Objetivou-se com este estudo compreender as dinâmicas das temperaturas corporais em fêmeas da raça Nelore, e relacionar os aspectos fisiológicos da termorregulação com as respostas celulares pela expressão de proteínas de choque térmico. O projeto foi desenvolvido no Laboratório de Biometeorologia e Etologia, da Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo, Pirassununga-SP. Foram utilizadas 20 vacas Nelore pluríparas, cíclicas, mantidas em sistema de pastejo. O período experimental precedeu a estação de monta de 2013 e terminou na inseminação artificial. Para classificar os animais quanto ao gerenciamento de calor foi monitorada a temperatura vaginal durante seis dias por meio de 16 data loggers (n=16). Com o intuito de melhor compreender o gerenciamento de calor, outras respostas fisiológicas, nomeadamente temperatura retal, caudal, ocular, frequência respiratória e taxa de sudação foram colhidas durante os quatro dias finais do monitoramento da temperatura vaginal. Para expressão gênica relativa colheram-se amostras de sangue (n=20). Foram realizados choques térmicos in vitro a 38ºC, 40ºC e 42ºC por duas horas. Após tratamento térmico obteve-se o pellet de leucócitos para posterior extração do RNA pelo método TRIzol. Foram escolhidas 10 vacas com os melhores resultados de concentração de RNA, 5 pertencentes ao grupo de vacas eficientes quanto ao gerenciamento de calor, e 5 não eficientes (n=10). A RT-PCR foi realizada com o kit SuperScript III. A reação polimerase em cadeia em tempo real (qPCR) ocorreu no aparelho StepOnePlus® Applied Biosystem utilizando como marcador fluorescente o SYBR® Green para os genes alvo validados HSPA1A, HSPD1, HSP90AA1, HIF1A e endógenos ACTB, RPL15 e PPIA. Os dados foram analisados por ANOVA, regressão e correlação do SAS 9.2. As vacas foram classificadas em eficientes e não eficientes por meio de coeficientes angulares, advindos da regressão das temperaturas vaginais durante períodos de ganho e perda de calor. As vacas eficientes apresentaram menores temperaturas retais 37,65ºC (P<0,01) e maior taxa de sudação 528,73 g. m-². h-¹ (P<0,06). A nível celular, o aumento programado in vitro da temperatura não aumentou quantidade de transcritos, promovendo manutenção à 38ºC e 40ºC e declínio à 42ºC. A ordem decrescente da abundância de transcritos foi HSPA1A, HSPD1 e HSP90AA1. Pode-se concluir que vacas Nelore com diferentes gerenciamentos de calor apresentam respostas termorregulatórias diferentes. A HSPA1A apresentou a maior abundância de transcritos sendo considerada como marcador molecular para termotolerância, por ser a mais sensível à temperatura e bem conservada. Não foi observado diferença nas expressões gênicas relativas das proteínas de choque térmico entre os animais classificados quanto ao gerenciamento de calor. / The aim of this study was to understand the dynamics of body temperatures in Nellore females, and relate the physiological aspects of thermoregulation with cellular responses by the expression of heat shock proteins. The project was developed in Biometeorology and Ethology Laboratory, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga-SP. Were used 20 Nellore pluriparous cows, cyclical, kept in grazing system. The experimental period preceded the 2013\' breeding season and ended up in artificial insemination. To classify animals in relation to heat management the vaginal temperature was monitored for six days through 16 data loggers (n=16). In order to better understand the heat management, other physiological responses, including rectal, tail and eye temperatures, respiratory rate and sweating rate were taken during the final four days of vaginal temperature monitoring. For gene expression was harvested blood samples (n=20). in vitro heat shocks were performed at 38ºC, 40ºC and 42ºC during two hours. After the heat treatment was obtained the leukocyte pellet for later RNA extraction by TRIzol method. Ten cows were chosen with the best RNA concentration results, five belonging to the group of cows efficient on heat management and five inefficient (n = 10). RT-PCR was performed with SuperScript III kit. The real-time polymerase chain reaction (qPCR) occurred in StepOnePlus® Applied Biosystems instrument using the SYBR® Green as fluorescent marker to the target validated genes HSPA1A, HSPD1, HSP90AA1, HIF1A and the endogenous ACTB, RPL15 and PPIA. Data were analyzed using ANOVA, regression and correlation SAS 9.2. The cows were classified as efficient and inefficient through angular coefficients, derived from regression of vaginal temperatures for the gain and heat loss periods. Efficient cows had lower rectal temperature 37.65ºC (P<0.01), higher sweating rate 528.73 g. m ². h-¹ (P<0.06). At cellular level, the increase of in vitro programmed temperature has not increased the transcripts amount, promoting the maintenance at 38ºC and 40ºC and decline at 42ºC. The decreasing order of transcripts amount was HSPA1A, HSPD1 and HSP90AA1. It can be concluded that Nellore cows with different heat managements has different thermoregulatory responses. The HSPA1A showed the highest transcripts abundance being considered as a molecular marker for thermotolerance, for being more sensitive to temperature and better conserved. There was no difference in gene expression for heat shock proteins between animals classified by heat management.

