LOPAP (Lonomia obliqua prothrombin activator protease) é uma serino protease isolada das cerdas da Lagarta Lonomia obliqua que induz efeitos sobre a coagulação sanguínea, semelhantes ao extrato de cerdas bruto. Recentemente, foi obtido um peptídeo do LOPAP, P4, que parece possuir efeitos similares à proteína. No sentido de complementar os dados sobre estas toxinas, este estudo investigou os efeitos do LOPAP ou do P4 sobre interações leucócito-endotélio in vivo; expressão de moléculas de adesão, síntese de mediadores inflamatórios, apoptose e necrose de leucócitos e célula endotelial, além de suas possíveis ações sobre a formação de novos vasos. Aplicação tópica de 30µg/mL ou 300µg/mL (10µL) de LOPAP ou de P4 na rede microvascular do mesentério de ratos Wistar machos não alterou o diâmetro de vênulas pós-capilares, as interações dos leucócitos ao endotélio, nem a reatividade microvascular frente à acetilcolina ou noradrenalina. Somente a concentração de 1000µg/mL aumentou o número de leucócitos aderidos à parede vascular, simultaneamente a estases intermitentes nos vasos da microcirculação. Ensaios de citometria de fluxo mostraram que a incubação de LOPAP ou P4 (300µg/mL) não modificou a expressão de L-selectina e β2-integrina em neutrófilos circulantes obtidos de ratos Wistar. Por outro lado, incubações do LOPAP ou do P4 com célula endotelial (cultura primária obtida do músculo cremaster de ratos Wistar; 300µg/mL) induziram a expressão da molécula ICAM-1 , e somente o P4 promoveu a expressão de VCAM-1. Diferentemente, nenhuma das toxinas alterou a expressão de PECAM-1. Ensaios imuinoenzimáticos realizados em sobrenadantes de neutrófilos e de célula endotelial incubadas com LOPAP ou P4 (300µg/mL) mostraram que as toxinas não alteraram a secreção de interleucina-6 (IL-6), interleucina-10 (IL-10), fator de necrose tumoral-α (TNF-α) por neutrófilos; as toxinas não induziram a secreção de TNF-α pela célula endotelial, mas aumentaram a secreção de IL-6 e IL-10. A concentração de óxido nítrico (NO), quantificado pela reação de Griess, estava aumentada nos sobrenadantes dos dois tipos celulares incubados com LOPAP ou P4. A incubação de LOPAP ou P4 com neutrófilos ou célula endotelial não alterou a viabilidade celular, mas preveniu a apoptose da célula endotelial ou neutrófilo provocada pela carência de soro bovino fetal. A incubação simultânea com L-NAME (1 mM) reduziu a proteção conferida pelo LOPAP ou pelo P4. A investigação dos efeitos das toxinas sobre a formação da rede microcirculatória foi realizada em camundongos Swiss, machos, utilizando o modelo da câmara dorsal por microscopia intravital. A aplicação tópica do LOPAP ou P4 (30µg/mL, 300µg/mL; 10µL; 3 doses a cada 96 horas) no leito microvascular dorsal reduziu significantemente o número de vasos na rede microcirculatória em condições basais. Adicionalmente, o tratamento com P4 inibiu a formação de novos vasos provocada pela aplicação tópica de suplemento de fatores de crescimento vascular. O tratamento de célula endotelial de microcirculação de camundongos imortalizadas (tENd) com LOPAP ou P4 (300µg/mL) reduziu a capacidade de migração destas células. Em conjunto, os dados obtidos até o momento mostram que o LOPAP e o P4 não induziram as interações leucócito-endotélio in vivo e que a secreção de mediadores por neutrófilos e célula endotelial, como o NO, podem estar envolvidos com suas atividades anti-apoptótica. Ademais, o LOPAP e o P4 inibem o crescimento de novos vasos, e um dos possíveis mecanismos pode ser a interferência nos mecanismos de migração da célula endotelial. / LOPAP (Lonomia obliqua prothrombin activator protease) is a serine protease isolated from the crude extract of Lonomia obliqua caterpillar, which induces effects on blood coagulation comparable to the extract. Recently it was obtained a peptide fragment from LOPAP, P4, with similar activities with the protein. This study aimed to complete data about these toxins, LOPAP and P4, on in vivo leukocyte-endothelial interactions; adhesion molecule expressions, synthesis of inflammatory mediators, necrosis and apoptosis of neutrophils and endothelial