Pré-condicionamento isquêmico em diferentes tempos e seu efeito na translocação bacteriana induzida por isquemia e reperfusão intestinal em ratos

Para avaliar os efeitos de diferentes tempos de pré-condicionamento isquêmico (IPC) em translocação bacteriana intestinal (BT). MÉTODOS: Trinta ratos Wistar pesando 280 ± 27g foram divididos em cinco grupos. No grupo IV (n = 6), a laparotomia foi realizada e a artéria mesentérica superior foi obstruído por um microclampe atraumática durante 30 minutos. Nos quatro grupos de pré-condicionamento (n = 6 cada) antes dos 30 minutos de isquemia-reperfusão (I / R), os ratos foram submetidos a IPC para duas, cinco, dez e 15 minutos, seguido pelo mesmo momento da reperfusão. A fim de avaliar se o tempo de pré-condicionamento influenciaram o surgimento de translocação bacteriana, as amostras de nódulos linfáticos mesentéricos, fígado e baço foram colhidas em condições estéreis, 24 horas após os procedimentos para a quantificação de unidades formadoras de colónias de bactérias por grama de tecido (CFU / g). O sangue foi recolhido para a medição de citoquinas. RESULTADOS: No grupo I / R, o total de CFU / g em gânglios linfáticos mesentéricos, baço, fígado, bem como o soro de TNF-a, IL-1A e IL-6 foram significativamente mais elevados do que nos outros grupos (p <0,05). Pré-condicionamento por 15 minutos significativamente atenuada BT e citocinas séricas quando comparado a outros períodos de pré-condicionamento (p <0,05). CONCLUSÃO: Nossos dados sugerem que o pré-condicionamento como um fator chave para reduzir a translocação bacteriana intestinal em I / R. Numa escala de dois a 15 minutos, o melhor tempo de pré-condicionamento isquémico pela atenuação da translocação bacteriana foi de 15 minutos

Estudo sobre a participação de rodedores na cadeia de transmissão de Leishmania infantum (Protozoa: Trypanosomatidae) no Rio Grande do Norte

American visceral leishmaniasis is a zoonosis caused by Leishmania infantum and transmitted by the bite of the sand flies Lutzomia longipalpis.The main domestic reservoir is the dog, while foxes and opposums are the known wild reservoirs. However, identification of natural infections with L. infantum in rodents appears for need of investigating the participation of these rodents how source of infection of the parasite. In the present work the Leishmania infantum infection was investigated in rodents captured in Rio Grande do Norte, aiming at to offer subsidies to the understanding of the epidemic chains of LVA in the State. Thirteen Galea spixii were distributed in four groups, being G1 the group control with four animals and the others, G2, G3 and G4, with three animals each. Those animals were intraperitoneally inoculated with 107 promastigotas of L. infantum and accompanied for, respectively, 30, 90 and 180 days. Weekly the animals were monitored as for the corporal weight and rectal temperature. At the end of each stipulated period the animals were killed. Blood were used for determination of the parameters biochemical and haematological, PCR, ELISA, microscopic examination and cultivation in NNN medium. Liver, spleen and lymph node were used in Giemsa-stained impression and cultivation in NNN medium. Liver and spleen fragments were still used in PCR and histopathological, respectively. At the same time 79 rodents of the species Rattus rattus, Bolomys lasiurus, Oligoryzomys nigripis, Oryzomys subflavus and Trichomys apereoides were captured in the Municipal districts of Brejinho, Campo Grande, Coronel Ezequiel, Passa e Fica and Vázea for identification of natural infection with L. infantum. Evidence of infection was checked by direct examination of Giemsa-stained impression of liver, spleen and blood and culture of these tissues in NNN medium. Antibodies were researched by ELISA. They were not found differences among the weigh corporal final, rectal temperature and biochemical and haematological parameters of the Galea spixii controls and infected. The rectal temperature of the animals varied from 36OC to 40OC. For the first time values of the haematocrit (33,6% to 42,8%), hemoglobin (10,2 to 14,5g/dl), erythrocyts number (4,67x106 to 6,90x106/mm3), total leukocytes (0,9x103 to 9,2x103/mm3), platelets (49x103 to 509x103/mm3) total proteins (1,56 to 6,06 g/dl), albumin (1,34 to 3,05 g/dl) and globulins (0,20 to 3,01 g/dl) of the Galea spixii were determined. The lymphocytes were the most abundant leucocytes. Infection for L. infantum was diagnosed in two animals euthanasied 180 days after the infection. In one of the animals was also identified antibodies anti-Leishmania. The parasite was not found in none of the five other species of rodents captured. Galea spixii are resistant to the infection for L. infantum and they are not good models for the study for visceral leishmaniose, although they can act as infection sources. More studies are necessary to determine the paper of the rodents in the epidemic chain of transmission of the visceral leishmaniose in the State of Rio Grande do Norte

