of this work was to investigate the effect of administration of the water extract of clove over a short-term to BALB/c mice on Th1 (IFN-gamma and IL-2) and Th2 (IL-4 and IL-10) cytokine production. After treatment, spleen cells were aseptically removed and cells were stimulated with concanavalin A. Supernatants of cell cultures were used for cytokine determination by ELISA. The chemical composition of the extract was also carried out, revealing that eugenol(4-allyl-2-methoxyphenol) was the major component in our sample. Although the anti-inflammatory action of clove has been mentioned, our data showed that clove administration to mice did not influence the Th1/Th2 cytokine balance. Further studies dealing with cytokine expression and production will provide a better understanding of clove's immunomodulatory and anti-inflammatory actions, using different extract concentrations and different intake periods.

The Effect of Propolis on Th1/Th2 Cytokine Expression and Production by Melanoma-bearing Mice Submitted to Stress

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Prevention of experimental diabetes by Uncaria tomentosa extract: Th2 polarization, regulatory T cell preservation or both?

Ethnopharmacological relevance: Uncaria tomentosa (Willd.) DC (Rubiaceae) is a species native to the Amazon rainforest and surrounding tropical areas that is endowed with immunomodulatory properties and widely used around the world. In this study we investigated the immunomodulatory potential of Uncaria tomentosa (UT) aqueous-ethanol extract on the progression of immune-mediated diabetes.Materials and methods: C57BL/6 male mice were injected with MLDS (40 mg/kg) and orally treated with UT at 10-400 mg/kg during 21 days. Control groups received MLDS alone or the respective dilution vehicle. Pancreatic mononuclear infiltrate and beta-cell insulin content were analyzed by HE and immunohistochemical staining, respectively, and measured by digital morphometry. Lymphocyte immunophenotyping and cytokine production were determined by flow cytometry analysis.Results: Treating the animals with 50-400 mg/kg of UT caused a significant reduction in the glycemic levels, as well as in the incidence of diabetes. The morphometric analysis of insulitis revealed a clear protective effect. Animals treated with UT at 400 mg/kg presented a higher number of intact islets and a significant inhibition of destructive insulitis. Furthermore, a significant protection against the loss of insulin-secreting presented beta-cells was achieved, as observed by a careful immunohistochemical evaluation. The phenotypic analysis indicated that the groups treated with higher doses (100-400 mg/kg) presented CD4(+) and CD8(+) T-cell values similar to those observed in healthy animals. These same higher doses also increased the number of CD4(+)CD25(+)Foxp3(+) regulatory T-cells. Moreover, the extract modulated the production of Th1 and Th2, with increased levels of IL-4 and IL-5.Conclusions: The extract was effective to prevent the progression of immune-mediated diabetes by distinct pathways. (C) 2011 Elsevier B.V. All rights reserved.