Expressão gênica dos receptores toll-símele e sua relação com citocinas potencialmente envolvidas em lesão de reperfusão durante a fase inicial do transplante pulmonar em humanos

Introdução: Imunidade inata é a primeira linha de defesa do hospedeiro contra microorganismos invasores, a qual é mediada por moléculas específicas que reconhecem patógenos, chamadas receptores toll-símile (TLRs). Os TLRs são também capazes de reconhecer ligantes endógenos, tais como conteúdos de células necróticas e proteínas de choque térmico (HSP), resultando na produção de citocinas e ativação do sistema imune adquirido. A função exata dos TLRs ainda é pouco entendida em transplante de órgãos. No entanto, tem sido sugerido que eles podem estar envolvidos na rejeição aguda ou crônica e atuar na resposta do enxerto a lesão por isquemia e reperfusão. Objetivo: Examinar as alterações na expressão gênica dos TLRs durante a fase inicial do transplante pulmonar em humanos e sua relação com citocinas potencialmente envolvidas na lesão por isquemia e reperfusão em transplante de órgãos. Métodos: Foram analisadas biópsias pulmonares de 14 pacientes submetidos a transplante pulmonar (LTx). Estas amostras foram coletadas no final do período de isquemia fria (TIF, n=14), no final do período de isquemia quente (TIQ, n=13),1 hora (n=12) e 2 horas (n=8) após a reperfusão do enxerto. RNA total foi isolado a partir de tecido pulmonar e os níveis de RNA mensageiro (mRNA) dos TLRs (1-10) bem como citocinas (IL-8, IL-6, IL-10, IFN-γ, IL-1β) e proteína de choque térmico 70 (HSP70) foram medidos por reação em cadeia pela polimerase em tempo real. Resultados: Foi detectada a expressão de mRNA de todos TLRs em tecido pulmonar. Nas amostras no TIF, os níveis de mRNA dos TLRs apresentaram-se com diferentes expressões gênicas. Os níveis de expressão dos TLRs, com exceção para o TLR3, estavam altamente correlacionados entre si no TIF e com os níveis de mRNA de IFN-γ, IL-10 e IL-1β e menos significativamente com os níveis de IL-6 e IL-8. Houve diminuição dos níveis de mRNA na grande maioria dos TLRs após reperfusão, o que foi diferente para a maioria das citocinas e HSP70, que apresentaram tendência a aumentar após transplante. A expressão gênica de TLR4 apresentou-se correlacionada com os níveis de IL-8 e IL-1β antes e após transplante (P<0.05). Pulmões de doadores que foram intubados por períodos acima de 72 horas (n=5) apresentaram níveis mais elevados de TLR2 e TLR10 (P<0.05). Conclusão: Pela primeira vez, foi demonstrado que a expressão dos TLRs altera-se durante o período de isquemia e reperfusão em transplante pulmonar em humanos. O tempo de intubação dos doadores pulmonares pode influenciar a expressão de receptores Toll-símile específicos. A correlação entre TLR4 e IL-8/IL-1β sugere que os TLRs pulmonares podem ter alguma função na resposta precoce do enxerto.

Partial protection against tissue cysts formation in pigs vaccinated with crude rhoptry proteins of Toxoplasma gondii

A vaccine containing crude Toxoplasma gondii rhoptry proteins incorporated in the immunostimulating complexes (ISCOM) adjuvant was tested in pigs for protecting against tissue cyst formation. For this, 38 mixed breed pigs were divided into four groups, G1 (vaccinated challenged, n = 10) received two doses (100 mu g/dose) of the rhoptry vaccine at days 0 and 21, G2 (vaccinated challenged, n = 10) received viable tachyzoites (7 x 10(7)) of the RH strain at day 0, G3 (unvaccinated challenged, n = 10) and G4 (unvaccinated unchallenged, n = 8). Pigs were challenged with 4 x 10(4) VEG strain oocysts 57 days later. The G1 pigs produced high IgG antibody levels in the indirect enzyme-linked immunosorbent assay (ELISA) after the second dose of rhoptry vaccine, but were not clinically protected against a high dose oocyst challenge. Partial protection was observed in G1 at the chronic phase of infection, when compared with G3. Pigs in group 2 developed high antibody levels and were protected against clinic signs. T gondii was not detected in two (G1) and three (G2) pigs by mouse bioassay. The results indicate partial protection in pigs vaccinated with a rhoptry vaccine. (c) 2005 Elsevier B.V. All rights reserved.

Acute 2,4-dichlorophenoxyacetic acid intoxication in broiler chicks

The acute toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D), a herbicide, was studied in chicks dosed with 100, 300, 500, or 600 mg 2,4-D/kg BW, by the oral route. Clinical, laboratory, and histopathological methods were used as indicators of toxicity. After acute exposure, the herbicide decreased motor activity and induced muscular weakness and motor incoordination; decreased weight gain; increased serum creatine kinase (CK) and alkaline phosphatase (AP) activities and serum uric acid (UA), creatinine (CR), and total proteins (TP) levels; and did not change serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) activities. These changes were time-and dose-dependent and reversible. The LD50 (lethal dose 50%) calculated for oral 2,4-D in chicks was 420 mg/kg BW (385 to 483). Chromatographic analysis of the serum of the intoxicated chicks showed the presence of the herbicide; the amount found was dose-and time-dependent, increasing from 2 to 8 h after exposure and decreasing afterwards. Histopathological post-mortem studies conducted on intoxicated chicks showed hepatic (vacuolar degeneration of the hepatocytes), renal (tubular nephrosis), and intestinal (hemorragic) lesions. Taken together, the observed alterations mainly reflected kidney and muscle tissue damage, although hepatic toxicity may also have occurred after acute 2,4-D intoxication.

Exposure to acute restraint stress reinstates nicotine-induced place preference in rats

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

Histological and immunohistochemical study of the expression of p53 and ki-67 proteins in the mucosa of the tongue, pharynx and larynx of rats exposed to cigarette smoke

Introduction: Head and neck cancers are linked to smoking. The most affected sites are the oral cavity, pharynx and larynx. Experimental studies show epithelial lesions caused by cigarette smoke. Objectives: To investigate in rats the effects of acute cigarette smoke exposure on the mucosa of the tongue, pharynx and larynx.Material and method: Wistar rats were allocated into two groups of 20 animals: CG (control) receiving food and water ad libitum and TG (Tobacco) exposed to the smoke of 40 cigarettes/day for 60 days. Biopsy of their tongues, pharynxes and larynxes were subjected to histopathological, histomorphometric and immunohistochemical studies of protein p53 and ki-67.Result: The histological analysis of tongue from the Tobacco group revealed epithelial hyperplasia (90%), basal cell hyperplasia (95%) and mild to moderate dysplasia (85%). In pharynx showed basal cell hyperplasia (85%), dysplasia (25%) and vascular congestion (95%). In larynx showed basal cell hyperplasia (70%), epithelial hyperplasia (55%), congestion (100%) and inflammatory infiltrate (25%). Morphometric analysis revealed that keratin layer thickness was greater in the tobacco group. P53 immunoexpression was negative in both groups. Ki-67 immunoexpression was positive in basal cell nuclei but in parabasal cell nuclei it was positive only in the Tobacco group.Conclusions: The exposure of animals to cigarette smoke for 60 days resulted in benign lesions. The duration of exposure was not enough to cause the development cancer, as confirmed by the negative expression of p53 protein in all slides examined. Analysis of ki-67 expression showed intense epithelial proliferation in response to damage.

Indigofera suffruticosa Mill as new source of healing agent: Involvement of prostaglandin and mucus and heat shock proteins

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

Acute exercise reverses aged-induced impairments in insulin signaling in rodent skeletal muscle

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

Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems

Phase diagrams of poly(ethylene glycol)/polyacrylate/Na2SO4 systems have been investigated with respect to polymer size and pH. Plasmid DNA from Escherichia coil can depending on pH and polymer molecular weight be directed to a poly(ethylene glycol) or to a polyacrylate-rich phase in an aqueous two-phase system formed by these polymers. Bovine serum albumin (BSA) and E. coil homogenate proteins can be directed opposite to the plasmid partitioning in these systems. Two bioseparation processes have been developed where in the final step the pDNA is partitioned to a salt-rich phase giving a total process yield of 60-70%. In one of them the pDNA is partitioned between the polyacrylate and PEG-phases in order to remove proteins. In a more simplified process the plasmid is partitioned to a PEG-phase and back-extracted into a Na2SO4-rich phase. The novel polyacrylate/PEG system allows a strong change of the partitioning between the phases with relatively small changes in composition or pH. (C) 2012 Elsevier B.V. All rights reserved.

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

INGAP-PP up-regulates the expression of genes and proteins related to K-ATP(+) channels and ameliorates Ca2+ handling in cultured adult rat islets

Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. Here, we investigated the effects of the pentadecapeptide INGAP-PP in adult cultured rat islets upon the expression of proteins constitutive of the K-ATP(+) channel, Ca2+ handling, and insulin secretion. The islets were cultured in RPMI medium with or without INGAP-PP for four days. Thereafter, gene (RT-PCR) and protein expression (Western blotting) of Foxa2, SUR1 and Kir6.2, cytoplasmic Ca2+ ([Ca2+](i)), static and dynamic insulin secretion, and Rb-86 efflux were measured. INGAP-PP increased the expression levels of Kir6.2, SUR1 and Foxa2 genes, and SUR1 and Foxa2 proteins. INGAP-PP cultured islets released significantly more insulin in response to 40 mM KCl and 100 mu M tolbutamide. INGAP-PP shifted to the left the dose-response curve of insulin secretion to increasing concentrations of glucose (EC50 of 10.0 +/- 0.4 vs. 13.7 +/- 1.5 mM glucose of the controls). It also increased the first phase of insulin secretion elicited by either 22.2 mM glucose or 100 mu M tolbutamide and accelerated the velocity of glucose-induced reduction of Rb-86 efflux in perifused islets. These effects were accompanied by a significant increase in [Ca2+](i) and the maintenance of a considerable degree of [Ca2+](i) oscillations. These results confirm that the enhancing effect of INGAP-PP upon insulin release, elicited by different secretagogues, is due to an improvement of the secretory function in cultured islets. Such improvement is due, at least partly, to an increased K-ATP(+) channel protein expression and/or changing in the kinetic properties of these channels and augmented [Ca2+](i) response. Accordingly, INGAP-PP could potentially be used to maintain the functional integrity of cultured islets and eventually, for the prevention and treatment of diabetes. (C) 2008 Elsevier B.V. All rights reserved.

Proteins of Leishmania (Viannia) shawi confer protection associated with Th1 immune response and memory generation

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

Toxicologic evaluation of acute and subacute oral administration of Cucurbita maxima seed extracts to rats and swine

The extract prepared from dried seeds of Cucurbita maxima was administered to rats and pigs. Following a single dose or 4 weeks of daily oral administration, the extract produced no changes in serum glucose, urea, creatinine, total protein, uric acid, GOT, GPT, LDH or blood counts. Urine analysis (urea, uric acid, creatinine, total protein, Na and K), as well as histopathological investigation, showed no abnormalities. These results taken as a whole indicate that the seeds of C. maxima as used in Brazilian folk medicine are not toxic for rats and swine.

Acute, subchronic and chronic 2,4-dichlorophenoxyacetic acid (2,4-D) intoxication in rats

The acute, subchronic and chronic toxicities of 2,4- dichlorophenoxyacetic acid (2,4-D) were studied in rats. Animals were exposed acutely (600 mg/kg), subchronically (200 ppm for 30 d) and chronically (200 ppm for 180 d) to 2,4-D by the oral route. Clinical, laboratory and histopathological methods were used as indicators of toxicity. After acute exposure, the herbicide decreased locomotor activity and induced ataxia, sedation, muscular weakness (mainly of the hind quarters) and gasping for breath; increased aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (AP), amylase activities and creatinine levels; decreased total protein (TP) and glucose levels; and increased hematocrit values. Subchronic and chronic 2,4-D exposures did not induce overt clinical signs or symptoms of intoxication. However, subchronic herbicide exposure increased AST activity and albumin and hematocrit values, and chronic exposure increased AST, AP and LDH activities, decreased amylase and glucose levels, but did not change hematocrit values. Chromatographic analysis of the serum of chronically exposed rats showed the presence of the herbicide; the amount found (3.76 ± 1.16 mg/ml) suggested the absence of 2,4-D accumulation within the body. Although macroscopic or histopathological lesions were not observed in acutely, subchronically or chronically 2,4-D exposed rats, the laboratory data obtained suggest tissue injuries after dosing, since the results are considered early indicators of primarily hepatic and muscle tissue damage.

Oral manifestations during chemotherapy for acute lymphoblastic leukemia: A case report

A 14-year-old, male patient was referred for the treatment of mucositis, idiopathic facial asymmetry, and candidiasis. The patient had been undergoing chemotherapy for 5 years for acute lymphoblastic leukemia. He presented with a swollen face, fever, and generalized symptomatology in the mouth with burning. On physical examination, general signs of poor health, paleness, malnutrition, and jaundice were observed. The extraoral clinical examination showed edema on the right side of the face and cutaneous erythema. On intraoral clinical examination, generalized ulcers with extensive necrosis on the hard palate mucosa were observed, extending to the posterior region. Both free and attached gingivae were ulcerated and edematous with exudation and spontaneous bleeding, mainly in the superior and inferior anterior teeth region. The tongue had no papillae and was coated, due to poor oral hygiene. The patient also presented with carious white lesions and enamel hypoplasia, mouth opening limitation, and foul odor. After exfoliative cytology of the affected areas, the diagnosis was mixed infection by Candida albicans and bacteria. Recommended treatment was antibiotics and antifungal administration, periodontal prophylaxis, topical application of fluor 1.23%, and orientation on and control of proper oral hygiene and diet during the remission phase of the disease.