O papel modulatório do ferro sobre a resposta imune na Leishmaniose Visceral

Iron is an essential element for many cellular functions, including the immune response against intracellular pathogens. In this study, we aimed evaluate the effect of iron on IRP2, IFN-γ, TNF-α, IL-6, IL-10, MIG and IP10 expression in PBMC and assess the effect of the spleen parasite load on the expression of these genes in the spleen of L. infantum naturally infected dogs. Blood sample from 7 DTH+ donor was collected and PBMC was obtained. The cells were cultivated in absence (iron chelator desferroximane, DFO 10 μM supplemented media) or in presence of iron (hemin 6 mM) for 1 h, followed by stimulation with Leishmania infatum antigen for 4 h. 44 dog spleen samples were obtained and parasite load in this organ was determinate by qPCR. Gene expression was analyzed by qPCR and cytokine production quantified by flow cytometry. In antigen stimulated cells, genes involved in immune response are significantly more expressed in presence of iron. T CD4+ and TCD8+ lymphocytes produces IFN-γ, TNF-α and IL-10 possibly in iron dependent pathway. Monocytes antigen stimulated reduced TNF-α, IL-6 and IL-10 production in presence of iron. We found spleen of infected dogs IRP2 expression increases according to parasite load in that organ, while an inverse profile was found for IFN-γ, TNF-α e IL-10 expression. These results suggest that T lymphocytes depends on iron to produce IFN-γ, TNF-α and IL-10, while iron seems to inhibit cytokine production in monocytes. So, we propose an immunoregulatory mechanism carried out by iron during L. infantum infection in humans and dogs

Estudo clínico-laboratorial e histopatológico de cães naturalmente infectados por Leishmania chagasi com diferentes graus de manifestação física

Canine Visceral Leishmania (CVL) is an important zoonotic disease that has a world wide distribution and has a large impact on public health on the American Continent, especially in Brazil, where the nature of endemic diseases in humans affects a large part of the nation. The influence of the prevalence of CVL in the increased rate of human cases in endemic areas and in the unleashing of epidemic outbreaks shows the need for a more profound understanding, that would generate significant advances in the current measures used to control the reservoirs of sickness that are practiced by the Programa Nacional de Vigilância e Controle da Leishmaniose Visceral. The present work describes and compares the clinical-laboratorial and histopathological findings of twenty-three dogs that were naturally infected by Leishmania chagasi, from endemic areas in metropolitan Natal, Rio Grande do Norte, Brazil. These animals, that were selected and given physical and serological exams (IFI and ELISA rK-39), were classified according to the degree of clinical severity and had blood samples drawn (whole blood and serum) for a complete hemogram and a coagulogram to be done as well as biochemical tests for kidney and liver function. The confirmation of infection by L. chagasi was done after the euthanasia of the animals, through the direct demonstration of the parasite in the impression of the spleen and liver crowned with GIEMSA and through a cultivation by means of NNN/Schneider. According to the clinical evaluation, the animals were classified as asymptomatic (7), oligosymptomatic (7) and polysymptomatic (9). Among the animals that were chosen to be autopsied, there were 2 asymptomatic, 3 oligosymptomatic and 3 polysymptomatic, for the purpose of studying their histopathology, having collected fragments of the spleen, liver, kidneys and skin and were fixed in 10% tamponed formol. The comparison between the average parameters of the clinical-laboratory tested animals in the groups was done through the Student t test (a<0.05). The main clinical signals observed were lymphadenomegaly, alopecy, dermatitis, exfoliation, cutaneous ulcers, onicogriphosis and emaciation. The main clinical-laboratorial alterations established, mainly in the polysymptomatic group, were anemia, hyperproteinemia, hyperglobulinemia, alterations in the albumin/globulin ratio and increased ALT activity. Renal alterations were not verified (urea and creatinine levels were normal). Thrombocytopenia was observed in three clinical groups. However, the other indicators of coagulation function (TAP and TTPA) did not have abnormal variations. There were inflammatory infiltrations and leishmania amastigotes in the skin of polysymptomatic dogs, however, they were not found in the skin of asymptomatic animals. Hypertrophy and hyperplasia of the phagocyte mononuclear system, leishmania amastigote parasites were found in the macrophages, extramedullary hematopoiesis and degenerative alterations were detected in the spleen and liver of 8 of the animals submitted to histopathological exams. In accord with these results, it was demonstrated that the expected alterations in the hematological and biochemical parameters in function of their viscerotropic nature of CVL are mainly observed in the more advanced stages of the disease. The absence of inflammatory infiltration and parasite load in the skin suggest that infected animals without symptoms may have an importance irrelevant to the infectiousness of the vector

