Biotech Application of Exopolysaccharides from Curvularia brachyspora: Optimization of Production, Structural Characterization, and Biological Activity.

C. brachyspora, a widespread dematiaceous fungus, was evaluated in this study to optimize the production of exopolysaccharides (CB-EPS). Optimization was performed using response surface methodology, and the best production yielded 75.05% of total sugar at pH 7.4, with 0.1% urea, after 197 h. The obtained CB-EPS showed typical signals of polysaccharides, which was confirmed by FT-IR and NMR. The HPSEC analysis indicated a polydisperse polymer, showing a non-uniform peak, with an average molar mass (Mw) of 24,470 g/mol. The major monosaccharide was glucose (63.9 Mol%), followed by mannose (19.7 Mol%), and galactose (16.4 Mol%). Methylation analysis encountered derivatives that indicated the presence of a ß-d-glucan and a highly branched glucogalactomannan. CB-EPS was tested on murine macrophages to verify its immunoactivity, and the treated cells were able to produce TNF-α, IL-6, and IL-10. However, the cells did not produce superoxide anions or nitric oxide nor stimulated phagocytosis. The results demonstrated an indirect antimicrobial activity of macrophages by stimulating cytokines, showing another biotech applicability for the exopolysaccharides produced by C. brachyspora.

XPR1 Mediates the Pancreatic ß-Cell Phosphate Flush.

Glucose-stimulated insulin secretion is the hallmark of the pancreatic ß-cell, a critical player in the regulation of blood glucose concentration. In 1974, the remarkable observation was made that an efflux of intracellular inorganic phosphate (Pi) accompanied the events of stimulated insulin secretion. The mechanism behind this "phosphate flush," its association with insulin secretion, and its regulation have since then remained a mystery. We recapitulated the phosphate flush in the MIN6m9 ß-cell line and pseudoislets. We demonstrated that knockdown of XPR1, a phosphate transporter present in MIN6m9 cells and pancreatic islets, prevented this flush. Concomitantly, XPR1 silencing led to intracellular Pi accumulation and a potential impact on Ca2+ signaling. XPR1 knockdown slightly blunted first-phase glucose-stimulated insulin secretion in MIN6m9 cells, but had no significant impact on pseudoislet secretion. In keeping with other cell types, basal Pi efflux was stimulated by inositol pyrophosphates, and basal intracellular Pi accumulated following knockdown of inositol hexakisphosphate kinases. However, the glucose-driven phosphate flush occurred despite inositol pyrophosphate depletion. Finally, while it is unlikely that XPR1 directly affects exocytosis, it may protect Ca2+ signaling. Thus, we have revealed XPR1 as the missing mediator of the phosphate flush, shedding light on a 45-year-old mystery.

Insulin Modulates Inflammatory Cytokine Release in Acute Stages and Augments Expression of Adhesion Molecules and Leukocytes in Lungs on Chronic Stages of Paracoccidioidomycosis.

Type 1 diabetesmellitus (T1D) is caused by partial destruction of the insulin-producing beta cells in the pancreas and is a major issue for public health care worldwide. Reduced or impaired immunological responses, which render patients more susceptible to infections, have been observed in T1D, and this dysfunction is often related to a lack of insulin in the blood. Paracoccidioidomycosis is an important systemic mycosis endemic in Latin America. To evaluate the effects of T1D on this fungal infection and the modulatory effects of insulin, we induced diabetes in C57Bl/6 male mice (alloxan, 60 mg/kg), infected the mice (Pb18, 1 x 106 cells), and treated the mice with neutral protamine Hagedorn (NPH) insulin (2 IU/600 mg/dL blood glucose). Twenty-four hours after infection, infected diabetic mice showed reduced secretion of interferon (IFN)-γ and interleukine (IL)-12 p70 compared to infected nondiabetic controls. On the 45th day of infection, infected diabetic mice presented higher IFN-γ levels, a higher tumor necrosis factor (TNF)-α:IL-10 ratio, and lower adhesion molecule expression levels than nondiabetic mice. In the in vitro experiments, alveolar macrophages from diabetic animals showed reduced phagocytic activity compared to those from control animals at 4, 12, and 24 h. In infected diabetic mice, treatment with insulin restored IL-12 p70 levels at 24 h of infection, reduced IFN-γ levels and the TNF-α:IL-10 ratio at 45 days, and restored vascular cell adhesion molecule (VCAM)-1 expression in pulmonary blood vessels, and this treatment reduced the diminished phosphorylation of extracellular signal-regulated kinases (ERK) and increased nuclear factor-kappa-B(iκb)-α and jun amino-terminal kinases (JNK) p46 levels in infected nondiabetic mice. In addition, insulin promoted increased phagocytic activity in the alveolar macrophages of diabetic mice. These data suggest that T1D mice are more susceptible to Pb18 infection and that insulin modulates this inflammation in diabetic mice by augmenting the expression of adhesion molecules and leukocytes in the lungs and by reducing chronic inflammation.