Claritromicina e clofazimina são utilizadas no tratamento da hanseníase e em infecções causadas pelo complexo Mycobacterium avium, comuns em portadores do HIV. Devido à escassez de dados sobre a toxicidade de esquemas terapêuticos que associam estes fármacos, este estudo teve por objetivo avaliar os efeitos adversos desta terapia, em ratos machos Wistar, por meio da determinação de parâmetros hematológicos, hemostáticos e bioquímicos e correlação destes parâmetros com a dose e concentração plasmática dos medicamentos, em regime de doses únicas e múltiplas. Para tanto foram realizados: a) contagem global e específica de leucócitos (método manual) e ensaios de fagocitose e burst oxidativo de neutrófilos (citometria de fluxo); b) contagem de plaquetas (método manual), tempo de protrombina, tempo de tromboplastina parcial ativada, níveis plasmáticos dos fatores VII e X (método automatizado); c) níveis séricos de gama-glutamiltransferase (método cinéticocolorimétrico) e bilirrubinas total e direta (método colorimétrico); d) concentrações plasmáticas dos fármacos (Cromatografia Líquida de Alta Eficiência). Não houve diferenças entre as concentrações plasmáticas dos fármacos administrados em monoterapia ou politerapia. Entretanto, tanto clofazimina como claritromicina tiveram redução das concentrações plasmáticas em regime de doses múltiplas, quando comparadas à dose única. Houve aumento do número de leucócitos (dose múltipla) e de células polimorfonucleares (doses única e múltipla) nos grupos tratados com claritromicina em monoterapia ou associada à clofazimina, e redução das células mononucleares, em doses única e múltipla, nos mesmos grupos. Os fármacos parecem inverter a proporção entre células mono e polimorfonucleares. Observou-se aumento do burst oxidativo nos animais tratados com os fármacos tanto em monoterapia como em regime de politerapia. Entretanto, não houve diferença entre os tratamentos com os fármacos em relação ao controle DMSO, em dose única. Em doses múltiplas, os tratamentos com clofazimina e claritromicina em monoterapia ou politerapia estimularam o aumento do burst oxidativo (p < 0,0001) em relação ao controle DMSO. Não foram verificadas diferenças na fagocitose entre os grupos tratados e controle, tanto em dose única como em doses múltiplas. Tempo de protrombina e tempo de tromboplastina parcial ativada não foram alterados com o uso dos fármacos. Os fatores VII e X da coagulação tiveram aumento de suas atividades quando os ratos foram tratados em regime de dose múltipla com claritromicina, em regime de mono e politerapia. Houve perda de cerca de 8 % do peso de ratos tratados com clofazimina e 18 % daqueles tratados com claritromicina ou com a associação dos dois fármacos, no esquema de doses múltiplas, entretanto não houve diferença entre os grupos quando foram avaliados os níveis de gama-glutamiltransferase e bilirrubinas total e direta. Concluindo, clofazimina e claritromicina provocam alterações hematológicas, hemostáticas e bioquímicas e os resultados de concentração plasmática são valiosos para avaliação de efeitos adversos em estudos comparativos de monoterapia e politerapia entre os medicamentos. / Clarithromycin and clofazimine have been used to treat leprosy and infections caused by Mycobacterium avium complex in HIV patients. Because there are few data about the toxicity of treatment regimens involving these drugs, this study aimed to evaluate the adverse effects of this therapy in male Wistar rats through the determination of hematological, haemostatic and biochemical parameters and correlate them with the dose and plasma concentrations of drugs, under a single and multiple dose regimen. Evaluation was performed as follows: a) Global and specific count of leukocytes (manual method), phagocytosis and oxidative burst of neutrophils assays (flow cytometry), b) platelet count (manual method), prothrombin time, activated partial thromboplastin time, plasma levels of factors VII and X (automated method), c) Gamma-glutamyltransferase (kinetic-colorimetric method) and total and direct bilirubin serum levels (colorimetric method), d) plasma concentrations of drugs (High-Performance Liquid Chromatography). There were no differences between plasma concentrations of the drugs administered in monotherapy or polytherapy. However, the concentrations of both clofazimine and clarithromycin have decreased in plasma in multiple dose regimen compared to single dose. There was an increase in the number of leukocytes (multiple dose) and polymorphonuclear cells (single and multiple doses) in the groups treated with clarithromycin in monotherapy or in association with clofazimine, and a decrease in the number of mononuclear cells in single and multiple doses, in the same groups. Both drugs seemed to reverse the proportion between mononuclear and polymorphonuclear cells. The