Vascular endothelial growth factor A (VEGFA) modulates bovine placenta steroidogenesis in vitro

Our objectives were to investigate the possible role of VEGFA in bovine placenta steroid synthesis and to determine whether cloned derived placental cells present similar responses as non-cloned ones. Placental cells from cloned (term) and non-cloned (days 90, 150, 210 and term) pregnancies were isolated and treated with VEGFA (50 ng/ml) for 24, 48 or 96 h. Progesterone (P-4) and estrone sulfate (E1S) were assessed by RIA, while aromatase P450-positive cells were quantified using the point counting test. The percentages of steroidogenic and non-steroidogenic populations were determined by flow cytometry. VEGFA augmented or decreased P-4 and E1S concentrations as well as aromatase P450-positive cell density, depending on gestational age and time in culture. The percentage of steroidogenic cells was lower than that of non-steroidogenic ones for each culture time (P < 0.05). VEGFA treatment did not change the proportion of steroidogenic and non-steroidogenic cells. Placental cells derived from cloned pregnancies presented higher concentrations of E1S and P4 than the non-cloned group. However, aromatase P450-positive cells were similar between groups (P > 0.05). VEGFA treatment altered P-4 and E1S levels in placental cells depending on type of gestation. These results suggest that VEGFA acts locally in the bovine placenta to modulate steroidogenesis during gestation, but in a different pattern between cloned and non-cloned derived placental cells at term. Therefore, this factor can be considered an important regulator of placental development and function. (C) 2012 Elsevier Ltd. All rights reserved.

Antiparasitical chemotherapy in Chagas' disease cardiomyopathy: current evidence

Chronic chagasic cardiomyopathy affects 20% of Chagas disease patients. At present, Chagas disease chemotherapy uses nitrofurans, benznidazole (Rochagan (R), Rodanil (R), Roche) or nifurtimox (Lampit (R), Bayer). Treatment during acute and recent chronic phases in childhood effects 71.5% and 57.6%, respectively, of parasitological cure. However, in clinical trials during the late chronic phase, only 5.9% of parasitological cure were achieved. This review focuses on the benefit from aetiological treatment to avoid, stop or revert myocarditis. Divergent data gathered from clinical practice are not convincing to support prescription of aetiological treatment as routine for indeterminate and cardiac chronic patients.

Thrombocytopenia and platelet activity in dogs experimentally infected with Rangelia vitalii

The aim of this study was to evaluate the platelet count, coagulation time and platelet activity in dogs experimentally infected with Rangelia vitalii during the acute phase of the disease. For this study, 12 young dogs (females) were used, separated in two groups. Group A (uninfected control) was composed by healthy dogs (n=5), and group B consisted of R. vitalii-infected animals (n=7). After being inoculated with R. vitalii-infected blood, animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite five days post-inoculation (PI). Blood samples were collected on days 0, 10, 20 and 30 PI. The material collected was placed in tubes containing EDTA for quantification of platelets, citrate anticoagulant platelet aggregation, and measuring the clotting time. Right after blood collection on days 10 and 20 PI, dogs were anesthetized for collecting bone marrow samples. A significant reduction (P<0.01) of the number of platelets was observed in R. vitalii-infected blood, when compared with uninfected dogs on days 10 and 20 PI. Additionally, macro-platelets were observed only in infected dogs. Prothrombin time and activated partial thromboplastin time did not differ between infected and uninfected dogs. The megakaryocyte count increased (P<0.01) significantly in infected dogs when compared with uninfected ones on days 10 and 20 PI. Platelet aggregation decreased (P<0.01) significantly in infected dogs in comparison to the control on days 10 and 20 PI. Therefore, rangeliosis in dogs causes a severe thrombocytopenia during the acute phase of infection. This platelets reduction probably occurred due to splenic sequestration and/or immune-mediated thrombocytopenia. (C) 2011 Elsevier B.V. All rights reserved.

