Repositório RCAAP

Conselho Nacional de Desenvolvimento Científico e Tecnológico

The Diabetes Mellitus (DM) is a group of metabolic diseases, with hiperglycemia resulting from defects in insulin secretion (type 1 DM) and / or action (type 2 DM), promoting the elevation of blood glucose levels, which can damage target organs (eyes, kidneys, nerves, heart). In the kidney presents as diabetic nephropathy (DN), characterized by increased levels of urinary albumin excretion (UAE), which could progress to end stage kidney disease. The renin-angiotensin-aldosterone system (RAAS) is an important mediator of the pathophysiological changes of DN. The pharmacological blockade of this system has been shown as a combined treatment of DN, decreasing urinary albumin excretion. The aim of this study was to evaluate the effects of triple RAAS blockade on kidney structure and function, as well as albuminuria in diabetic Wistar rats with DN in development. Induction of diabetes in rats was performed with intravenous administration of alloxan (50mg/kg) diluted in 0.9% saline and glycemia observed 24h after the induction of diabetes; animals with blood glucose greater than 200 mg/dL were considered diabetic and were divide into 5 groups: I) control group - no treatment (CG); II) experimental group 1 - treated with enalapril (EN) (angiotensin converting enzyme inhibitor - ACEI), III) experimental group 2 - treated with losartan (LO) (angiotensin receptor blocker - ARB), IV) experimental group 3 - treated with aliskiren (AL) (direct renin inhibitor - DRI); V) experimental group 4 - treated with triple blockade ACEIARB- DRI (EN+LO+AL). After 90 days of treatment, animals were placed in metabolic cages to collect 24-hour urine and thereafter for determination of blood, proteinuria, and glomerular filtration rate by creatinine clearance. Then, the animals were anesthetized and had their kidneys removed for histological and immunohistochemical studies (&#945;-SMA and PCNA). After induction, all animals that became diabetic, remained with high glycemic levels throughout the treatment period. Histology showed on CG glomerular collagen deposition and tubulointerstitial, tubular dilation, space capsule expansion and inflammatory infiltration; those changes were attenuated by treatments with EN, LO, AL and EN+LO+AL. The treatments reduced the expression of &#945;-SMA glomerular (EN p<0.01; LO p<0.01; AL p<0.05, EN+LO+AL p<0.01) and tubulointerstitial (EN p<0.05, AL p<0.01, EN+LO+AL p<0.05) compared to CG. There were no significant differences in the number of PCNA glomeruli positive cells and in the analysis of plasma concentrations of sodium, potassium and urea between groups. However, EN and LO preserved a largest number of proliferating tubulointerstitial cells. Rats treated with AL (p<0.05) and EN+LO+AL (p<0.05) had a higher glomerular filtration rate (GFR) when compared to CG, EN and LO. AL and EN+LO+AL significantly reduced proteinuria in these animals when compared to CG (p<0.01). The treatment with EN+LO+AL reduced the expression of &#945;-SMA glomerular and tubulointerstitial, preserved a greater number glomeruli, GRF and UAE. However, administration of AL resulted in similar data.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects about 8 million people worldwide. The disease is manifested in two phases, acute, characterized by high parasitaemia, and chronic by control of infection by the host. An adequate immune response is required to control infection. The response profile to infection by T. cruzi is the T helper type 1 (Th1). In recent studies, it was shown that the cytokines IL-3, IL-7 and IL-10 are highly expressed during the chronic phase of experimental infection by T. cruzi. The IFN-γ and IL-10 cytokines have been extensively studied to infection by T. cruzi, and IFN-γ plays a fundamental role in controlling parasitemia in the acute phase. The rP21 protein based on the P21 of T. cruzi induces nonspecific phagocytosis, polymerize actin cytoskeleton and seems to be matter in the passage to the chronic phase of the disease. Thus, in this study we analyzed the role of IFN-γ, IL-3, IL-7, IL-10 cytokines and the rP21 protein on in vitro infection by T. cruzi, and analyzed the role of these cytocines in the polymerization of actin. It was observed on peritoneal macrophages and myoblasts C2C12, that the cytokines IFN-γ, IL-7 and rP21 protein, induce the polymerization of actin cytoskeleton. Moreover, in macrophages and myoblasts infected with G and Y strain was seen differential effect of cytokines and rP21 in the parasite multiplication. However, in all analyzed groups IFN-γ and rP21 decreased parasite multiplication. This effect may be due to the induction of actin polymerization, along with induction of nitric oxide (NO) and reactive oxygen species (ROS) for the groups treated with IFN-γ. Meanwhile, the observed effect of rP21 seems to be linked by only with the actin cytoskeleton where the polymerization thereof, act in forming a mechanical barrier for parasite multiplication. Therefore, it is important to investigate the potential role of the actin cytoskeleton during intracellular multiplication of the parasite and its interaction with the host\'s immune response once an understanding of the modulation of such properties, would provide a basis for better understanding of the biology of the parasite.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Saint Louis Encephalitis Virus (SLEV) is a flavivirus that infects humans through Culex mosquito bite. It causes Saint Louis Encephalitis (SLE), a disease that affects mainly the elderly with a mortality rate of 17% and various severity clinical presentations. In order to reach the central nervous system (CNS) from circulating blood the pathogen must cross a barrier formed by endothelial cells and astrocytes, the blood-brain-barrier (BBB). Astrocytes are the most common glial cell type in CNS, their role is to keep neural tissue homeostasis, control neuroinflamation and immune response. Althought astrocytes can be infected by flavivirus, there are few studies to evaluate its infection and cell alterations post SLEV exposure. For astrocyte plays an essential role in CNS functioning, they were investigated for morphologic alterations and activation (astrogliosis), immunological response via MHC-I and apoptosis induction via caspase-3 activation post-infection. A comparative study using plaque forming units (PFU) and MTT salt assay was performed to determine viral titer revealing that both methods can be used to access viral titres of a sample with similar results. VERO cells shown higher cytotoxicity and mortality rates than astrocytes when infected with SLEV. Besides, flaviviral exposure unleashed astrocyte immune response marked by raise in MHC-I expression and astrogliosis, characterized by intense GFAP expression, and increase in number and length of cytoplasmic processes. When exposed to growing viral concentrations, it has been observed a proportional increase of caspase-3 expression, also showing nuclear envelope destruction. Immune staining with anti-SLEV antibody revealed that the virus displayed a perinuclear location during the replication process. The great SLEV resistance of astrocytes suggests that other infection mechanisms, as BBB breakdown must be associated in the neuroinvasive form of SLEV infection. Also astrocyte might play a role in chronic infection of CNS.

TNF-α is a key cytokine involved in process of carcinogenesis, including prostate cacrinogenesis, being TNFR-1 the responsible for the majority of its answers. This receptor triggers two opposite pathways: cell death or cell survival. So, due to this paradoxal role, a TNFR-1 knockout model is interesting in order to clarify the real effect of TNF in prostate cancer. Thus, this investigation had as a general propose to evaluate the role of TNF signalling pathway in prostate carcinogenesis by chemical induction in mice. Therefore, C57bl/6 wild type (WT) and TNFR-1 knockout mice (KO) were treated with mineral oil or MNU in association with testosterone (MNU+T) during 6 months. After this period of induction, prostate samples were processed for histological and biochemical analysis. It was observed that the treatment with MNU+T led to development of benign and malign lesions in both WT e KO animals. However, the incidence of malign lesions was significantly higher in the former, indicating the involvement of TNFR-1 with cellular survival and proliferation pathaway. These data were supported by proliferative profile analysis, which showed that proloferation was significantly lower in KO, even after carcinogenesis. Moreover, knockout animals had smaller prostate amounts of mTOR after treatment with MNU+T in comparison to WT, suggesting the potential of TNFR-1 in activating this proliferative pathway. It was also observed a decrease in AR quantities in prostate after carcinogenesis in both KO and WT. Since AR expression may be regulated as a result of PI3K/AKT/mTOR pathway activation, it is suggested that the decrease of AR in KO may have occurred as a result of smaller quantities of mTOR in these animals. Moreover, it s suggested that TNF-α /TNFR-1 has key role in MMP2 expression, reducing the content of this enzyme in its absence, and that the decrease of MMP2 may be responsible to the accumulation of fibronectin in KO. Finally, it was observed a similar increase in apoptosis in WT and KO group after carcinogenesis, showing clearly that TNFR-1 in this model was not involved in the induction of cell death. Therefore, it is concluded that the cytokine TNF-α, through its receptor TNFR-1 promoted cell proliferation and cell survival by activation of the AKT/mTOR pathway, which implicates its role in prostate carcinogenesis in this experimental model.

In cattles, most of pregnancy losses occurs at the beginning of gestation, notably from the 7th to the 16th day of the cycle, a period in which, the embryo depends entirely on the uterine environment to survive and to start their preimplantation growth. During this period, the embryonic death after embryo transfers performed in vitro (TE-IVP) or in vivo (TE-OM) is on average nearly twice as high as that produced by natural mating or artificial insemination (AI). The recipient sensitization against the paternal and maternal MHC molecules of allogeneic embryo might be one of the causes of high rates of pregnancy loss observed after TE. Studies in humans and in various species have pointed that the sensitization with conceptus antigens may affect the reproductive performance facilitating the recognition and the maternal acceptance of allogeneic embryo through induction of cytokyne and immunoregulatory cells in the the uterine microenvironment. The purpose of this study is to determine whether simultaneous or separate administration of paternal and maternal antigens in the uterus of the cows embryo recipientes, during the estrus, increases the expression of genes which can facilitate recognition and development of allogeneic embryos during early pregnancy. Forty-five crossbed cows were evaluated. The animals were divided in four treatments: T0: control; T1: Semen; T2: PBMCs and T3: PBMCs+Semen. The cows were estrus synchronized and received antigens in the uterine body on the estrus day. Uterine biopsies were collected in vivo on D0 for control, and after seven (D7) and fourteen (D14) days after the estrus and administration of antigens in order to evaluate the treatment effect on the uterine environment of the receiving at the moment of the anovoluation procedure would occur in TE-IVP, and during the period in which the bovine embryo would have their preimplantation growth, respectively. The gene expression was evaluated in real time PCR, and then transcribed from FOXP3, IDO, IL-10 and CSF-1 were detected in all RNA samples extracted from uterine biopsies. Semiquantitative analyses of relative gene expression among the control and the treat groups demonstrated that none of the treatments significantly incresed those gene expressions. Furthermore, at D14 all the treatments leaded to a decline in amount CSF-1 transcripts and, further, treatment with both antigens also to a drop in the abundance of IL-10 transcripts. In conclusion, the isolated or simultaneous antigens admnistration in the in the uterus of IVP embryo recipient cows seems not to increase the maternal tolerance to alloantigens embryo nor benefit conditions for their growth and preimplantation development, at least with regard to the effect mediated by FOXP3, IDO, IL- 10 and CSF-1 on D7 and D14 in the estrous cycle.

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

High-fat diets induce the formation of inflammatory signals in the hypothalamus which reduces their ability to respond to the hormones leptin and insulin, involved with the control of satiety and food intake. The hypothalamus contains a large amount of glial cells highlighting the astrocytes, which are incorporated in heterogeneous groups of neurons that participate in the control circuit of energy homeostasis, additionally, they are involved in various processes, including the neuroinflammation. In this sense, the present study aimed to investigate the role of hypothalamic astrocyte responses in front of excess fatty acids and the interaction of these cells with neurons of the hypothalamus. For that, we used primary cell cultures of hypothalamic astrocytes and primary cultures of hypothalamic neurons. The medium removed from astrocyte cultures treated with fatty acid (estereato or palmitate) or LPS (positive control) was used on neuronal cultures for further in vitro study of synaptogenesis, also, measurement of cytokines present in conditioned medium was performed with the CBA kit mouse-Th1 /Th2/Th17 by flow cytometry with the aim of analyzing the profile of inflammatory response considering the different treatments. In front of this result, we have seen that the treatment with fatty acid promoted a profile of Th2 response (anti-inflammatory), especially when compared to Th1 profile (pro-inflammatory) of the LPS control. After the treatment, the total RNA of astrocytes was extracted for the sequencing of transcripts by RNA-Seq. Through the analysis of the genes differentially expressed between the groups, it was observed that treatment with fatty acid induced an inflammatory response, however, with lesser intensity in relation to treatment with LPS, and, at the same time, some genes related to neuroprotection were more expressed in astrocytes treated with fatty acid, collaborating with the data of the CBA. The culture of neuron was standardized in our laboratory and, subsequently, treated with conditioned medium. By immunostaining, we observed that treatment with the conditioned medium in astrocytes treated with fatty acids reduced the total synaptic density, primarily affecting excitatory synapses, on the other hand, the number of neurons was similar between treatments, however, with the presence of a reduced number of pyknotic nuclei after the treatment with fatty acids. These results suggest an active role of astrocyte in inflammation induced by the excess of fatty acids, participating in the modulation of the inflammatory response, apparently, with neuroprotective effects.

