Repositório RCAAP

Toxoplasma gondii is an obligate intracellular parasite that presents different strain types and degrees of virulence. The strains are classificated like clonal or typical and exotic or atypical. This parasite presents several evasion mechanisms, which guarantees the successful replication in the host cell and, between these mechanisms, there is ability to modulate apoptosis of infected cells as well of cells in the microenvironment of infection. The present work aimed to evaluate de ability of two atypical strains of T. gondii (Udi1CH-05 and Udi2CH-05) on modulation of apoptosis in BeWo cells, the susceptibility of these cells by infection, as well as the influence of those strains on the standard of secretion of cytokines by the trophoblastic cells lineage. These parameters were evaluated from the perspective of the immediate origin of the strains derived directly from cell culture or from animals and maintained for a short period in culture (here called Calomys callosus / culture). The Udi1CH-05 culture category upregulated apoptosis from 12 hours post infection, and from 2 hours of infection by Udi2CH-05, culture category, apoptosis was uncontrolled in BeWo cells. The condition C. callosus / culture of both strains didn t control apoptosis when compared to uninfected control. BeWo cells infected with Udi2CH-05, C. callosus / culture category produce high levels of IL-12 in the early stages of infection, suggesting a typical inflammatory response. Levels of TNF-α were higher in the early stages of infection and with a gradual decrease production, coinciding with failure to induce apoptosis when cells were infected BeWo Udi2CH-05, C. callosus / culture category. The levels of IL-10 found during infection of BeWo cells by Udi1CH-05 indicate that other cytokines may be involved in the control of infection, whereas this cytokine acts in controlling the immune response induced by infection of cells by BeWo Udi2CH-05, C. callosus / culture category. The production of TGF-β detected by infection Udi1CH-05 C. callosus / culture or Udi2CH-05 of both categories, shows that this cytokine acts in the immunoregulation of infected cells, whereas infection by Udi1CH-05, culture category, suggests that other anti-inflammatory cytokines can participate in immunoregulation, since the increase the apoptosis rate coincided with the decreased production of TGF-β.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Ethanol consumption is one of the biggest risk factors for world morbidity and mortality, once its dependence is a serious public health problem. In laboratory animals, some behaviors related to ethanol dependence can be investigated, like conditioned place preference (CPP) and the symptoms of withdrawal syndrome. Stress is a factor of major importance for beginning, maintenance and reinstatement of abuse psychoactive drugs and can regulate neurochemical alterations induced by chronic administration of these substances. The objective of this study was investigate the effects of forced swimming stress, concomitant to ethanol chronic administration (in liquid diet), on CPP and ethanol withdrawal syndrome, and its relation to alterations of dopaminergic and serotoninergic neurotransmission in mice. When we evaluate anxiety parameters during ethanol withdrawal, it was not found alterations in both elevated plus maze and open field (OF). However, exposure to ethanol alone induced reduction of exploratory activity in OF, correlating to increase of dopaminergic turnover (ratio between dopaminergic metabolites by dopamine) observed in prefrontal cortex (PFC), due to the same treatment. PFC alteration can still be responsible by increase of ethanol seeking, analyzed in the CPP procedure. Nevertheless, in nucleus acumbens (NAc), it was found reduction of dopaminergic turnover in animals exposed to stress alone, which may be predisposed to increase of ethanol CPP in this experimental group. In animals exposed concomitantly to ethanol and stress, it was not found alterations in both CPP as in dopaminergic turnover in PFC or NAc. Absence of alterations in this group may be related with increase of dopaminergic storage in amygdala. Thus, our study indicates that stress as well as ethanol induces dopaminergic alterations in different encephalic regions and this can be responsible by characteristic behaviors of withdrawal syndrome and ethanol seeking. Therefore, stress has an important role for ethanol addiction development and it is necessary more studies to understand the neurochemical pathways responsible by this interaction.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Chagas´ disease affects 8-10 million people in the Americ. In the chronic phase of this disease, patients can develop alterations in the gastrointestinal tract, and one of them is called megacolon. It is accepted that the disturbance of immune system and enteric nervous system (ENS) is associated with this form development. Some substances act in both systems and fulfill a link between the nervous and immune systems. One of these substances is the serotonin (5-HT) that composes the group of biogenic amines (neurotransmitters). About 90% of serotonin existent in the human body is produced in the intestine. Previous data indicated that the intestinal levels of serotonin may provide a balance between ENS and immune system. To evaluate whether serotonin intestinal levels are related with regulation of immune system, we investigated the relation among 5-HT expression, intensity of inflammatory process and denervation in colon samples from patients with megacolon. We evaluated, by immunohistochemistry technique, samples of colon from patients submitted to necropsy or surgical procedures at Federal University of Goias (Goiânia, Minas Gerais, Brazil). This work was approved by UFU Research Ethics Committee (ETIC n° 110/11). It was used antibodies linked with immunofluorescent markers to measure the presence of serotonin, CD8 lymphocytes and peripherin (neuronal marker). Our results indicated that low levels of serotonin are associated with intense inflammatory process, high degree of inflammation and with constipation intestinal. These data suggested that serotonin may act in the intestine as a regulator of inflammation process and avoid the neuronal destruction.