Análise da estabilidade genética de células-tronco mesenquimais humanas

Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren´t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient

Comparação de efeitos dos extratos de Hypericum perforatum (Hipérico) e de Mentha crispa (Hortelã) em diferentes modelos experiemtais

Several clinic evaluations have been possible with radiobiocomplexes labeled with technetium-99m (99mTc). Some natural and synthetic drugs are capable of to interfere on the labeling of blood constituents with 99mTc, as well as on the biodistribution of radiobiocomplexes. Authors have also reported about the toxicity of several natural products. The aim of this study was to compare the effects of the Mentha crispa (hortelã) and of the Hypericum perforatum (hipérico) in different experimental models. On the labeling of red blood cells (RBC) and plasma and cellular proteins with 99mTc, both extracts were capable of to decrease the radioactivity percentage on the cellular compartment and on the fixation on plasma and cellular proteins. On the morphometry of the RBC, only the hortelã was capable to alter the shape and the perimeter/area ratio of the RBC. On the biodistribution of the radiobiocomplex sodium pertechnetate (Na99mTcO4), the hortelã increased the Na99mTcO4 distribution in the kidney, spleen, liver and thyroid, meanwhile the hipérico decreased the Na99mTcO4 distribution in the bone, stomach, lungs and thyroid, and increased the Na99mTcO4 distribution in the pancreas. On the bacterial cultures survival, the hipérico was capable of to protect the bacteria against the stannous chloride (SnCl2) effect. The hipérico did not alter the topology of plasmidial DNA and did not protect the plasmidial DNA against the SnCl2 action. Probably, the effects presented by both extracts could be due to chemical compounds of the extracts that could alter the morphology of the RBC and the plasma membrane ions transport, and/or by phytocomplexes that could be formed with different effects dependent on the biological system considered

Estudo de efeitos de um extrato aquoso de farinha de casca de maracujá (Passiflora edulis f. flavicarpa) na marcação de constituintes sangüíneos com Tecnécio 99m, na morfometria das hemácias e na biodisponibilidade do radiofármaco Pertecnetato de Sódio em ratos Wistar

Blood constituents labelled with technetium-99m (99mTc) has been used with radiobiocomplexes in several procedures in nuclear medicine. Some natural and sintetic drugs are capable to interfere on the labeling of blood constituents with 99mTc, on the morphology of red blood cells (RBC) and on the biodistribution of radiobiocomplexes. The aim of this study was evaluate the effect of an extract of Passiflora edulis f. flavicarpa on the labeling of blood constituints with 99mTc, on the morphology of RBC and on the biodistribution of the radiopharmaceutical sodium perthecnetate in Wistar rats. On the in vitro studies the Passiflora edulis f. flavicarpa decreased significantly (p<0.05) the %ATI on plasma proteins and on the in vitro morhology of RBC, the passion fruit peel flour altered the shape and the perimeter/área ratio. On the in vivo estudies the extract did not altered the %ATI in blood constituents, and did not altered the shape of RBC. Although, on the biodistribution of the radiobiocomplex sodium perthecnetate (Na99mTcO4) this extract decreased significantly (p<0.05) the uptake in duodenum, spleen, pâncreas and blood, and increased the uptake in stomach. It can be suggested that the effects presented by this extract could be a result of some substances contained in this extract that could alter the binding of 99mTc to plasma proteins, the morphology of RBC and the biodistribution of the radiobiocomplex sodium perthecnetate. This study was multidisciplinary experimental research. It was developed with the contribution of different Departments and Services of the Hospital Universitário Pedro Ernesto of the Universidade Estadual do Rio de Janeiro

Avaliação do efeito de um adoçante comercial com sucralose na marcação de constituintes sanguíneos com tecnécio-99m, na morfologia das hemácias e na biodisponibilidade dos radiofármacos pertecnetato de sódio e ácido dietilenotriaminopentacético-tecnécio-99m em ratos Wistar.