Angiogenesis and inflammation act simultaneously in several pathophysiological processes. The interactions with the extracellular matrix (ECM) are required in inflammatory angiogenesis. The non-enzymatic disintegrins comprise a family of peptides, low molecular weight, derived from the venom of snakes, which strongly inhibit the functions of integrins. In this study we evaluated the therapeutic effect of disintegrin DisBa-01, derived from the venom of the snake Rhinocerophis alternatus in inflammatory angiogenesis induced by synthetic implants in mice. Treatment with DisBa-01 inhibited the main key components of fibrovascular tissue induced by implants as inflammation and angiogenesis, without changing the fibrogenic component. This experimental model is the implementation of a synthetic matrix in the subcutaneous tissue of the animal, which induces the formation of fibrovascular tissue rich in inflammatory cells, blood vessels and MEC. The model of synthetic implants, also allows the simultaneous evaluation of inflammation, angiogenesis and tissue repair. The inhibitory effects of DisBa-01 were observed by MPO activity (representing activated neutrophils), NAG activity (representing activated macrophage), the chemokines CXCL -1, MCP-1 and the cytokine TNF- α. The anti-angiogenic activity was observed through the reduction of hemoglobin content, number of vessels, blood flow intra-implant by laser doppler and the levels of VEGF and FGF cytokines. The evaluation of DisBa-01 in multiple parameters provides additional evidence to the therapeutic potential of this disintegrin.

Currently obesity is a worldwide public health problem, characterized by excess body fat. It is known that obesity can cause musculoskeletal abnormalities that can negatively influence the tendons. The achilles tendon, tougher human body has a function to transmit power generated in the muscle to bone. Besides being influenced by the overweight, the tendon also has the ability to respond to physical exercise. This study aimed to observe morphological and biochemical changes in calcaneal tendon of mice subjected to a high-fat diet and exercise. Adults Swiss mice were used (n = 36), were divided into 4 groups: 1- sedentary normal diet (ND), 2- Normal diet and swimming (DNEX), 3- sedentary fat diet (DH) 4- fat diet and swimming (DHex). The diet was started from the 8th. week of life and training from the 15th. week, it is held for 8 consecutive weeks in aquarium with fixed dimensions. In order to verify that the high fat diet caused an overweight, food consumption, body weight and the weight of visceral adipose tissue were followed over these eight weeks .The calcaneal tendons were removed and for routine histological techniques and analyzes Biochemical. Cuts with 7 mm thick were stained with HE, Picrosirius and Von Kossa and later analyzed in ordinary light microscope. For biochemical analysis, the determination of proteins was performed by the Bradford method. The DH group had higher values of body weight and visceral adiposity, showing that high fat diet fed to animals was efficient. However, DHex group was observed a reduction of these values, indicating that physical activity has reversed these values. Morphological analysis showed no statistical differences in the number of fibroblasts in both groups. However, the exercise groups showed higher amounts of collagen and non-collagenous proteins. It was also observed the presence of calcifications in the tendons of mice fat diet group without exercise.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

The skin covers the entire body, because it is the largest organ of the body is composed of three distinct layers, it is extremely important physiological. The present study was conducted surgical incisions in rats and tested the effectiveness of two drugs, comfrey and silver sulfadiazine in tissue repair analyzing extracellular matrix, level of re-epithelialization of skin collagen quantification, counting of mast cells,Count of blood vessels, Determination of hemoglobin Dosage ,Dosage of NAG activity, and activity myeloperoxidase. Soon this work aims and main scope is to provide a more thorough analysis and scientific content of Symphytum officinale (comfrey) widely used in popular culture for its healing properties anti-inflammatory and healing in dermatological treatment comparing well with agent widely used silver sulfadiazine 1%. Concludes that the comfrey plays a key role in wound healing with its anti-inflammatory and certainly opens up new possibilities for action, based on the mechanism of degranulation of mast cells and neutrophils during the repair phase, decreased angiogenesis best arrangement of collagen and has the potential to provide better performance for the healing of wounds.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Fundação de Amparo a Pesquisa do Estado de Minas Gerais