cells, besides possible actions on angiogenesis. Topical applications of 30μg/mL or 300µg/mL (10µL) of LOPAP or P4 on microvascular network of mesentery of Male Wistar rats did not affect the diameter of postcapillary venules, leukocyte-endothelial interactions, either vascular reactivity to acetylcholine or norepinephrine. Only topical application of 1000µg/mL (10 µL) promoted increment on the number of leukocytes adhered to vessel wall, simultaneously to intermittent blood stasis in the microvascular network. Flow cytometry assays showed that incubations with LOPAP or P4 (300µg/mL) did not modify the expression of L-selectin or β2-integrin in neutrophils from Male Wistar rats. On the other hand, incubations of LOPAP or P4 with primary cultured endothelial cells evoked expressions of ICAM-1, and only incubation with P4 promoted expression of VCAM-1. Differently, the treatments did not affect PECAM-1 expression in endothelial cells. Immunoenzimatic assays carried out in supernatants of neutrophils and endothelial cells incubated with LOPAP or P4 (300µg/mL) showed that both toxins did not alter the basal secretion of interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) by neutrophils; both toxins did not promote secretion of TNF-α by endothelial cells, however they enhanced the secretion of IL-6 and IL-10. Concentrations of nitric oxide (NO), quantified by Griess reaction, were enhanced in the supernatant of neutrophils or endothelial cells incubated with LOPAP or P4. LOPAP or P4 treatments did not alter the viability of neutrophils or endothelial cells, but prevented the apoptosis of both type cell caused by fetal bovine serum deprivation. Simultaneous incubation with L-NAME (1 mM) and LOPAP or P4 reduced the protection on apoptosis exerted by the toxins. The action of toxins on formation of microcirculatory network was investigated in Male Swiss mice. Topical application of LOPAP or P4 (30µg/mL, 300µg/mL; 10µL; 3 times each 96 hours) in the microvascular network significantly reduced the number of vessels in basal conditions. Additionally, treatment with P4 inhibited the new vessel formation provoked by topical application of vascular growth factor supplement. Immortalized endothelial cells of mice (tEnd) incubated with toxins presented lower migration than cells incubated with saline. Together, data herein obtained showed that LOPAP and P4 did not evoke in vivo leukocyte-endothelial interactions and secretions of chemical mediators by neutrophils and endothelial cells, as NO, seem to be related to anti-apoptotic activities. Additionally LOPAP or P4 inhibit the microcirculatory network formation, by interfering, at least in part, by altering the migration of endothelial cells.

INTRODUÇÃO: A interação entre leucócitos e plaquetas é fundamental para o início e a progressão da reestenose e da aterosclerose. Recentemente foi evidenciado em estudos in vitro que a integrina Mac-1 dos leucócitos se liga à glicoproteína Ibalfa (GP Ibalfa) das plaquetas e que esta interação possui uma função central na firme adesão e transmigração de leucócitos em locais de deposição de plaquetas. Entretanto, não há estudos in vivo que avaliam a importância da interação entre a integrina Mac-1 dos leucócitos e a GP Ibalfa das plaquetas (alfaMbeta2-GP Ibalfa) em modelo experimental de lesão vascular. MÉTODO: Um peptídeo denominado M2 ou anticorpo anti-M2 foi desenvolvido para bloquear a interação da integrina Mac-1 dos leucócitos com a GP Ibalfa das plaquetas, visando, deste modo, inibir a adesão de leucócitos na superfície do vaso coberta por plaquetas, a proliferação celular e a hiperplasia neointimal. Este peptídeo foi injetado e comparado com anticorpo-controle em camundongos C57B1/6J submetidos à lesão vascular da artéria femoral com corda-guia. Um dia (controle: n= 6; anti-M2: n= 6), 5 dias (controle: n= 9; anti-M2: n= 9) ou 28 dias (controle: n= 9; anti-M2: n= 9) após a lesão vascular, as artérias femorais foram retiradas para a realização de morfometria e imunohistoquímica. RESULTADOS: O bloqueio da interação alfaMbeta2-GP Ibalfa promoveu redução estatisticamente significativa de 75% do número de leucócitos na camada média no primeiro dia após a lesão vascular (controle: 7,9 ± 5,0% do total de células na camada média; versus anti-M2: 2,0 ± 1,6%; p=0,021), bem como determinou diminuição estatisticamente significativa de 42% em 5 dias (controle: 42,3 ± 12,9% do total de células na neoíntima; versus anti-M2: 24,6 ± 10,8%; p=0,047) e de 58% em 28 dias do acúmulo de leucócitos na neoíntima em desenvolvimento (controle: 7,9 ± 3,0% versus anti-M2: 3,3 ± 1,3%; p=0,012). A proliferação celular na camada média do vaso em 5 dias pós-lesão vascular apresentou redução estatisticamente significativa de 64% com o bloqueio da interação alfaMbeta2-GP Ibalfa (controle: 5,0 ± 2,9% do total de células na camada média; versus anti-M2: 1,8 ± 0,5%; p=0,043), assim como houve diminuição significativa de 47% da proliferação celular na camada íntima do vaso em 28 dias (controle: 3,8 ± 1,7% do total de células na camada íntima; versus anti-M2: 2,0 ± 1,2%; p=0,047). O bloqueio da interação alfaMbeta2-GP Ibalfa também determinou redução estatisticamente significativa de 56% do espessamento intimal em 28 dias (controle: 10.395 ± 3.549um2; versus anti-M2: 4.561 ± 4.915um2; p=0,012). CONCLUSÕES: O recrutamento de leucócitos após a lesão vascular é dependente da interação alfaMbeta2-GP Ibalfa e a neutralização desta interação inibe a proliferação celular e a formação neointimal. / INTRODUCTION: The interaction between leukocytes and platelets is fundamental for the beginning and the progression of restenosis and atherosclerosis. Recent in vitro studies have shown that the leukocyte integrin Mac-1 binds to the platelet glycoprotein (GP) Ibalfa, and this interaction plays a central role in the leukocyte firm adhesion and transmigration at sites of platelet deposition. However, there is no in vivo study evaluating the importance of the integrin Mac-1 and GP Ibalfa (alfaMbeta2-GP Ibalfa) interaction in experimental models of vascular injury. METHODS: A peptide termed M2 or anti-M2 antibody was developed to block the leukocyte Mac-1 and platelet GP Ibalfa interaction, aiming to inhibit the adhesion of leukocytes to the platelet-coated surface of vessels as well as the cellular proliferation and the neointimal hyperplasia. The peptide was injected and compared with a control-antibody in C57B1/6J mice subjected to wire-induced femoral artery injury. One day (control: n= 6; anti-M2: n= 6), 5 days (control: n= 9; anti-M2: n= 9) or 28 days (control: n= 9; anti-M2: n= 9) after vascular injury, the femoral arteries were harvested for morphometry and immunohistochemistry. RESULTS: The alfaMbeta2-GP Ibalfa interaction blockade promoted a statistically significant 75% reduction in leukocytes in the medial layer on the first day after vascular injury (control: 7.9 ± 5.0% out of the total cells in the medial layer versus anti-M2: 2.0 ± 1.6%; p=0.021), as well as determined a statistically significant 42% decrease 5 days later (control: 42.3 ± 12.9% out of the total cells in the neointima versus anti-M2: 24.6 ± 10.8%; p=0.047), and a 58% decrease in leukocyte accumulation in the developing neointima 28 days later (control: 7.9 ± 3.0% versus anti-M2: 3.3 ± 1.3%; p=0.012). The cellular proliferation in the vessel medial layer 5 days after vascular injury presented a statistically significant 64% reduction by the alfaMbeta2-GP Ibalfa interaction blockade (control: 5.0 ± 2.9% out of the total cells in the medial layer versus anti-M2: 1.8 ± 0.5%; p=0.043), and there was also a significant 47% decrease in the vessel intimal layer cellular proliferation 28 days later (control: 3.8 ± 1.7% out of the total cells in the intimal layer versus anti-M2: 2.0 ± 1.2%; p=0.047). Furthermore, the alfaMbeta2-GP Ibalfa interaction blockade determined a statistically significant 56% reduction in the intimal thickening 28 days after vascular injury (control: 10,395 ± 3,549um2 versus anti-M2: 4,561 ± 4,915um2; p=0.012). CONCLUSIONS: The leukocyte recruitment after vascular injury depends on the alfaMbeta2-GP Ibalfa interaction, and its neutralization inhibits cellular proliferation and neointimal formation.

Este estudo teve por objetivos: a) caracterizar o influxo de leucócitos (LC) induzido pelo veneno de Bothrops moojeni (VBm); b) avaliar o papel dos mastócitos (MC) e da histamina neste evento; c) analisar a liberação de TXA2, PGD2, LTB4, MCP-1 e KC e d) avaliar a capacidade do VBm desgranular MC in vivo e in vitro. A injeção intraperitoneal de VBm, em camundongos, causou o recrutamento de LC e neutrofilia. O tratamento dos animais com cromoglicato aboliu o influxo de LC enquanto a difenidramina, ranitidina e a tioperamida, antagonistas da histamina, reduziram o influxo de neutrófilos. Ainda, o veneno induziu a liberação de PGD2, TXA2, LTB4, MCP-1 e de KC e causou a desgranulação de MC in vivo e a liberação de -hexosaminidase de MC in vitro. Em conclusão, o VBm induz influxo de LC para o local de sua injeção. Este efeito depende da histamina, via receptores H1, H2 e H4 e da desgranulação de MC, que decorre de ação direta do veneno nestas células. A neutrofilia e o TXA2, LTB4, MCP-1 e KC devem contribuir para o influxo de leucócitos causado pelo VBm. / In this study the effects of Bothrops moojeni venom (BmV) on the cellular component of inflammatory responses and the mechanisms involved in this effect were investigated. The effects of venom on peritoneal and circulating leukocyte numbers and on the release of inflammatory mediators, such as LTB4, TXA2, PGD2, MIP-1 and KC, were assessed. The role of both mast cells and histamine receptors in leukocyte recruitment induced by BmV was assessed by selected pharmacological treatments. BmV caused a marked infiltration of leukocytes (3-24 h) when injected into the peritoneal cavity of mice. Neutrophils (PMN) were the predominant cell type in the early stages of response whereas macrophages (MN) were accumulated from 3 up to 24 h. Moreover, BmV increased blood neutrophil numbers at 3 h after injection. The BmV-induced leukocyte influx was abrogated by cromoglicate and significantly reduced either by difenidramine or ranitidine or tioperamide, histamine H1, H2 and H4 receptor antagonists, respectively, at 6 h after injection. Significant increments in peritoneal levels of LTB4, TXA2, PGD2, MIP-1 and KC were detected at distinct periods of time after venom injection. In addition, BmV induced mast cell degranulation both in vivo and in vitro. In conclusion, obtained data demonstrated the ability of BmV induce leukocyte recruitment into the site of its injection. This effect is dependent on mast cell activation and degranulation, which may be due to a direct effect of venom on these cells, and is mediated at least in part by histamine via H1, H2 and H4 receptors. Moreover, the ability of venom to mobilize leukocytes from bone marrow reserve compartments and to release the chemotactic mediators TXA2, LTB4, MCP-1 and KC may be relevant for leukocyte infiltration.

In conclusion, gene expression profiling in blood may have potential to be an adjuvant marker for early detection of AD. Expression marker panel is more informative than single gene expression signature. Further validation in prospective studies will substantiate its clinical application and explore its potential to differentiate AD from other dementias and to predict the progression from MCI to AD. (Abstract shortened by UMI.) / In the study, the profile of 12 target gene expression levels in peripheral blood cells were determined in 96 AD, 145 MCI and 167 normal controls (NC) by quantitative real-time RT-PCR. The genes were identified with (i) high expression in blood and brain; (ii) differential expression between AD and control; (iii) AD related candidate genes. Then, a list of genes were selected including CTSB, CTSD, DDT, ITPKB, NDUFA6, NRD1, PIN1, SNX2, TSC1, UQCRC1, CNR2, GSTM3. Seven genes were found to be differentially expressed between AD and NC group, with upregulation of CTSB, CTSD, DDT, TSC1 and UQCRC1, and downregulation of ITPKB and PIN1 in AD patients. Expression levels of two genes were increased in the MCI compared with NC group, including CTSB and CTSD. In addition, an upregulation of CTSD, UQCRC1, NRD1 and downregulation of ITPKB were observed in AD subjects in comparison to the MCI group (Mann-Whitney U test, p<0.05). After adjusting for confounding factors of age, gender, education level, ApoE4 status and the total CIRS score, expression level of any single gene was not associated with the classfication of AD or MCI (Logistic regression, p>0.05). A five gene biomarker panel, including DDT, ITPKB, PIN1, TSC1 and UQCRC1 was identified with logistic regression analysis. The function of Logit(P)= ln(P/(1-P))= b0+b1RatioDDT+ b2RatioITPKB + b3Ratio PIN1 +b4 RatioTSC1+b5Ratio UQCRC1 were defined as the probability of a subject to be diagnosed as "AD" or "MCI' by using 5-gene biomarker panel. ROC analysis showed that the AUC for the 5-gene biomarkers panel in differentiating between AD and NC, between MCI and NC, between AD and MCI were 0.79 (95% CI, 0.72-0.86; p<0.001), 0.61 (95% CI, 0.53-0.69; p=0.007) and 0.68 (95% CI, 0.60-0.76; p<0.001) respectively. The 5-gene combination was found to discriminate AD subjects from normal controls with good sensitivity and specificity of 70.7% and 86.7% respectively at an optimal cut-off point of 0.486. Low sensitivity (42.4%) and acceptable specificity (76.2%) were observed at a cut-off threshold of 0.505 when differentiating MCI from NC subjects. Between AD and MCI subjects, gene combination showed a sensitivity of 61% and specificity of 73.7% at a cut-off value of 0.496. / Several genes including CTSD, DDT, NDUFA6, TSC1 and UQCRC1 were found in association with the cognitive and psychiatric symptoms, indicating the role of genetic factors in moderating the presence of cognitive and NP profiles in demented individuals. / The aim of the present study is to evaluate the gene expression profiling of peripheral leukocytes in Chinese subjects with Alzheimer's disease (AD) and explored its potential of clinical application. Behavioral phenotypes of cognitive performance and neuropsychiatric assessment were also investigated in association with gene expression in AD. Persons with mild cognitive impairment (MCI), as an at-risk state between normal aging and clinical dementia, was also assessed in consideration that the information may provide a better understanding of the mechanisms involved in clinical progression of AD. / The genes identified in this study were involved in processes implicated in neurodegneration, including protein isomerization (PIN1), calcium disequilibrium and mitochondria insufficiency (ITPKB and UQCRC1), increased inflammatory response (DDT), apoptosis (CTSB and CTSD) and neurogeneration (NRD1 and TSC1). / Fu, Yan. / Adviser: Chiu Wa Lam. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 132-168). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Koblasa,Vladimír - Comparison of control measurements on various types of haematology analyzers used by ÚKBLD CHLTC at University Hospital in Prague First Faculty of Medicine, Charles University in Prague, Praha 2, Kateřinská 32 Head of the work: prof. MUDr. Jan Kvasnička, DrSc. Supervisor -consultant: Mgr. Ivana Malíková Blood cell count is essential testing method in hematology, where it is necessary to ensure properquality control. Aim of this study was to compare the results of measurement control materials with defined parameters and the same samples at different haematological analyzers to obtain evidence for the expression of measurement uncertainties. There are used more types of blood analyzers in ÚKBLD CHLTC at University Hospital in Prague, which operate on different principles. For comparsion were selected analyzers using the impedance working principle, where individual blood cell passes between two electrodes controlled by low voltage. Variation of this voltage is recorded and accurately defined for each type of blood cells. It was also chosen analyzer that works with optical detection. Analyzer illuminates the individual blood cell by light beam. A cell that enters into the path of light rays, reduce its optical density incident on the photocell. The change of the light density...

Este estudo apresenta um painel de dados hematológicos, bioquímicos, morfológicos e citoquímicos para um grupo de serpentes dos gêneros Bothrops e Crotalus mantidas em cativeiro no Instituto Butantan. Para tanto, amostras de sangue foram colhidas da veia coccígea ventral e processadas de acordo com métodos padronizados. Os resultados alcançados foram analisados para determinar diferenças interespecíficas e sexuais. A avaliação citoquímica incluiu Sudan Black B (SBB), Ácido Periódico de Schiff (PAS) e Benzidina Peroxidase (BP). Os resultados hematológicos e bioquímicos mais relevantes indicaram um número significativamente mais alto de basófilos em B. jararaca e níveis mais altos de cálcio nas fêmeas da maioria das espécies. Os azurófilos marcaram positivamente para todas as colorações. A diferenciação entre trombócitos e linfócitos foi facilmente obtida com PAS. Os basófilos marcaram positivamente apenas com PAS em Bothrops alternatus. A marcação em heterófilos variou consideravelmente entre as espécies. Ultra-estruturalmente, os leucócitos foram semelhantes ao já descrito em literatura com algumas variações no tipo dos grânulos dos basófilos e heterófilos. Os grânulos dos basófilos apresentaram-se redondos em sua maioria, com tamanho e densidade variadas, enquanto que os nos heterófilos foram heterogêneos em forma, tamanho e densidade. / This study reports a panel for hematological, biochemical, morphological and cytochemical data for a group of captive snakes belonging to the genus Bothrops and Crotalus mantained at Instituto Butantan, São Paulo, Brazil. Blood samples were collected from ventral coccigeal vein and were processed according to standard protocols. Cytochemical staining including Sudan Black B (SBB), Periodic Acid-Schiff (PAS) and Benzidine peroxidase (BP) were conducted. Blood values were evaluated to determine interspecific and sex differences. We found a significant increase of basophils in Bothrops jararaca and higher levels of calcium in females. Azurophils stained positively for all stains and a differentiation between lymphocytes and thrombocytes was easily obtained with PAS stain. Basophils stained positively only with PAS in Bothrops alternatus. Heterophils staining varied between species. The ultrastructure of leucocytes were similar within described in literature, with some variations on granules of basophils and heterophils. Basophils granules were round, with heterogeneous size and density; whereas, heterphils granules were heterogeneous in shape, size and density.

A irradiação do sangue humano é usada para evitar a TA-DECH (\"transfusão associada a doença do enxerto contra hospedeiro\"), um raro mas devastador efeito adverso dos leucócitos presentes em componentes de sangue de doadores. Normalmente esta prática de irradiação é executada para a eliminação física de leucócitos. A implementação de procedimentos permitirá que a dose apropriada, dentro de uma faixa de 25 Gy a 50 Gy, seja absorvida pelas bolsas de sangue coletadas em um banco de sangue. Os estudos para estabelecer os procedimentos de BPF (Boas Práticas de Fabricação) foram desenvolvidos baseados na norma ISO 11137 Esterilização de produtos médicos Requisitos para validação e controle de rotina Esterilização por radiação. Dois sistemas dosimétricos foram usados para o mapeamento de dose durante os estudos da qualificação do irradiador, carregamento de produto, validação de processo de irradiação e auditoria. O dosímetro CaSO4:Dy apresentou dificuldades em relação à incerteza na medição da dose, estabilidade, rastreabilidade e calibração. Os dosímetros PMMA e Gafchromic mostraram uma melhor performance e foram adotados para estudos de qualificação de irradiadores necessários para a implantação de BPF. Os testes de irradiação foram realizados em um irradiador Gammacell 220. Os procedimentos desenvolvidos podem ser adaptados para diferentes tipos de irradiadores gama, permitindo a implantação de um programa de garantia da qualidade e BPF para irradiação de sangue. / The irradiation of human blood is used to avoid the TA-GVHD (transfusion-associated graft-versus-host-disease), a rare but devastating adverse effect of leukocytes present in blood components for a immunocompetent transfusion recipients. Usually this irradiation practice is performed to a physical elimination of leukocytes. The implementation of the procedures will assure that the properly dose in a range of 25 Gy to 50 Gy will be delivered to the blood in the bag collected in a blood tissue bank. The studies of the procedures in order to establish a GMP (Good Manufacturing Practices) were developed under the guidelines of the standard ISO 11137 Sterilization of health care products Requirements for validation and routine control Radiation sterilization. In this work, two dosimetric systems were used for dose mapping during the studies of irradiator qualification, loading pattern, irradiation process validation and auditing. The CaSO4:Dy dosimeter presented difficulties concerning to uncertainty on dose measurement, stability, traceability and calibration. The PMMA and Gafchromic dosimetric systems have shown a better performance and were adopted on studies of irradiators qualification that are necessary to implementation of GMP. The irradiation tests have been done in a Gammacell 220 irradiator. The developed procedures can be adapted for different kinds of gamma irradiators, allowing implanting a quality assurance program and a GMP for blood irradiation.

Hidroquinona (HQ) é um dos metabólitos do benzeno responsáveis pelos efeitos tóxicos da exposição ao solvente, além de ser componente da dieta, medicamentos, cigarro e poluente do meio ambiente. Considerando a imunotoxicidade desta substância, o grupo de pesquisa do laboratório investiga o papel da exposição à HQ por período prolongado de tempo sobre respostas inflamatórias agudas (RIA). Neste contexto, o presente trabalho avaliou os efeitos desta exposição sobre os mecanismos vasculares e celulares da RIA de diferentes doses diárias de HQ (5, 10 ou 50 mg/kg) em ratos Wistar machos, via i.p., por 17 ou 22 dias, uma vez ao dia, com intervalos de 2 dias a cada 5 doses. Animais controles receberam o veículo (salina com 5% etanol). A resposta inflamatória aguda inata foi induzida pela administração de glicogênio de ostra (1% em PBS, 5 mL) na bolsa subcutânea dorsal ou pela instilação de lipopolissacarídeo de Salmonella abortus (LPS; 100 µL de solução 100 µg/mL); A resposta inflamatória aguda adquirida foi provocada pela inalação de ovalbumina (10 mL de solução de OA 1% PBS, 15 min) em animais previamente sensibilizados (10 µ/100mg AI(OH)3 no décimo dia de exposição). Os resultados obtidos mostraram que: 1) o aumento do número de leucócitos na bolsa dorsal de animais expostos a 50 mg/kg de HQ é dependente, pelo menos em parte, da maior interação de leucócitos circulantes à parede vascular da microcirculação, mas não é decorrente de alterações na reatividade microvascular; o aumento de expressão de moléculas de adesão (β2 integrina), que pode ser a responsável pelo aumento de interaçãoleucócito-endotélio e migração celular; 2) a redução da migração de leucócitos para o pulmão inflamado pelo LPS em animais expostos a HQ, nas menores doses, não foi decorrente de modificações na interação leucócito-endotélio, nem da expressão de moléculas de adesão nos leucócitos circulantes ou na célula endotelial do tecido pulmonar; 3) a redução da migração celular para o pulmão durante a RIA alérgica em animais expostos a 5, 10 ou 50 mglkg de HQ é dependente, pelo menos em parte, da menor concentração de anticorpos anafiláticos circulantes e conseqüentemente da desgranulação reduzida de mastócitos teciduais, visualizados no leito mesentérico após desafio in situ pela OA. A menor produção de anticorpos anafiláticos pode ser decorrente da ação