oxidative burst was observed in animals treated with drugs in polytherapy or in monotherapy, however there was no difference between the treatment with drugs and the control with DMSO in single dose. In multiple doses, treatment with clofazimine and clarithromycin in monotherapy or polytherapy stimulated the increase of oxidative burst (p <0.0001) compared to control. There were no differences in phagocytosis between the treated and control groups in single and multiple doses. Prothrombin time and activated partial thromboplastin time have not changed with the use drugs. In contrast, the activities of factors VII and X of coagulation have increased when rats were treated with multiple doses regimes with clarithromycin alone or in association with clofazimine. There was weight loss of 8% in rats treated with clofazimine and 18% in those treated with clarithromycin or with association of the drugs in the multiple doses regimen. However, there was no difference between the groups when gammaglutamyltransferase and total and direct bilirubin levels were analyzed. Therefore, clofazimine and clarithromycin induce hematological, hemostatic and biochemical changes and the results of plasma concentration is valuable for assessing adverse effects in comparative studies of monotherapy and polytherapy of these drugs.

Chronic stress exposure is hypothesized to increase the risk for developing a mental or physical disease, which can be summarized as an overall increased vulnerability to adverse health conditions. This thesis shows one potential pathway of stress exposure entering the body via activation of the hypothalamus pituitary adrenal (HPA) axis and the interaction between HPA axis and immune activation. Chronic stress caused by work overload or the experience of stressful life events was associated with altered levels of cortisol, the main effector hormone of the HPA axis. Further, chronic stress was shown to effect the distribution of neutrophils, a leukocyte subtype known for its defence properties as well as for its side effects of tissue damage. Additional analyses support a theory that HPA axis and immune cells work in synergy to adapt the organism to chronic stress exposure and therefore represent one pathway how stress can cause altered immune defence toward an increased vulnerability to disease.

Leukocytes express a family of high molecular weight glycoproteins called leukocyte common antigens (CD45) which have tyrosine phosphatase activity and are involved in phosphotyrosine signal transduction. Antibodies to different CD45 isoforms distinguish functionally different CD4+ T cell subsets in humans, rats, and mice. Selected protein isoforms are expressed through a process of exon splicing which is cell-type and differentiation-state specific. Splicing of the three variable exons, A, B, and C, which encode amino acids located near the extracellular amino terminus of the protein, potentially results in generation of eight different mRNA transcripts. The purpose of this study was to determine the relative levels of all eight different CD45 transcripts present in a panel of murine CD4+ T cell lines and normal murine and human CD4+ T cell subsets separated with antibodies to CD45 variable exons. I show, as expected, that the broad features of CD45 surface isoform expression in these cells can be accounted for by the relative amounts of the eight differentially spliced transcripts. Unexpectedly, all the differences in CD45 isoform expression among the CD4+ T cell subpopulations that I measured could be accounted for by differences in the overall level of variable exon expression. I did not see differences among T cell populations in the relative expression of particular variable exons. Exon B was always found in greater abundance than exons C or A. Of the dual exon species, only AB and BC were found in CD4+ T cells. The AC species was undetectable. Human CD4+ T cells, especially those in the naive subset, express higher levels of CD45 variable exons than murine CD4+ T cells. In unrelated studies, I have generated a rat-mouse hybridoma which secretes a rat IgG antibody reactive with mouse CD45. I show that the monoclonal antibody, 25D10, defines a novel epitope consistent with a post-translational modification of CD45, similar but distinct from the epitope recognized by monoclonal antibody RA3.6B2 (anti-B220). This conclusion is based on evidence that it precipitates similar molecular weight bands from cells as does a framework monoclonal antibody to CD45, yet has a distinct cell surface expression as determined by flow cytometric analysis. It stains activated Th cell lines at a higher intensity than resting Th cells, stains 60-70% of splenocytes, and 25-30% of lymph node cells. It stains all class II positive cells but not freshly isolated CD4+, CD8+ T cells or CD45 transfected fibroblasts.