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Aspectos clínicos e imunológicos da infecção por Leishmania Infantum Chagasi em cães do Rio Grande do Norte / Paula Vivianne Souza de Queiroz. Natal/RN

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Influência da esplenectomia na biodistribuição do ácido dimercaptosuccínico-tecnécio-99m (99mTc-DMSA) em ratos Wistar

A esplenectomia é indicada em doenças hematológicas, hiperesplenismo grave, traumas e tem sido causa de importantes alterações metabólicas e imunológicas. O objetivo do presente trabalho é avaliar se a retirada do baço altera a biodistribuição do radiofármaco DMSA Tc99m em ratos Wistar e se há correlação com possíveis alterações da função renal. Foram usados dois grupos: esplenectomia (n=6) e controle (n=6) animais não operados. Após 15 dias, foi administrado 0,1 ml de DMSA-Tc99m via plexo orbital (0,66 MBq). Trinta minutos depois, foram retiradas amostras do rim, coração, pulmão, tireóide, estômago, bexiga, fêmur, sangue. Após pesadas as amostras, foi determinado o percentual de radioatividade/g (% ATI/g) em cada uma delas, com o Wizard Gama Counter Perkin-Elmer . Dosadas uréia e creatinina sérica, hematócrito, plaquetas e leucócitos. Estatística pelo teste t, com significância 0,05. Foi observada redução significante no %ATI/g no rim e sangue (p<0,05) dos animais esplenectomizados, aumento significante (p<0,05) da uréia (88,8±18,6mg/dL) e creatinina (0,56±0,08), comparado aos controles (51,5±1,6 e 0,37±0,02mg/dL, respectivamente) assim como leucocitose, aumento de plaquetas e redução de hematócrito. Em conclusão, a esplenectomia provocou comprometimento da função renal, repercutindo na alteração da captação de DMSA 99mTc pelo rim de ratos. Uma parceria entre a Unidade de Medicina Nuclear da Liga Norteriograndense Contra o Câncer e o Núcleo de Cirurgia Experimental da Universidade Federal do Rio Grande doNorte tornou possível a realização deste trabalho, que contou com a participação de profissionais de diferentes áreas como: Biologia, Biomedicina, Medicina Nuclear, Cirurgia Geral, Bioquímica e Estatística, atestando dessa forma o caráter multidisciplinar do trabalho

Biodistribuição do fitato de sódio marcado com tecnécio-99m em ratos esplenectomizados

O baço, como maior órgão linfóide do corpo humano, desempenha funções imunológicas relevantes, tais como depuração de bactérias da corrente sangüínea, produção de anticorpos e interação com a função hepática. A esplenectomia tem sido evitada sempre que possível, mas quando realizada pode provocar uma série de efeitos indesejáveis. O radiofármaco 99mTc-fitato é usado no diagnóstico de doenças, especialmente no fígado, através de exames de imagem, na dependência de sua biodistribuição. Algumas drogas e intervenções cirúrgicas podem interferir na biodistribuição de radiofármacos e inexistem na literatura dados sobre efeitos da esplenectomia no metabolismo do 99mTc-fitato. O objetivo do trabalho foi avaliar se a esplenectomia interfere na biodistribuição hepática do 99mTc-fitato e na função do fígado em ratos Wistar. Sob anestesia e técnica asséptica, os animais do grupo SP (n=6) foram esplenectomizados. No grupo C (controle; n=6) os animais não foram operados. Após 15 dias de observação, foi injetado de 0,1ml de 99mTc-fitato via plexo orbital (0,66MBq) em todos os animais. Após 30 minutos, foram retiradas amostras hepáticas para determinação do percentual de radioatividade/grama (% ATI/g), usando-se contador gama Wizard Perkin-Elmer. Realizou-se dosagem sérica de ALT, AST e LDH, e leucometria. Estatística pelo teste t, com significância de p>0,05. Observou-se diferença significativa (p=0,034) comparando-se o %ATI/g no fígado dos ratos esplenectomizados (0,990,2) com os controles (0,400,2). ALT, AST e LDH tiveram dosagens significativamente menores e houve leucocitose nos esplenectomizados (p=0,01), comparando-se com os controles. Concluiu-se que, em ratos, a esplenectomia provavelmente provocou alteração na captação de 99mTc-fitato pelo fígado, coincidindo com alterações na função hepática. A realização deste estudo teve caráter multidisciplinar, envolvendo pesquisadores de diversas áreas como Medicina Nuclear, Cirurgia, Análises Clínicas e Estatística. Este aspecto preencheu os requisitos da multidisciplinaridade do Programa de Pós-graduação em Ciências da Saúde