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Gestational diabetes is defined as any degree of reduction of glucose tolerance, whose early detection occurs during pregnancy and may or may not persist after delivery. Studies indicate that the offspring of diabetic mothers display abnormal changes in development as well as metabolic disorders. The prostate gland is androgen-dependent and highly sensitive to hormonal disorders as well as to changes in blood glucose. Recent studies have shown that diabetes causes drastic atrophy in the prostate of rodents, interferes with the proliferation and apoptosis of epithelial cells, alters expression of androgen receptor and leads to significant stromal remodeling. However, little is known about the effects of diabetes later in embryonic development of the prostate. This study was important because there are no studies that show the effects of diabetes on the development of prostate rats whose mothers were diabetic. The aim of this study was to evaluate the organization of epithelial and stromal prostate of adult rats whose embryonic development was under the influence of gestational diabetes. Diabetes was induced in Wistar pregnant females rats by intraperitoneal injection of alloxan (100mg/kg body weight). Male rats offspring of diabetic (PD) and normal mothers (PC) were killed at 12 weeks of age and the ventral prostate was removed, weighed and fixed. The prostate was evaluated for general histology, frequency of collagen fibers, cell death (TUNEL), immunohistochemistry (fibroblasts, fibronectin, smooth muscle cells, cell proliferation - PCNA), western blotting (PCNA, metalloproteinases - MMP-2 and -9, androgen receptors, AKT), zymography and total antioxidant activity and catalase. The prostate of PD animals showed lower weight compared to the PC group and these animals also exhibited hyperglycemia and a significant reduction of testosterone. Cell proliferation as well as the levels of AKT and AR were higher in the offspring of diabetic mothers. The stromal analysis showed that the PD group showed a reduction of -actin, while vimentin and fibronectin did not change. The activity of MMP-2 is increased in PD animals, although the distribution of this MMP was lower in this group. This finding was associated with a significant decrease in collagen distribution. The prostate of the offspring of diabetic rats also showed reduced activity of catalase and total antioxidant activity. The results indicate that rats that developed in a diabetic environment have glycemic and hormonal changes that impact on structural changes in the ventral prostate. Among them, there is greater cell proliferation triggered by AR and AKT and stromal remodeling, which was characterized by increased activity of MMP-2 and collagen degradation. This situation may interfere with tissue architecture and homeostasis glandular. Moreover, the stroma is negatively affected by a decrease in smooth muscle cells, possibly impairing the contraction of the gland as well as the epithelium-stroma interaction stimulated by these cells. This work also shows that gestational diabetes may increase oxidative stress by reducing the activity of enzymes control.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Saliva is an oral fluid abundant with molecules, being non-invasive, easy collection and with minimal discomfort to the animal, with great potential for the overall monitoring of health and disease, with huge prognostic values. The Immunoglobulin A is the first in line of immunological attack of mucosal tissue. The Canine Gastroenteritis is the most common case series in medical veterinary clinic originating through various sources, as the viral the most common. The main objective of the present study is the quantification of IgA in the saliva of dogs affected by gastroenteritis and to compare the results with the amounts found in healthy dogs as well as quantifies the hematologic and serum biochemistry, confirming if they represent standards of differential diagnosis for the disease here investigated. The GEV group presented eosinopenia, neutropenia, monocytopenia, serum hypoproteinemia, hypoalbuminemia serum and salivary hyperproteinemia. The serum IgA showed a decrease when comparing with the control group. For group GEG presented eosinopenia, neutrophilia with left shift. Also showed microcytosis, hypoproteinemia, hypoalbuminemia and salivary hyperproteinemia but with no statistical difference for these parameters. We conclude that the measurements of total leukocytes along with the segmented neutrophils, eosinophils, dosage and serum albumin PT and also the determination of PT showed increased salivary IgA and quantification decreased, suggesting parameters for diagnosing disease. However, because the definition of reference value missing, most studies dosing canine IgA should be performed.