High Glucose Environments Interfere with Bone Marrow-Derived Macrophage Inflammatory Mediator Release, the TLR4 Pathway and Glucose Metabolism.

Macrophages may be a crucial aspect of diabetic complications associated with the inflammatory response. In this study, we examined how hyperglycaemia, a common aspect of diabetes, modulates bone marrow-derived macrophages (BMDMs) under an inflammatory stimulus. To perform this study, BMDMs from non-diabetic and diabetic (60 mg/kg alloxan, i.v.) male C57BL/6 mice (CEUA/FCF/USP-488) were cultured under normal (5.5 mM) and high glucose (HG, 25 or 40 mM) conditions and stimulated or not stimulated with lipopolysaccharide (LPS, 100 ng/mL). Compared to the BMDMs from the normoglycaemic mice, the LPS-stimulated BMDMs from the diabetic mice presented reduced TLR4 expression on the cell surface, lower phagocytic capacity, and reduced secretion of NO and lactate but greater oxygen consumption and greater phosphorylation of p46 SAPK/JNK, p42 ERK MAPK, pAKT and pPKC-δ. When the BMDMs from the non-diabetic mice were cultured under high-glucose conditions and stimulated with LPS, TLR4 expression was reduced on the cell surface and NO and H2O2 levels were reduced. In contrast, the diabetic BMDMs cultured under high glucose conditions presented increased levels of lactate and reduced phosphorylation of AKT, PKC-δ and p46 SAPK/JNK but enhanced phosphorylation of the p46 subunit of SAPK/JNK after LPS stimulation. High glucose levels appear to modify macrophage behaviour, affecting different aspects of diabetic and healthy BMDMs under the same LPS stimulus. Thus, hyperglycaemia leaves a glucose legacy, altering the basal steady state of macrophages.

Insulin Modulates Paracoccidioides brasiliensis-Induced Inflammation by Restoring the Populations of NK Cells, Dendritic Cells, and B Lymphocytes in Lungs.

Paracoccidioidomycosis, a key issue for Brazilian health service, can be aggravated in patients with impaired immunological responses, such as diabetic patients. We evaluated the role of insulin in inflammatory parameters in diabetic and nondiabetic mice using a systemic mycosis Paracoccidioides brasiliensis (Pb) model. Diabetic C57BL-6 mice and controls were infected with Pb18 and treated with insulin for 12 days prior to experiments. After 55 days, infected diabetic mice exhibited fewer leukocytes in both peritoneal lavage fluid (PeLF) and bronchoalveolar lavage fluid and reduced secretion of interleukin- (IL-) 6 in lungs. In addition, diabetic mice presented a reduced influx of TCD4+ cells, TCD8+ cells, B lymphocytes, NK cells, and dendritic cells compared to control infected groups. Insulin treatment restored the leukocyte number in PeLF and restored the presence of B lymphocytes, dendritic cells, and NK cells in lungs of diabetic animals. The data suggest that diabetic mice present impaired immunological response to Pb18 infection and insulin modulates inflammation by reducing IL-6 levels in lung and CINC-1 levels in spleen and liver homogenates, restoring leukocyte concentrations in PeLF and also restoring populations of dendritic cells and B lymphocytes in lungs of diabetic mice, permitting the host to better control the infection.