Biodistribuição do pertecnetato de sódio após cirurgia do Switch Duodenal

The biliopancretic diversion with duodenal switch is one of the mixing techniques used in the treatment of morbid obesity. The duodenal switch reduces the stomach capacity and leaves only 50-100 cm of small intestine for nutrition and absorption. The surgery produces hormonal, structural and biochemical changes, which can influence on the result of scintigraphic examinations in operated patients. With the objective of evaluate the postoperative biodistribution of sodium pertechnetate (Na99mTcO4) in brain, thyroid, heart, lung, liver, spleen, kidney, stomach, duodenum, pancreas, small intestine, bladder, muscle and bone of Wistar rats. The rats were randomly allocated into 3 groups of 7 rats each: the duodenal switch group (DS), the control group (C) and the sham group (S). They were operated under anesthesia and aseptic technique. In the tenth postoperative day, 0.1mL of sodium pertechnetate was injected via orbital plexus. After 30 min the animals were killed with overdose of anesthetic and samples of liver, spleen, pancreas, stomach, duodenum, small intestine, thyroid, lung, heart, kidney, bladder, muscle, bone and brain were harvested, washed with saline and weighed. The detention of radioactivity was made using the automatic Gamma Counter Wizard, PerkinElmer and the percentage of activity per gram of tissue (%ATI/g) was calculated. There was no early or late mortality in either rats groups. The values of percent radioactivity per gram of tissue (%ATI/g), showed no significant difference in liver, stomach, small bowel, duodenum, kidney, heart, bladder, bone and brain, when compared the DS rats with sham and controls rats. A postoperative significant increase in mean %ATI/g levels was observed in spleen, pancreas and muscle in group DS rats, as compared to group S and C rats (p<0.05). In the lung there was an increase and in thyroid a decrease in mean %ATI/g of DS rats, when compared to sham rats (p>0.05). In conclusion, the biliopancreatic diversion with duodenal switch in rats modified the biodistribution of sodium pertechnetate in thyroid, lung, pancreas, spleen and muscle. The study had the participation of the departments and laboratories researches, as Nucleus of Experimental Surgery, Department of Surgery, Laboratory of Radiobiology, Department of Pathology and Service of Nuclear Medicine, certifying the character of a multidisciplinary research

Biodistribuição do pertecnetato de sódio (Na99mTcO4) em ratos submetidos à ressecção extensa de intestino delgado

Massive resection of the small intestine results in short bowel syndrome with anti-absorptive effect and repercussions on the metabolism. Morphologic and functional evaluation may be necessary in order to control wrapped organs. Scintigraphy an examination with little invading and no biologic damage can be used. The purpose were to assess the biodistribution of sodium pertecnetate in organs of rats subjected to massive resection of the small intestine, the intestinal adaptation of the remnant intestinal mucosa and weight curve evaluation in the postoperative period. Twenty-one Wistar rats were randomly allocated into three groups (n = 7). The operated group named short bowel (SB) after anesthetized was subjected to massive resection of the small intestine; the control group (C), and sham group (SHAM). On the 30th postoperative day, 0.l mL of sodium pertechnetate was injected into the venous orbital plexus. After 30 minutes, the rats were killed with an overdose of anesthetic, and fragments of the liver, spleen, pancreas, stomach, duodenum, small intestine, thyroid, lung, heart, kidney, bladder, muscle, femur and brain were harvested. The percentage of radioactivity per gram of tissue (%ATI/g) was determined using Gama Counter WizardTM 1470, PerkinElmer to all samples. Biopsies of 3 cm remaining jejunum were removed to histological analyses. Analysis of variance (ANOVA) and the Tukey test for multiple comparisons were used, considering p<0.05 as significant. The study had the participation of some departments and laboratories, as Nucleus of Experimental Surgery, Department of Surgery, Laboratory of Radiobiology, Department of Pathology and Service of Nuclear Medicine, certifying the character of a multidisciplinary research. The results were no significant differences in %ATI/g of the sodium pertechnetate in the organs of the groups studied (p>0.05). An increase in the weight of the SB rats was observed after the second postoperative week. The jejunal mucosal thickness of the SB rats was significantly greater than that of C and sham rats (p<0.05. The biodistribution of sodium pertechnetate was not affected by massive intestinal resection in rats. An adaptive response by the intestinal mucosa probably contributed to the reversion of weight loss and the biodistribution of sodium pertechnetate was not affected by the surgery

Identificação dos princípios ativos presentes no extrato etanólico de cereus jamacaru e avaliação em ratos dos possíveis efeitos tóxicos e/ou comportamentais da exposição prolongada

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Análise em conjunta de testes cosmológicos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior