The bank vole (Microtus arvalis) develops glucose intolerance both when kept in captivity and in the wild state. Glucose intolerant bank voles kept in captivity exhibited polydipsia, polyuria, hyperglycemia, hyperinsulinemia, islet autoantibodies and a markedly changed islet structure resembling so–called hydropic degeneration. Islets showing hydropic degeneration have reduced β–cell mass. However, the relative islet size to total pancreas area was not changed. Pancreatic islet isolated from glucose intolerant bank voles had an altered islet function showing signs of being exposed to an increased functional demand on their β–cells. Also, islets from male bank voles seem more affected than the islets from females. Islets isolated from glucose tolerant male bank voles cultured for 5 days at 28 mM glucose did not reveal any change in insulin gene expression or insulin biosynthesis rate. However, islets from female bank voles displayed a glucose concentration dependent response. This suggests that there is gender difference in that, islets of female more easily than islets of males adapt to elevated glucose concentration. Furthermore, islets isolated from glucose tolerant males had reduced insulin gene expression after exposure to proinflammatory cytokines for 48 hrs. This effect seemed to be NO-independent since only a minor elevation of nitrite accumulation in the medium was seen, and the use of iNOS inhibitor could not counteract the cytokine effect. The observed response seen in bank vole islets upon exposure to various glucose concentrations or proinflammatory cytokines is similar to those seen in studies of human islets. The bank vole may therefore represent a novel animal model for the study of diabetes. An unresolved issue is the role of the Ljungan virus which is found in the bank vole colony. Bank voles developing glucose intolerance display features of both human type 1 and type 2 diabetes, where environmental factors seems to play an important role as determinant. Our findings suggest that bank voles bred in the laboratory may develop more of a type 2 diabetes. However, bank voles caught in nature instead may rather develop a type 1 form of the disease.

! BACKGROUND: Exposure to Environmental Tobacco Smoke (ETS) is elevating blood levels of inflammatory mediators and chemoattractants which seem to play an important role in the development of several diseases (e.g. Chronic Obstructive Pulmonary Disease). First evidences showed that men and women might differ in their proneness for these diseases. The aim of this study was to investigate whether there are effects of ETS during pregnancy on inflammatory cytokines in cord blood and in mother’s blood and if there are any differences between male and female newborns. METHODS: Within the LiNA (Lifestyle and environmental factors and their influence on Newborn Allergy Risk) study, whole blood samples of 460 mother-child pairs were analyzed for the concentrations of IL-6, IL-8, IL-10, IL-12, IL-4, IL-5, INF-gamma, TNF-alpha and MCP-1 using cytrometic bead asseys. The association between ETS exposure and cytokines was calculated using the Mann-Whitney-U-test and adjusted with a multiple regression model for parental atopy, parental education status and cat ownership. The exposure assessment is based on questionnaire data on smoking behaviour of the parents and measurement of indoor benzene concentration. RESULTS: Female newborn, being exposed in utero to 10 cigarettes a day or more, had significantly higher blood concentrations of IL-8, IL-6 and MCP-1 whereas there have been no elevations in male newborn being exposed to the same amount of cigarettes. Furthermore a significantly decreased amount of INF-gamma was found in cord blood of male newborns but not in female newborns. General increasing levels of TNF-alpha in cord blood where found for daily smoke exposure without relating it to the exact number of cigarettes. CONCLUSION: The data of this study refer to gender-specific differences in the susceptibility to ETS exposure. The induction of inflammatory signals in cord blood in response to cigarette smoke exposure is stronger in female than in male newborn. / Die vorliegende Arbeit ist Teil einer umweltepidemiologischen Kohortenstudie (LiNA), in der der Einfluss von Umwelt- und Lebensbedingungen auf die Entwicklung von Immunsystem und Allergien bei Neugeborenen unter Einbezug der vorgeburtlichen Zeit untersucht wird. In welchem Maße sich eine Rauchbelastung während der Schwangerschaft auf die Zytokinmuster der Neugeborenen im Nabelschnurblut auswirkt und inwiefern dies mit dem Zytokinmuster der Mutter korreliert, sollte das Ziel dieser Dissertation sein. Dafür wurden Daten von insgesamt 629 Mutter-Kind-Paaren erhoben, Zytokin- und Chemokinbestimmungen, sowie die des Gesamt-IgE aus den Blutproben der 34. SSW und denen der Nabelschnur vorgenommen. Interessanterweise konnten geschlechterspezifische Unterschiede im Zytokinspektrum der Neugeborenen gefunden werden. Bei den weiblichen Neugeborenen zeigte sich eine deutliche Erhöhung proinflammatorischer Marker, wenn deren Mütter dem Rauch von mehr als 10 Zigaretten pro Tag ausgesetzt waren. Dieser Anstieg war weder im Blut der männlichen Neugeborenen noch im Blut der Schwangeren in der 34. SSW zu beobachten. Zusätzlich konnte beobachtet werden, dass auch einzig die männlichen Neugeborenen stark negativ mit ihrer IFN-gamma-Produktion auf die passive Rauchbelastung reagieren. Die mit dieser Arbeit ermittelten Daten, dass das Immunsystem beim Neugeborenen geschlechterspezifisch unterschiedlich auf Tabakrauch zu reagieren scheint, sind erstmals in der Literatur zu finden. Die Erforschung des Immunsystems und dessen Beteiligung an zahlreichen Erkrankungen, besonders den chronisch Inflammatorischen, ist durchaus relevant im medizinischen Alltag. Diese Arbeit trägt einen weiteren Baustein dazu bei und gibt Anstoß für weitere Studien.

A successful immune response depends on prompt and sufficient recruitment of leukocytes from the circulation to infected or injured sites. Mobilization of leukocytes to hypoxic tissues is vital for angiogenesis, i.e. the formation of new blood vessels from preexisting vasculature, and thus crucial for tissue growth and regeneration. Deviations from normal leukocyte recruitment drive a variety of pathologies, including chronic inflammation, autoimmune diseases and cancer, for which therapeutic options are limited or unspecific. Understanding the mechanisms by which the body controls leukocyte recruitment is therefore critical for the development of novel therapeutic strategies. The present investigations focused on delineating the mechanisms behind leukocyte mobilization from the bloodstream to afflicted sites, by means of in vivo imaging techniques and in vitro assays. We demonstrate that, in response to inflammation, increased vascular permeability enhances transendothelial transport of tissue-released chemokines. Within the vasculature, chemokines form a chemotactic gradient sequestered on heparan sulfate, which directs crawling neutrophils and expedites their extravasation to the inflamed tissue. Consequently, gradient formation grants efficient bacterial clearance. Citrullination of chemokines by leukocyte-derived PAD enzymes in the inflamed tissue prevents chemokine transport into blood vessels, which dampens further neutrophil recruitment and thereby controls the amplitude of the inflammatory response. Moreover, the mechanisms of neutrophil recruitment in response to proangiogenic factors released during hypoxia are revealed to differ from those observed during classical inflammation. Particularly, VLA-4 integrin and VEGFR1 expressed on a defined subset of neutrophils, along with endothelial VEGFR2, are required for efficient neutrophil recruitment to hypoxia. Rather than stimulus-induced phenotypic changes on neutrophils, specific neutrophil subtypes with innate proinflammatory or proangiogenic functions (respectively, CD49d-VEGFR1lowCXCR4low and CD49d+VEGFR1highCXCR4high) coexist in the circulation of humans and mice. In summary, this dissertation provides relevant information on specific steps of neutrophil recruitment to inflamed or hypoxic tissues, which may represent future means to down-regulate aberrant immune responses during chronic inflammation and autoimmune diseases; to increase angiogenesis during ischemia; or to limit pathological angiogenesis, a characteristic of tumor growth and of several chronic inflammatory disorders.

Submitted by LUISA CUNHA CARNEIRO null (luisacunhacarneiro@hotmail.com) on 2016-02-29T22:54:55Z No. of bitstreams: 1 Tese_Luisa_Cunha_Carneiro.pdf: 2347194 bytes, checksum: 385e788cab291232de3fc02da72024b3 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-03-01T14:26:24Z (GMT) No. of bitstreams: 1 carneiro_lc_dr_jabo.pdf: 2347194 bytes, checksum: 385e788cab291232de3fc02da72024b3 (MD5) / Made available in DSpace on 2016-03-01T14:26:24Z (GMT). No. of bitstreams: 1 carneiro_lc_dr_jabo.pdf: 2347194 bytes, checksum: 385e788cab291232de3fc02da72024b3 (MD5) Previous issue date: 2016-02-03 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / De modo geral, o objetivo deste estudo foi determinar se as células endometriais bovinas responderam a análogos virais de padrões moleculares associados a patógenos (PAMPs) mediante a produção de citocinas pró-inflamatórias após ativadas pelos receptores “Toll-Like” (TLRs) no endossoma celular e no citoplasma celular pelo genes indutores de ácido retinóico tipo I (RIG-I). No primeiro experimento, amostras uterinas de vacas de corte mestiças pós-púberes foram dissectadas para obtenção de células endometriais epiteliais e estromais. Um controle negativo e quatro PAMPs: LPS, ssRNA, Poly I:C (LMW), Poly (I:C) HMW foram utilizados. Dois grupos de tratamentos (transfectados e não transfectados) foram analisados durante 24 horas. Em outro experimento, células endometriais foram tratadas apenas com o PAMP Poly (I:C) LMW e um grupo Controle Negativo. Neste, os grupos foram incubados às 0, 2, 6, 12, 24, 36, 48 e 72 horas. Sobrenadantes foram colhidos para desenvolver o teste de ELISA para IL-6 e IL-8. Células epiteliais produziram IL-6 em resposta ao Poly I:C (HMW) quando comparadas com o Controle (Grupo DOTAP positivo; P< 0.05), enquanto que o LPS induziu produção de IL-6 e IL-8 em células estromais (P< 0.05). O uso de um reagente de transfecção entre as células e tratamentos demonstrou efeito (P> 0.05). Ainda, células estromais tratadas por Poly I:C (LMW) demonstraram uma maior produção de IL-6 às 48 e 72 horas (P< 0.05), e para o IL-8 às 6, 12, 24, 36, 48 e72 horas quando comparadas com o grupo Controle (P< 0.05). No segundo experimento, outras amostras uterinas de vacas de corte pós-púberes foram utilizadas. A obtenção de células endometriais estromais e epiteliais foram isoladas pelo mesmo protocolo do primeiro experimento. O PAMP Poly (I:C) LMW e um controle negativo foram utilizados. Proteínas para o RIG-I e p65 foram colhidas após 12, 24, 48 e 72 horas de tratamento. Em resposta a Poly (I:C) LMW, células estromais ativaram o RIG-I às 48 hours (P< 0.05) quando comparadas com o grupo controle. Enquanto que, as células epiteliais não foram suficientemente estimuladas pelo Poly (I:C) LMW na ativação do RIG-I em nenhum momento testado (P> 0.05). A proteína p65 depois de estimulada pela Poly (I:C) LMW foi ativada às 12 horas pelas células estromais (P< 0.05) e às 24 horas pelas células epiteliais (P< 0.05). Conclui-se que, células endometriais bovinas foram essenciais na ativação das vias exercidas tanto pelos TLR como RIG-I com função de iniciar uma defesa imunológica contra infecções virais. / In general, the objective of this study was to determine if bovine endometrial cells replied to virus analogs of pathogen associated molecular pattern (PAMPs) by production of proinflammatory cytokines after Toll-Like Receptor (TLR) activation in the cell endosome and after retinoic acid inducible gene – I (RIG-I) stimulation in the cell cytoplasm. In the first experiment, uterine samples from post pubertal cross-breed beef cows were dissected using a protocol to obtain epithelial and stromal cells. A negative control and four different PAMPs: LPS, ssRNA, Poly I:C (LMW), Poly (I:C) HMW were used. Two treatments (transfected and non-transfected) groups were investigated during 24 hours. In the other experiment, endometrial cells were treated with only Poly (I:C) LMW and a negative Control group. All incubated at 0, 2, 6, 12, 24, 36, 48 and 72 hours. Supernatants were collected to develop Elisa for IL-6 and IL-8. Epithelial cells produced IL-6 in response do Poly I:C (HMW) compared to Control (P< 0.05), otherwise, LPS induced IL-6 and IL-8 in stromal (P< 0.05). The transfection Reagent differ between cells and treatments (P> 0.05). Still, in stromal cells treated by Poly I:C (LMW) the production of IL-6 was higher at 48 and 72 hours (P< 0.05), and for IL-8 at 6, 12, 24, 36, 48 and 72 hours when compared to the Control (P< 0.05). In the second experiment, uterine samples from others post pubertal mixed-breed beef cows were used. To obtain stromal and epithelial cells, uterine samples were dissected with the same protocol as the first experiment. The PAMP Poly (I:C) LMW and a negative control were used. Proteins for RIG-I and p65 were collected after 12, 24, 48 and 72 hours. In response to Poly (I:C) LMW induction, stromal cells activated RIG-I at 48 hours (P< 0.05) were compared to the Control group. On the other hand, epithelial cells were not sufficient stimulated Poly (I:C) LMW to activate RIG-I at any time point evaluated (P> 0.05). The protein p65 after stimulated by Poly (I:C) LMW was activated at 12 hours by stromal (P< 0.05) and at 24 hours by epithelial cells (P< 0.05). In conclusion, bovine endometrial cells were elemental factors in the activation of both TLR and RIG-I pathway in order to start an immune defense against viral infection. / CNPq: 140625/2013-5

A sepse é caracterizada por uma produção excessiva de mediadores inflamatórios, acompanhada de taquicardia e hipotensão. Experimentalmente, a administração de endotoxina (Lipopolissacarídeo, LPS) em doses relativamente elevadas induz choque endotoxêmico, sendo um bom modelo de estudo da sepse. Diversos grupos têm demonstrado ações antiinflamatórias e antitumorais do citral, um composto do óleo essencial de Cymbopogon citratus. Nosso laboratório demonstrou ação antipirética do citral em modelo de febre induzida por LPS, acompanhada de redução nos níveis de citocinas plasmáticas e de prostaglandina E2 (PGE2) no plasma e área pré óptica do hipotálamo (POA), importante região termorregulatória. A hipótese testada neste trabalho foi a de que o citral atenua a hipotensão provocada pela endotoxina, além de amenizar as alterações termoregulatórias. Todos os procedimentos foram executados de acordo com os princípios éticos de experimentação animal, aprovados pelo comitê de ética local (CEUA 2015.1 1214 58-2). Foi realizado o implante de cânulas na artéria e veia femoral para registro da pressão arterial e administração de LPS (1,5 mg/kg) ou salina apirogênica 0,9% além do implante de datalogger na cavidade peritoneal de ratos Wistar, para registro da temperatura corporal. No dia do registro, 30 minutos antes da administração de LPS ou salina, os animais receberam citral (100 mg/kg) ou tween 80 a 1% (veículo) por via oral. Os parâmetros cardiovasculares e temperatura corporal foram registrados por 300 minutos após os respectivos tratamentos. Os valores de pressão arterial média (PAM) e frequência cardíaca (FC) foram coletados a cada 10 minutos após o tratamento e a temperatura corporal foi registrada pelo datalogger em intervalos de 5 minutos. Em outro protocolo foi realizado apenas o implante de cânula na veia femoral dos animais de todos os grupos para administração de LPS ou salina, coleta de sangue para dosagem de interleucina 6, PGE2, nitrito e nitrato e corticosterona e coleta do encéfalo para dosagem de PGE2 e PGD2. As diferenças estatísticas entre os grupos foram analisadas pelo teste ANOVA two-way seguido por pós teste de Newman-Keuls, com o nível de significância adotado de p < 0,05. A administração de LPS provocou queda na PAM eaumento na FC. Tais respostas não foram afetadas pela administração prévia de citral. O LPS também induziu febre e aumento nas concentrações plasmáticas de interleucina - 6 (IL-6), óxido nítrico (NO), PGE2 e corticosterona. Esses parâmetros não foram alterados pela pré- administração de Citral. No entanto, o citral provocou redução na produção de PGD2 naPOA, sem alterar a de PGE2 nesta região. Podemos concluir que o citral não previne as alterações nos parâmetros cardiovasculares no modelo de endotoxemia em ratos, porém reduz a produção de um mediador termorregulatório e inflamatório do sistema nervoso central (a PGD2), sem alterar a produção de outros mediadores inflamatórios a nível periférico (no plasma). Portanto, em um modelo mais agressivo de inflamação sistênica o citral não se mostrou suficiente para proteger o organismo das ações deletérias do LPS. / Sepsis is characterized by the overproduction of inflammatory mediators, accompanied by tachycardia and hypotension. Experimentally, administration of endotoxin (Lipopolysaccharide, LPS) in relatively high doses induces endotoxemic shock, a widely used model of sepsis in rats. Several groups have demonstrated anti-inflammatory and antitumor roles of citral, an essential oil compound of Cymbopogon citratus. Emilio-Silva et al. (2017) have shown an antipyretic role of citral in a model of LPS-induced fever, accompanied by a reduction of cytokines and prostaglandin E2 plasma levels and in the preoptic area of hypothalamus (POA), the hierarchically most important thermoregulatory region. We hypothesized that citral attenuates the LPS-induced hypotension, besides mitigating the thermoregulatory adjustments in rats. All procedures were performed in agreement with ethical guidelines for animal experimentation aproved by the local ethical committee (CEUA 2015.1 1214 58-2). Femoral artery and vein were implanted with cannulas for blood pressure recording and LPS (1.5 mg/kg) or 0.9% apyrogenic saline injection. In a second surgical procedure a datalogger was implanted into the peritoneal cavity for measurements of body temperature. All surgical procedures were performed under Ketamin/xilazin (100/10 mg/kg) anesthesia. One day after arterial catheterization, 30 minutes prior to LPS or saline administration, the animals received either citral (100 mg / kg) or 1% tween 80 (vehicle) oralstarly. The cardiovascular parameters and body temperature were recorded for 300 minutes after the respective treatments. Mean blood pressure (MAP) and heart rate (HR) were collected every 10 minutes after treatments and body temperature was recorded by the datalogger at 5 minute intervals. Blood samples were obtained in another set of rats for interleukin-6, PGE2, nitrite and nitrate and corticosterone analyses. The brain was removed for PGE2 and PGD2 analyses. Statistical differences between groups were analyzed by the two-way or one-way ANOVA test followed by Newman-Keuls post-test, with significance level adopted at p <0.05. As expected, LPS administration caused a decrease in MAP and an increase in HR, and these responses were not affected by citral. LPS also induced fever and increased plasma levels of interleukin - 6 (IL - 6), nitric oxide (NO), prostaglandin E 2 andcorticosterone. These parameters were also not altered by citral. On the other hand, citral caused a reduction in prostaglandin D2 concentration in the POA, but failed to alter PGE2 levels in this region. Our data are consistent with the notion that citral does not affect changes in cardiovascular and thermoregulatory parameters. Consistently, citral also caused no changes in both LPS-induced peripheral inflammatory mediators (in plasma) and in the POA, except PGD2. Therefore, in our model which mimetic a fairly critical situation, citral may not be sufficient to protect the organism from the deleterious actions of LPS. Financial support: FAPESP / CAPES.