A insulina regula as vias de sinalização induzidas por LPS em macrófagos derivados de medula óssea / Insulin regulates LPS-induced signaling pathways in macrophages derived from bone marrow

Pacientes diabéticos apresentam alterações no sistema imunológico que promovem, em parte, maior suscetibilidade de infecções bacterianas. O tratamento com insulina melhora a sobrevida e reduz o número de infecções recidivas no paciente com diabetes mellitus do tipo 1 (DM1). Pouco se sabe sobre os efeitos do diabetes e a ação da insulina nos macrófagos. Neste trabalho, investigamos a proteína fosfatidilinositol-3-quinase (PI3K), proteína quinase B (Akt) e as quinases ativadas por mitógenos (MAPK) em macrófagos derivados de medula óssea (BMDM) e sua participação no estímulo por lipopolissacarídeo (LPS) na presença ou não do tratamento com insulina através da secreção dos mediadores inflamatórios fator de necrose tumoral (TNF)-α, interleucina (IL)-6 e IL-10. Observamos que os BMDM de animais com DM1 apresentam aumento da expressão da subunidade catalítica PI3K p110alpha com redução na subunidade reguladora PI3K p55 e maior expressão da fosforilação das proteínas Akt (Serina-473 e Treonina-308), quinase regulada por sinal extracelular (ERK) 1/2 e quinase ativada por estresse/quinase Jun-amino-terminal (SAPK/JNK) MAPK. Observou-se alteração na concentração das citocinas TNF-α, IL-6 e IL-10 no sobrenadante da cultura de BMDM dos animais diabéticos após estímulo com LPS, menor taxa de metabolismo mitocondrial, no entanto, sem resultar em morte celular, tampouco na expressão do receptor do tipo Toll 4 na membrana celular. Já o reestímulo destas células com LPS promoveu aumento na concentração de TNF-α sem alteração das demais citocinas. Além disto, o tratamento com insulina, simultaneamente ao estímulo com LPS, dos BMDM oriundos de animais diabéticos aumentou a concentração de TNF-α, IL-6, da fosforilação de p38, ERK 1/2 e SAPK/JNK MAPK, PI3K p55 e da Akt (Serina-473), o que não ocorreu nos BMDM dos animais não diabéticos sob a mesma condição. Este efeito foi abolido pela inibição farmacológica da PI3K e da ERK 1/2, resultando em novo aumento da concentração de TNF-α e IL-6. A análise conjunta destes resultados indica que a insulina, através da modulação das vias PI3K, Akt, ERK 1/2 e SAPK/JNK, amplifica o aumento da concentração de TNF-α e IL-6 sob estímulo com LPS

Diabetic patients present alterations in the immune system that promote in part a greater susceptibility of bacterial infections. Insulin treatment improves survival and reduces the number of recurrent infections in patients with type 1 diabetes mellitus (DM1). Little is known about the effects of diabetes and the action of insulin on macrophages. In this work we investigated the phosphatidylinositol-3-kinase (PI3K) / protein kinase B (Akt) and mitogen-activated kinase (MAPK) proteins in bone marrow-derived macrophages (BMDM) and their participation in lipopolysaccharide (LPS) or treatment with insulin through the secretion of inflammatory mediators tumor necrosis factor (TNF) -α, interleukin (IL) -6 and IL-10. We observed that BMDM of animals with DM1 increased PI3K p110alpha catalytic subunit expression with a reduction in the PI3K p55 regulatory subunit and increased expression of the phosphorylation of the Akt (Serine-473 and Threonine-308), extracellular signal regulated kinase (ERK) 1/2 and Jun-amino-terminal stress-kinase / kinase (SAPK / JNK) MAPK. Changes in the concentration of TNF-α, IL-6 and IL-10 cytokines in the supernatant of the BMDM culture of diabetic animals after stimulation with LPS were observed, possibly due to a lower rate of mitochondrial metabolism, however, without resulting in cell death , so little in the expression of the Toll 4 receptor on the cell membrane. The re-stimulation of these cells with LPS promoted an increase in TNF-α concentration without alteration of the other cytokines. In addition, insulin and simultaneously LPS stimulation of BMDM from diabetic animals increased the concentration of TNF-α, IL-6, phosphorylation of p38, ERK 1/2 and SAPK / JNK MAPK, PI3K p55 and Akt (Serine-473), which did not occur in the BMDM of non-diabetic animals under the same condition. This effect was abolished by pharmacological inhibition of PI3K and ERK 1/2, resulting in a further increase in the concentration of TNF-α and IL-6. The analysis of these results indicate that insulin by modulating the PI3K, Akt, ERK 1/2 and SAPK / JNK pathways amplifies the concentration levels of TNF-α and IL-6 under stimulation with LPS