Simultaneous circulation of all four dengue serotypes in Manaus, State of Amazonas, Brazil in 2011

Introduction: Manaus, the capital city of the state of Amazon with nearly 2 million inhabitants, is located in the middle of the Amazon rain forest and has suffered dengue outbreaks since 1998. Methods: In this study, blood samples were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), aimed at identifying dengue virus serotypes. Results: Acute phase sera from 432 patients were tested for the presence of dengue virus. Out of the 432 patients, 137 (31.3%) were found to be positive. All the four dengue virus serotypes were observed. Conclusions: The simultaneous circulation of the four dengue serotypes is described for the first time in Manaus and in Brazil.

Deficient Regulatory T Cell Activity and Low Frequency of IL-17-Producing T Cells Correlate with the Extent of Cardiomyopathy in Human Chagas' Disease

Background: Myocardium damage during Chagas' disease results from the immunological imbalance between pro-and production of anti-inflammatory cytokines and has been explained based on the Th1-Th2 dichotomy and regulatory T cell activity. Recently, we demonstrated that IL-17 produced during experimental T. cruzi infection regulates Th1 cells differentiation and parasite induced myocarditis. Here, we investigated the role of IL-17 and regulatory T cell during human Chagas' disease. Methodology/Principal Findings: First, we observed CD4(+)IL-17(+) T cells in culture of peripheral blood mononuclear cells (PBMC) from Chagas' disease patients and we evaluated Th1, Th2, Th17 cytokine profile production in the PBMC cells from Chagas' disease patients (cardiomyopathy-free, and with mild, moderate or severe cardiomyopathy) cultured with T. cruzi antigen. Cultures of PBMC from patients with moderate and severe cardiomyopathy produced high levels of TNF-alpha, IFN-gamma and low levels of IL-10, when compared to mild cardiomyopathy or cardiomyopathy-free patients. Flow cytometry analysis showed higher CD4(+)IL-17(+) cells in PBMC cultured from patients without or with mild cardiomyopathy, in comparison to patients with moderate or severe cardiomyopathy. We then analyzed the presence and function of regulatory T cells in all patients. All groups of Chagas' disease patients presented the same frequency of CD4(+)CD25(+) regulatory T cells. However, CD4(+)CD25(+) T cells from patients with mild cardiomyopathy or cardiomyopathy-free showed higher suppressive activity than those with moderate and severe cardiomyopathy. IFN-gamma levels during chronic Chagas' disease are inversely correlated to the LVEF (P = 0.007, r = -0.614), while regulatory T cell activity is directly correlated with LVEF (P = 0.022, r = 0.500). Conclusion/Significance: These results indicate that reduced production of the cytokines IL-10 and IL-17 in association with high levels of IFN-gamma and TNF-alpha is correlated with the severity of the Chagas' disease cardiomyopathy, and the immunological imbalance observed may be causally related with deficient suppressor activity of regulatory T cells that controls myocardial inflammation.

Imagens contrastadas por manganês na fase aguda da epilepsia induzida por pilocarpina

As imagens de ressonância magnética são úteis no estudo de modelos experimentais de epilepsia do lobo temporal. A técnica manganese-enhanced MRI (MEMRI) é de interesse por combinar os efeitos provocados pelo manganês no aumento do contraste de populações celulares ativadas, ao competir com o cálcio na transmissão sináptica. Assim, o propósito deste estudo foi investigar a evolução temporal do contraste provocado pelo manganês na fase aguda da epilepsia do lobo temporal induzida por pilocarpina sistêmica e compará-las à expressão da proteína c-Fos. Nessa fase, a intensidade do sinal MEMRI foi analisada em três diferentes pontos temporais (5, 15 ou 30 minutos) após o início do status epilepticus (SE). O grupo que foi mantido em status epilepticus por 30 minutos mostrou diminuição na intensidade de sinal no CA1 e giro denteado (GD). Não houve diferenças entre o Grupo Controle e os outros grupos tratados com pilocarpina. A expressão da proteína c-Fos, nos mesmos animais, mostrou que, mesmo no status epilepticus de curta duração (5 minutos) já há ativação celular máxima nas sub-regiões do hipocampo (GD, CA1 e CA3). Nas condições experimentais testadas, nossos dados sugerem que o sinal MEMRI não foi sensível para identificar variações detectáveis da ativação celular na fase aguda do modelo de pilocarpina. Nossos achados não são consistentes com a ideia que o contraste por manganês reflete primariamente alterações na atividade celular durante o SE quando outros elementos modificadores do sinal podem atuar.