Changes in the chromatin of sperm have been linked to subfertility in several species of mammals. One of the factors that leads to these changes is protamine deficiency. In this study we analyzed the location of changes of chromatin in the sperm of bulls and the relationship with the markup of protamine immunocytochemistry. Semen samples of fertile and subfertile bulls were examined through computational analysis of images of smears stained with toluidine blue (TB), DAPI and marked with protamine 1 (PR1) antibody. For the evaluation TB blades were used in two separate analyses: a conventional, where we obtained the value of decompression percentage (DESC%) and average chromatin heterogeneity (HET) of sperm, and an alternative, which labels changes according to the location of the change in the head of the sperm and percentage of sperm with each type of change in the samples. In both was compared samples from fertile and subfertile bulls. Conventional analysis showed a significant increase (p = 0.01) both DESC% and HET in subfertile samples. On the other hand, alternative analysis showed an increase in subfertile samples of changes in basal half (AMB) p = 0.05 and totally changed heads (CTA) p = 0.04. This alternative computational analysis was also applied to staining with DAPI, where the subfertile samples showed an increase in base alterations (AB) p = 0.02. The dispersed changes (AD) were higher with p = 0.02 in the fertile samples. On labeling with anti-PR1, changes in the base (AB) were higher in subfertile samples with p = 0.01. With these results we conclude that, with the alternative technique was possible to propose a classification of changes in staining and immunolabelling, but this technique showed a low efficiency to distinguish fertile from subfertile bulls. DAPI staining assessed in confocal microscopy is presented as a possible alternative for staining with AT. The conventional technique with AT showed the best result for differentiation between fertile and subfertile groups. PR1 Immunostaining showed that this protein is distributed throughout the bull sperm head, and that chromatin alterations in these cells have no relation with the amount of PR1.

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Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Snakes venoms are a complex mixture of proteins, organic and inorganic compounds. Some of the protein, enzymatic or non-enzymatic compounds are able to interact with platelet receptors, causing hemostatic disorders such as blood incoagulability, hemorrhage and thrombosis, as well as to interact with components of the extracellular matrix, activating or inhibiting cell growth. Because of its medical importance, these compounds acting on hemostasis have been isolated from the venom of snakes and studied as potential therapeutic in the treatment and diagnosis of diseases such as thrombosis and cancer. The present study aimed to purify and to characterize a toxin present in the venom of Bothrops alternatus. The toxin called Bothalternina was purified using columns size ion-exchange on DEAE-Sephacel and Reversed Phase (C2/C18). The analysis of two-dimensional electrophoresis showed that Bothalternina has an apparent molecular mass of 26 kDa and pI to 4.6. The amino acid sequence of the N-terminal region was carried out by Edman degradation method and revealed the following sequence: (IISPPVCGNELLEVGEECDCGTPENCQNECCDA), which showed identity with the PIII class SVMPs. Bothalternina had a specific inhibitory effect on platelet aggregation induced by ristocetin in platelet rich human plasma. On the other hand, the toxin did not inhibit the ADP and epinephrine-induced aggregation. Bothalternina was also able to inhibit the growth of HeLa tumor cells. This toxin can be of medical interest because it was shown to inhibit platelet aggregation and tumor cell growth.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Galectin-3 (Gal-3) is a protein of the lectin-family, has affinity for β-galactose-containing carbohydrates, and can be localized in nucleus, cytoplasm, membrane associated or secreted. This protein is involved in many immunoregulatory processes, such as DC/T lymphocyte adhesion, inflammatory responses and cell migration toward inflammatory foci and cell proliferation. It was also seen that the T. cruzi infection, that is the etiological agent of Chagas disease, increases the expression of Gal-3. Thus, in this paper we aim to explore the biological activities of galectin-3 in acute and chronic T. cruzi experimental infection. Mice C57/BL6 Wild-Type (WT) and galectin-3 knockout (Gal-3KO) were infected intraperitoneally and was evaluate parasitaemia, recruitment of inflammatory cells in the peritoneal cavity, production of cytokines in spleen and heart and cardiac fibrosis. The data presented here demonstrate that the lack of Galectin-3 enhanced the parasitaemia and reduced the recruitment of leukocytes. In heart samples, we observed an increased secretion of TNF-α and IFN-γ in WT while in galectin-3 knockout mice we detected increased production of IL-1β and IL-4 during the acute phase. This scenario may have accounted to the higher infection rate during acute infection observed in knockout mice. We observed that in the chronic phase of infection the heart tissue of WT mice showed an immune response to Th2 profile, important to control tissue damage, with basal levels of IFN-γ and TNF-α, decrease in the concentration of IL-1β and increased IL-4. The increase in IL-4 is important for reducing heart damage and was not observed in animals Gal-3 KO. In chronic phase we observed an increased recruitment of mastocyte in Gal-3 KO animals and larger fibrosis of the heart. Therefore, the Gal-3 showed chemotactic and immunoregulatory functions that are needed to control the acute phase of infection and decreased chronic heart damage.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

After peripheral nervous injuries several modifications cellular and molecular develop to promote axonal regeneration. The axons, disconnected from their cell bodies, suffer from Wallerian degeneration. Products of this degeneration are eliminated by Schwann cells and macrophages. In the CNS, in the cell bodies, changes called plastic responses of the neuron and glia occurs in the spinal cord, highlighting the astrocytes plays an important role in this plasticity. Studies have shown that Angiotensin-(1-7), a System Renin-Angiotensin metabolite, participates in actions involving central behavioral aspects, learning and memory, by influencing the neuroplasticity. In this sense, the present work investigated the functional recovery after peripheral lesion of the sciatic nerve in rodents treated with the Ang-(1-7). For that, we procedured with the nerve crushing injury, with preservation of the epineurium. During two weeks after injury, was carried out to test the functional index of the sciatic nerve that evaluates the footprint during gait of the animals. In addition, the nerve was assessed axonal regeneration and the activity of Schwann cells by immunohistochemistry for neurofilament and S-100. In front of these results, we believe that treatment with Ang-(1-7) interferes negatively, possibly, delaying the responses of Schwann cells, which reflects a less axonal organization together with a slower return of nerve function. The lumbar spinal cords, where are the neuronal cell bodies that had their axons injured, were also stained with GFAP, synaptophysin and Mas antisera, for assessing, respectively, the astroglial activity, synaptic density and the Ang-(1-7) receptor. The treatment with Ang-(1-7) showed, in the spinal cord, reduction of the astrocytic activity and lower synaptic density after injury. Also, it was observed that the expression of Mas is changed by axonal injury, but treatment with Ang-(1-7) reduces this expression after injury by comparing the control animals with the treated animals. In this way, we can conclude that the treatment with Ang-(1-7) can influence the peripheral and central responses after nerve injury, apparently influencing the glial cells responsible, at least in part, by neuroinflammation reaction that occurs after axonal damage.