da HQ em diferentes tipos celulares, uma vez que foi observada expressão reduzida de moléculas co-estimulatórias em linfócitos do baço (CD45R e CD6), menor atividade microbicida de macrófagos peritoniais frente a Candida albicans e menor secreção de interferon-γ por células do peritônio. Em conjunto, os resultados apresentados mostram os mecanismos da HQ sobre a resposta do organismo ao trauma de diferentes origens, interferindo com tipos celulares distintos envolvidos nas reações. / Hydroquinone (HQ) is one of the metabolites of benzene responsible for the toxic effects of exposure to solvent, as well as being part of the diet, medicines, tobacco and polluting the environment. Considering the immunotoxicity of this substance, our laboratory has investigated the role of exposure to HQ by prolonged period of time on acute inflammatory responses (AIR). In this context, this study evaluated the effects of this exposure on the vascular and cellular mechanisms of AIR of different daily doses of HQ (5, 10 or 50 mg/kg) in male rats, via ip, for 17 or 22 days, a once a day, with intervals of 2 days every 5 doses. Control animais received the vehicle (saline with 5% ethanol). Innate AIR was induced by the administration of oysters glycogen (1% in PBS, 5 mL) into subcutaneous back pouch or by instillation of lipopolysaccharide of Salmonella aborius (LPS, 100 µL of solução100 µg/mL); Allergic AIR gained was caused by inhalation of ovalbumin (10 mL solution of 1% OA in PBS, 15 min) in previously sensitized animal (10 µ/100mg AI (OH)3 on the tenth day of exposure). Results showed that: 1) the increased number of white blood cells back into the pouch of animais exposed to 50 mg/kg of HQ is dependent, at least in part, of higher interaction of circulating leukocytes to the vascular wall of the microcirculation, but is not due to changes in microvascular reactivity; an increase of expression of molecules of accession (β2 integrin), which may be responsible for the enhanced interaction of leukocyteendothelial and cell migration 2) the reduced migration of leukocytes into the lung inflamed by LPS in animals exposed to 5 or 10 mg/kg was not due to changes in the interactions of leukocyte to endothelium, either the expression of adhesion molecules in circulating white blood cells or in cell endothelial lung tissue, 3) the reduced cell migration to the lungs during the AIR allergic to animais exposed to HQ, in lower doses, is dependent on lower concentration of· circulating anaphylactics antibodies which may be responsible for the mast cell desgranulation. The lower amount of anaphylatic antibodies in the circulation may be due to action of HQ in different cells, as reduced expression of co-stimulatory molecules by Iymphocytes (CD45R and CD6), lower microbicide activity of macrophages and reduced secretion of interferon-γ by peritoneal cells were detected. Together, the results show the toxicity of HQ on the body\'s response to trauma from different sources, interfering with different cell types involved in the reactions.

The cytokine environment of early pregnancy is known to be a key determinant of the development of the pre-implantation embryo, and its subsequent implantation and growth. Factors in male seminal fluid have been identified as regulators of the expression of cytokines in the female tract of mice, humans and other mammalian species, with insemination eliciting a cascade of molecular and cellular events, reminiscent of a classic inflammatory response. In humans, perturbations in seminal fluid signalling have been proposed to predispose to pathologies of pregnancy including implantation failure, recurrent miscarriage and pre-eclampsia. Seminal transforming growth factor-beta (TGFβ) is identified as one key molecule present in seminal fluid responsible for inducing the female post-mating cytokine response in mice. Research in humans however, has shown the seminal TGFβ content of fertile versus infertile couples to be similar, while the content of other known seminal constituents such as interferon-gamma (IFNγ), correlate with reproductive success. This project aimed to investigate the nature of active factors present in seminal fluid in mice, and their interactions in regulating the uterine cytokine environment during early pregnancy, utilising a variety of in vitro and in vivo experimental strategies. Further, the effect of perturbation in the peri-conception cytokine environment on short and long term pregnancy and postnatal outcomes was investigated. Evaluation of uterine fluids from estrous and mated mice showed a marked upregulation of a number of cytokines following mating, including granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-6 (IL-6) and the chemokine KC (rodent IL-8 homologue). Increased production of factors such as GM-CSF and subsequent generation of a receptive uterine environment is thought to be crucial for optimal embryo development and placentation. It has previously been shown that seminal factors such as TGFβ contribute to the uterine post-mating inflammatory response, however other moieties present in seminal fluid, for instance cytokines induced in response to infection such as IFNγ or products from the mucosal microflora, may also play a regulatory role. Using uterine epithelial cells cultured in vitro, it was shown that a variety of immune modulators including the cytokines TGFβ and IFNγ, as well as bacterial products, gram negative lipopolysaccharide (LPS) and gram positive lipoteichoic acid (LTA), can alter basal cytokine production. IFNγ, a pro-inflammatory cytokine secreted by activated natural killer cells and T-cells, is known to interfere with TGFβ signalling in other contexts. Independently TGFβ, LPS and LTA stimulate GM-CSF production while differentially regulating IL-6 and KC production. Conversely IFNγ inhibits GM-CSF production, without effecting IL-6 or KC. Pair wise combinations of TGFβ, LPS and LTA resulted in additive stimulation of GM-CSF, while addition of IFNγ to cultures in conjunction with any of these molecules downregulated GM-CSF and KC stimulation. These in vitro studies indicate factor-specific interactions between seminal fluid constituents and highlight the complex nature of seminal fluid signalling. Consequently we propose that the relative ratio of seminal signalling factors is likely to be more important than the absolute concentration of various regulators, in determining the optimal female reproductive tract response. Using the mouse as an in vivo model, I have in addition demonstrated that LPS and LTA instilled into an estrous uterus can elicit cytokine production comparable to that observed following insemination. Further, these studies have shown that IFNγ instilled into the uterus of a recently mated mouse can reduce the post-copulatory GM-CSF and KC surge. However administration of IFNγ had no effect on near term pregnancy outcomes including fetal or placental weights, fetal crown-rump length, or implantation or resorption rates. The ‘developmental origins of adult disease hypothesis’ proposes the idea that the early uterine environment encountered by the conceptus contributes toward the risk of metabolic disorders in adulthood, hence a long term study of progeny conceived after IFNγ administration was also undertaken. Neo-natal outcomes, such as birth weight, litter size and gestation length were unaltered, as was growth trajectory to 22 weeks of age. Adult metabolic markers, glucose tolerance, organ weight, muscle weight, adiposity and systolic blood pressure were not affected by the perturbation of peri-conceptual cytokine parameters. This work has examined the potential regulatory role of a number of seminal fluid signalling agents in directing the post-mating cytokine response, and has furthermore shown the relatively resilient nature of the early cytokine environment to subtle perturbation. Delineating the identity and roles of seminal fluid factors in early pregnancy brings us closer to an understanding of the key physiological events of early pregnancy and assists in identifying potential risk factors for human pregnancy pathologies. / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Coronary artery stent placement has been a significant advance in the percutaneous treatment of atherosclerotic disease, and tissue engineered vascular grafts may provide a viable alternative to autologous segments for small diameter vessels. However, in-stent restenosis remains an important limitation, and tissue engineered grafts have poor patency and high risk of thrombus formation due to their inability to maintain a confluent, adherent, and quiescent endothelium. While animal models provide insight into the pathophysiology of these situations, elucidation of the relative importance of stent or graft components, hemodynamic factors, and molecular factors is difficult. Very little research has focused on bridging gaps in knowledge concerning blood/biomaterial interactions, blood/endothelial cell interactions, and endothelial cell/smooth muscle cell cross-talk. The work presented within this thesis will do just that. The objective of this thesis research was to elucidate the influence of biomaterial-induced activation of leukocytes on endothelial cell or smooth muscle cell phenotype, as well as endothelial cell/smooth muscle cell cross-talk in co-culture systems. Towards this goal, two complimentary in vitro endothelial cell/smooth muscle cell co-culture models with divergent smooth muscle cell phenotype were developed and characterized. Using these systems, it was found that the presence of more secretory smooth muscle cells (as would be seen in wound healing or disease) in general enhanced endothelial cell activation in response to biomaterial-pretreated monocytes, while the presence of less secretory smooth muscle cells (to model more quiescent smooth muscle cells found in uninjured healthy vessels) suppressed endothelial cell activation in response to biomaterial-pretreated monocytes (and neutrophils to a small degree). Additionally, biomaterial-pretreated monocytes and neutrophils amplified a smooth muscle cell phenotypic shift away from a more quiescent state. It is likely that the compounding effect of secretory smooth muscle cells and biomaterial-activated leukocytes are responsible for altered vascular wound healing upon implantation of stents or vascular grafts. Understanding the specific signals causing these effects, or signals delivered by contractile smooth muscle cells that limit these effects help to provide design criteria for development of devices or grafts capable of long term patency.