The effect of beta-blockade on myocardial remodelling in Chagas' cardiomyopathy

OBJECTIVE: Chagas' disease has spread throughout Latin America because of the high rate of migration among these countries. Approximately 30% of Chagas' patients will develop cardiomyopathy, and 10% of these will develop severe cardiac damage leading to heart failure. Beta-blockade improves symptoms and survival in heart failure patients; however, its efficacy has not been well established in Chagas' disease. We evaluated the role of carvedilol in cardiac remodeling and mortality in a Chagas' cardiomyopathy animal model. METHODS: We studied Trypanosoma cruzi infection in 55 Syrian hamsters that were divided into three groups: control (15), infected (20), and infected + carvedilol (20). Animals underwent echocardiography, electrocardiography, and morphometry for collagen evaluation in ventricles stained with picrosirius red. RESULTS: The left ventricular diastolic diameter did not change between groups, although it was slightly larger in infected groups, as was left ventricular systolic diameter. Fractional shortening also did not change between groups, although it was slightly lower in infected groups. Collagen accumulation in the interstitial myocardial space was significantly higher in infected groups and was not attenuated by carvedilol. The same response was observed in the perivascular space. The survival curve showed significantly better survival in the control group compared with the infected groups; but no benefit of carvedilol was observed during the study. However, in the acute phase (up to 100 days of infection), carvedilol did reduce mortality. CONCLUSION: Carvedilol did not attenuate cardiac remodeling or mortality in this model of Chagas' cardiomyopathy. The treatment did improve survival in the acute phase of the disease.

Liver accumulation of Plasmodium chabaudi-infected red blood cells and modulation of regulatory T Cell and dendritic cell responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (PciRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.

Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows

In beef cattle, the ability to conceive has been associated positively with size of the preovulatory follicle (POF). Proestrus estradiol and subsequent progesterone concentrations can regulate the endometrium to affect receptivity and fertility. The aim of the present study was to verify the effect of the size of the POF on luteal and endometrial gene expression during subsequent early diestrus in beef cattle. Eighty-three multiparous, nonlactating, presynchronized Nelore cows received a progesterone-releasing device and estradiol benzoate on Day–10 (D 10). Animals received cloprostenol (large follicle-large CL group; LF-LCL; N ¼ 42) or not (small follicle-small CL group; SF-SCL; N ¼ 41) on D 10. Progesterone devices were withdrawn and cloprostenol administered 42 to 60 hours (LF-LCL) or 30 to 36 hours (SF-SCL) before GnRH treatment (D0). Tissues were collected at slaughter on D7. The LF-LCL group had larger (P < 0.0001) POF (13.24 0.33 mm vs. 10.76 0.29 mm), greater (P < 0.0007) estradiol concentrations on D0 (2.94 0.28 pg/mL vs. 1.27 0.20 pg/mL), and greater (P < 0.01) progesterone concentrations on D7 (3.71 0.25 ng/mL vs. 2.62 0.26 ng/mL) compared with the SF-SCL group. Luteal gene expression of vascular endothelial growth factor A, kinase insert domain receptor, fms-related tyrosine kinase 1, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid deltaisomerase 7 was similar between groups. Endometrial gene expression of oxytocin receptor and peptidase inhibitor 3, skin-derived was reduced, and estrogen receptor alpha 2, aldo-keto reductase family 1, member C4, and lipoprotein lipase expression was increased in LF-LCL versus SF-SCL. Results support the hypothesis that the size of the POF alters the periovulatory endocrine milieu (i.e., proestrus estradiol and diestrus progesterone concentrations) and acts on the uterus to alter endometrial gene expression. It is proposed that the uterine environment and receptivity might also be modulated. Additionally, it is suggested that increased progesterone secretion of cows ovulating larger follicles is likely due to increased CL size rather than increased luteal expression of steroidogenic genes.