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The manual differential count of leukocytes is a common analysis in the hematological laboratory. It is used for morphological assessment of the blood cells and can get valuable information according to diagnosis of hematological diseases. The microscopy assessment is dependent of a good staining result of the blood smear in order to get the best conditions to differentiate the different cell types and detect morphological or pathological findings.The aim of this study was to determine if it was possible to change the staining method without any compromises to the quality of the staining results. For this study 25 whole blood samples were collected, and blood smears was made and stained with four different staining protocols, including the one currently used in the routine practice at laboratoriemedicin Sundsvall. The samples were examined by three biomedical scientists and the staining quality of the cells was graded on a four-point scale. The statistical results with Friedmans and Wilcoxons signed-rank test showed differences between the methods on the nuclear and cytoplasm of lymphocytes and the nuclear of monocytes and neutrophils. The recommended staining protocol from the manufacturer was the method that had highest frequency of statistically significant differences compared to the other methods for those cell types. The differences were in favour for the other methods, and the current method showed the best performance. In conclusion it’s not recommended to continue the study with the manufacturer´s staining protocol, but its valuable to continue compare the best performed staining protocols.

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BACKGROUND: Atherosclerotic vascular disease is a significant medical and socioeconomic problem and contributes to mortality in multiple diseases including myocardial infarction (MI), stroke, renal failure, and peripheral vascular disease. Atherosclerosis, as well as other vascular diseases including post-intervention restenosis and allograft vasculopathy, is known to be driven by chronic inflammation and, consequently, pro- and anti-inflammatory cell signaling molecules have been an important target of cardiovascular research. Interleukin (IL)-19 is a recently discovered member of the IL-10 family of anti-inflammatory cytokines. IL-19 is expressed in injured vascular cells, including vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), where it exerts an anti-inflammatory effect. In VSMCs, IL-19 signaling results in inhibition of proliferation, migration, spreading, production of reactive oxygen species (ROSs), and expression of pro-inflammatory genes. In ECs, IL-19 signaling is pro-angiogenic and results in increased EC proliferation, migration, and spreading. AIMS and HYPOTHESIS: The goal of the present study was to explore the hypothesis that IL-19 mediates anti-inflammatory effects on vascular cells by inhibiting the expression of pro-inflammatory genes, such intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, IL-1&beta;, IL-8, and monocyte chemotactic protein (MCP)-1, through modulation of the mRNA stability factor HuR by post- transcriptional (e.g., microRNA) and post-translational (e.g., serine phosphorylation) mechanisms. METHODS and RESULTS: We found that IL-19 can significantly inhibit tumor necrosis factor (TNF)-&alpha;-driven ICAM-1 and VCAM-1 mRNA and protein abundance in cultured human coronary artery ECs (p &lt; 0.01). IL-19 treatment of ECs, but not monocytes, significantly inhibited monocyte adhesion to cultured EC monolayers (p &lt; 0.01). In wild-type mice, systemic administration of IL-19 significantly reduced TNF-&alpha;-induced leukocyte rolling and adhesion as quantitated by intravital microscopy (p &lt; 0.05). IL-19 failed to inhibit TNF-&alpha;-induced nuclear factor (NF)-&kappa;B activation in ECs. IL-19 inhibited nuclear-to-cytoplasmic translocation of HuR and significantly reduced mRNA stability of ICAM-1 and VCAM-1 (p &lt; 0.01 ). IL-19 significantly inhibited serine-phosphorylation of HuR, which is required for its translocation, and significantly increased expression of the putative HuR regulator microRNA (miR)-133 in VSMCs. CONCLUSIONS: These data are the first to report that IL-19 can reduce leukocyte-EC interactions, and to propose reduction in HuR-mediated mRNA stability of ICAM-1 and VCAM-1 as a mechanism. We conclude that expression of IL-19 by ECs and VSMCs may represent an auto-regulatory mechanism to promote resolution of the vascular response to inflammation. These results suggest that IL-19 is anti-inflammatory in vascular cells and, therefore, may be of therapeutic value in atherosclerotic vascular disease. / Physiology