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Made available in DSpace on 2014-06-11T19:32:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-11-26Bitstream added on 2014-06-13T18:43:57Z : No. of bitstreams: 1 lopes_fcm_dr_arafcf.pdf: 1909059 bytes, checksum: 974b3ea32c4f5b47c9236b3e9490981c (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / Produtos naturais têm contribuído intensamente para o desenvolvimento da terapêutica moderna. As plantas produzem um vasto número de substâncias, que em estado natural ou após sofrerem transformações químicas, possuem diversas atividades farmacológicas. Fungos endofíticos, organismos que vivem no interior das plantas, também podem representar novas fontes de produtos biologicamente ativos. Atualmente, a relação causal entre inflamação, imunidade inata e câncer é largamente aceita. O envolvimento de mediadores inflamatórios, como óxido nítrico (NO) e citocinas, gerados por macrófagos ativados, na patogênese das doenças inflamatórias já está bem estabelecido. Além disso, a inibição da angiogênese tem sido reconhecida como uma promissora abordagem terapêutica para o controle do crescimento tumoral, das metástases e das doenças inflamatórias crônicas. Alchornea glandulosa Poepp & Endl. (Euphorbiaceae) é uma planta com conhecida atividade antiinflamatória que está distribuída do sudeste ao sul do Brasil, principalmente na Mata Atlântica e no Cerrado. O potencial antiinflamatório, antitumoral e antiangiogênico dos compostos obtidos a partir da planta (fração acetato de etila e os compostos puros isoquercitrina, afzelina, ácido gálico, pteroginina e pteroginidina) e de fungos endofíticos presentes no interior das suas folhas (extratos acetato de etila ALG-A, ALG-02 e ALG-03) foram estudados por meio de experimentos utilizando-se culturas de macrófagos murinos, linhagens tumorais murinas de câncer de mama (LM2) e pulmão (LP07) e culturas de células endoteliais de veia umbilical humana (HUVEC). Ensaios de determinação de óxido nítrico (reagente de Griess), citocinas pró-inflamatórias TNF-α, IL-1β, IL-6 e IL-12 (ELISA), atividade citotóxica (MTT) e avaliação da taxa de inibição do crescimento de tumores tratados com injeção... / Natural products have contributed enormously to the development of important therapeutic drugs used currently in modern medicine. Plants produce a vast number of compounds that, either directly or after chemical modifications, exert pharmacological activities. Endophytic fungi, organisms which live in plants, are also being recognized as new sources of biological active substances. Nowadays, the relationship among inflammation, innate immunity and cancer are widely accepted. Inflammatory mediators as nitric oxide (NO) and cytokines produced by activated macrophages are involved in the pathogenesis of inflammatory diseases. Besides that, angiogenesis inhibition has been accepted as a promising therapy for the control of tumor growth, metastasis and also chronic inflammatory conditions. Alchornea glandulosa Poepp & Endl. (Euphorbiaceae) is a plant that demonstrates anti-inflammatory activity. It can be found in Brazil, distributed from southeast to south, mainly in the Atlantic Forest and Cerrado. The anti-inflammatory, antitumor and antiangiogenic potential of the compounds obtained from this plant (ethyl acetate fraction and the pure compounds isoquercitrin, afzelin, gallic acid, pterogynine and pterogynidine) and from the endophytic fungi present in its leaves (ALG-A, ALG-02 and ALG-03 ethyl acetate extracts) were studied using macrophage cultures, tumor cell lines (LM2 and LP07) and human umbilical vein endothelial cells (HUVEC). To evaluate anti-inflammatory and antitumor activity, in vitro assays were utilized to determine NO (Griess reagent), TNF-α, IL-1β, IL-6 and IL-12 proinflammatory cytokines (ELISA) and cytotoxicity (MTT). Tumor growth inhibition rate was also studied in vivo. Apoptosis (TUNEL assay), proliferation (bromodeoxiuridine – BrdU), invasion (double-chamber assay), capillary-like structures formation (matrigel) and NFκB activity (ELISA) were realized to study... (Complete abstract click electronic access below)