The role of Nox2-derived ROS in the development of cognitive impairment after sepsis

BACKGROUND: Sepsis- associated encephalopathy (SAE) is an early and common feature of severe infections. Oxidative stress is one of the mechanisms associated with the pathophysiology of SAE. The goal of this study was to investigate the involvement of NADPH oxidase in neuroinflammation and in the long-term cognitive impairment of sepsis survivors. METHODS: Sepsis was induced in WT and gp91phox knockout mice (gp91phox-/-) by cecal ligation and puncture (CLP) to induce fecal peritonitis. We measured oxidative stress, Nox2 and Nox4 gene expression and neuroinflammation in the hippocampus at six hours, twenty-four hours and five days post-sepsis. Mice were also treated with apocynin, a NADPH oxidase inhibitor. Behavioral outcomes were evaluated 15 days after sepsis with the inhibitory avoidance test and the Morris water maze in control and apocynin-treated WT mice. RESULTS: Acute oxidative damage to the hippocampus was identified by increased 4-HNE expression in parallel with an increase in Nox2 gene expression after sepsis. Pharmacological inhibition of Nox2 with apocynin completely inhibited hippocampal oxidative stress in septic animals. Pharmacologic inhibition or the absence of Nox2 in gp91phox-/- mice prevented glial cell activation, one of the central mechanisms associated with SAE. Finally, treatment with apocynin and inhibition of hippocampal oxidative stress in the acute phase of sepsis prevented the development of long-term cognitive impairment. CONCLUSIONS: Our results demonstrate that Nox2 is the main source of reactive oxygen species (ROS) involved in the oxidative damage to the hippocampus in SAE and that Nox2-derived ROS are determining factors for cognitive impairments after sepsis. These findings highlight the importance of Nox2-derived ROS as a central mechanism in the development of neuroinflammation associated with SAE.

Intervention to reduce C-reactive protein determination requests for acute infections at an emergency department

Introduction C-reactive protein (CRP) levels rise during inflammatory processes and have been ordered for rheumatic disease follow-up since the 1950s. The number of tests ordered in the emergency setting has increased, but without evident improvement in medical care quality. Objective To determine the pattern of CRP determinations in the emergency department (ED) of a university hospital in Sao Paulo, Brazil, and to evaluate the effect of an intervention with staff and students about the best use of the test in the ED. Methods Data regarding CRP testing requests, related diagnoses and the number of monthly consultations in the hospital ED were analysed before and after the intervention. Because of an increase in CRP measurement requests from 2007 to 2009, the author started discussing the role of CRP determinations in the medical decision-making process in early 2010. Staff and faculty members openly discussed the pattern of requests in the hospital and related current medical literature. During 2010, the medical staff worked as multipliers to change the behaviour of new students and residents. The results of the first 4 months after the intervention were presented at another general meeting in July 2010. Results From 2007 to 2009, there were 11 786 CRP measurement requests with a clear exponential trend. After the intervention, during the calendar year 2010, there was a 48% reduction in adjusted annual CRP requests. Pneumonia, fever and urinary tract infections were the most common reasons for CRP requests. Discussion Inexpensive, well-directed, interactive educational interventions may affect professional behaviour and curb rates of laboratory tests.

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective ATI receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P) H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT(1)-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. (c) 2012 Elsevier Inc. All rights reserved.

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na2SO4, (NH4)(2)SO4, MgSO4, LiSO4, Na2HPO4, sodium phosphate buffer (pH 7.0), NaH2PO4, K2HPO4, potassium phosphate buffer (pH 7.0), KH2PO4, C6H8O7, Na3C6HSO7, KCHO2, NaCHO2, NaCO3, NaHCO3, C2H4O2, sodium acetate buffer (pH 4.5), and NaC2H3O2] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents. (C) 2011 Elsevier Inc. All rights reserved.

Maternal immune activation in late gestation enhances locomotor response to acute but not chronic amphetamine treatment in male mice offspring: Role of the D1 receptor

Exposure to elevated levels of maternal cytokines can lead to functional abnormalities of the dopaminergic system in the adult offspring, including enhanced amphetamine (AMPH)-induced locomotion. Therefore, it seems reasonable to consider that offspring of challenged mothers would behave differently in models of addictive behavior, such as behavioral sensitization. Thus, we sought to evaluate the effects of prenatal exposure to lipopolysaccharide (LPS) on the locomotor response to acute and chronic AMPH treatment in male mice offspring. For this purpose, LPS (Escherichia coli 0127:B8; 120 mu g/kg) was administered intraperitoneally to pregnant Swiss mice on gestational day 17. At adulthood, male offspring were studied under one of the following conditions: (1) locomotor response to acute AMPH treatment (2.5 or 5.0 mg/kg) in an open field test; (2) behavioral sensitization paradigm, which consists of a daily injection of AMPH (1.0 mg/kg) for 10 days and observation of locomotion in the open field on days 1, 5, 10 (development phase), 15 and 17 (expression phase). The LPS stimulated offspring showed enhancement of the locomotor-stimulant effect after an acute AMPH challenge in comparison to baseline and saline pre-treated mice. They also showed development of behavioral sensitization earlier than the saline pre-treated group, although no changes between saline and LPS pre-treated groups were observed on development or expression of locomotor behavioral sensitization to AMPH. Furthermore, there was up-regulation of D1 receptor protein level within striatum in the LPS-stimulated offspring which was strongly correlated with increased grooming behavior. Taken together, our results indicate that motor and dopaminergic alterations caused by maternal immune activation are restricted to the acute AMPH challenge, mostly due to up-regulation of the D1 receptor within the mesolimbic and nigrostriatal pathways, but no locomotor differences were observed for behavioral sensitization to AMPH. (C) 2012 Elsevier B.V. All rights reserved.

Protective effects of aerobic exercise on acute lung injury induced by LPS in mice

Abstract Introduction The regular practice of physical exercise has been associated with beneficial effects on various pulmonary conditions. We investigated the mechanisms involved in the protective effect of exercise in a model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Methods Mice were divided into four groups: Control (CTR), Exercise (Exe), LPS, and Exercise + LPS (Exe + LPS). Exercised mice were trained using low intensity daily exercise for five weeks. LPS and Exe + LPS mice received 200 µg of LPS intratracheally 48 hours after the last physical test. We measured exhaled nitric oxide (eNO); respiratory mechanics; neutrophil density in lung tissue; protein leakage; bronchoalveolar lavage fluid (BALF) cell counts; cytokine levels in BALF, plasma and lung tissue; antioxidant activity in lung tissue; and tissue expression of glucocorticoid receptors (Gre). Results LPS instillation resulted in increased eNO, neutrophils in BALF and tissue, pulmonary resistance and elastance, protein leakage, TNF-alpha in lung tissue, plasma levels of IL-6 and IL-10, and IL-1beta, IL-6 and KC levels in BALF compared to CTR (P ≤0.02). Aerobic exercise resulted in decreases in eNO levels, neutrophil density and TNF-alpha expression in lung tissue, pulmonary resistance and elastance, and increased the levels of IL-6, IL-10, superoxide dismutase (SOD-2) and Gre in lung tissue and IL-1beta in BALF compared to the LPS group (P ≤0.04). Conclusions Aerobic exercise plays important roles in protecting the lungs from the inflammatory effects of LPS-induced ALI. The effects of exercise are mainly mediated by the expression of anti-inflammatory cytokines and antioxidants, suggesting that exercise can modulate the inflammatory-anti-inflammatory and the oxidative-antioxidative balance in the early phase of ALI.