Introdução. Embora necessário ao desenvolvimento econômico e bastante disseminado na sociedade atual, o trabalho em turnos noturnos fixos contribui para consequências sociais negativas e prejuízos à saúde dos trabalhadores deste turno de trabalho. Objetivo. Investigar possíveis efeitos do trabalho em turnos fixos noturnos sobre o sono e biomarcadores hormonais e inflamatórios selecionados. Métodos. Estudo transversal em indústria de metais sanitários da cidade de São Paulo, de que participaram 17 trabalhadores do sexo masculino do turno noturno (das 21h às 6h) e 21 trabalhadores de um dos turnos diurnos (das 7h às 17 h). Dados sociodemográficos, de saúde e de condições de trabalho e sobre fadiga, sonolência, estresse e matutinidade-vespertinidade foram obtidos por questionário. O ciclo atividade/repouso foi avaliado por actimetria e por protocolo diário de atividades durante 10 dias consecutivos, abrangendo ao menos um ciclo de turnos completo. Amostras de saliva coletadas em momentos diferentes durante três dias de trabalho foram analisadas por ELISA para determinação das concentrações dos cinco biomarcadores. Todos os dados obtidos foram então submetidos às análises matemáticas e estatísticas mais adequadas a cada caso. Resultados. O trabalho noturno foi associado a uma diminuição na secreção da melatonina e a alterações em seu ritmo de secreção. Foram observadas também alterações no ritmo de secreção do cortisol e no padrão de variação diário das citocinas salivares TNF, IL-1 e IL-6. Nos trabalhadores diurnos, a privação parcial do sono foi associada a aumento significativo da concentração da IL-6 na saliva. Um maior grau de dessincronização crônica entre os trabalhadores noturnos estaria associado a um maior encurtamento do sono diurno em dias de trabalho, a uma grande variabilidade no ritmo circadiano de secreção do cortisol e a uma maior variabilidade no padrão de variação diário da produção da IL-1 salivar. Conclusões. Os resultados indicam que o trabalho noturno em turnos fixos pode vir a ocasionar prejuízos à saúde dos trabalhadores deste esquema de turnos, tornando-o desaconselhável desse ponto de vista. Diferentes graus de sincronização do sistema de temporização endógeno estariam associados a maior ou menor adaptação ao trabalho em turnos noturnos fixos, com possíveis reflexos, em médio e longo prazos, sobre a tolerância ao trabalho em turnos e os efeitos à saúde nesses trabalhadores. Turnos diurnos com um horário de início associados à privação parcial do sono também poderiam acarretar prejuízos futuros à saúde desses trabalhadores / Introduction. Despite its relevance for the economic development and widespread employment in modern societies, night work contributes to social distress and health injuries of workers. Aim. Assess the effects of permanent night work on sleep and selected hormonal and inflammatory biomarkers. Methods. This cross-sectional study in a sanitary metals industry in São Paulo, Brazil, included only male workers. Demographic, health and working conditions data were obtained for seventeen permanent night workers (shift hours: 21:00 to 6:00) and twenty one permanent day workers (shift hours: 7:00 to 17:00). They also answered questionnaires about fatigue, sleepiness, stress and morningness-eveningness. Activity/rest cycle was evaluated by actimetry and activity protocols for ten consecutive days, thus covering at least an entire shift work cycle. Saliva samples collected in different moments for three workdays were submitted to ELISA analysis for the assessment of the salivary levels of the five biomarkers. All data were further submitted to the appropriate mathematical and statistical analyzes. Results. Night work was associated with lower levels of salivary melatonin and with a disturbed rhythm of secretion. It was also associated with disturbances in the cortisol rhythm and in the daily variation pattern of salivary cytokines TNF, IL-1, and IL-6. Partial sleep deprivation was associated with significant elevated levels of salivary IL-6. Higher levels of chronic dessinchronization among night workers were associated with a shorter day sleep during workdays, great variability in the cortisol rhythm and to more variability in the daily variation pattern of salivary IL-1. Conclusions. Permanent night work might cause health injuries and should be avoided. Distinct levels of desynchronization seem to be associated with a better or worse adaptation to permanent night work, which could be later reflected in the tolerance to shift work and general health of night workers. An early morning start of day shifts is associated with partial sleep deprivation and could cause later health injuries; for this reason, they should also be avoided

Introdução. Embora necessário ao desenvolvimento econômico e bastante disseminado na sociedade atual, o trabalho em turnos noturnos fixos contribui para consequências sociais negativas e prejuízos à saúde dos trabalhadores deste turno de trabalho. Objetivo. Investigar possíveis efeitos do trabalho em turnos fixos noturnos sobre o sono e biomarcadores hormonais e inflamatórios selecionados. Métodos. Estudo transversal em indústria de metais sanitários da cidade de São Paulo, de que participaram 17 trabalhadores do sexo masculino do turno noturno (das 21h às 6h) e 21 trabalhadores de um dos turnos diurnos (das 7h às 17 h). Dados sociodemográficos, de saúde e de condições de trabalho e sobre fadiga, sonolência, estresse e matutinidade-vespertinidade foram obtidos por questionário. O ciclo atividade/repouso foi avaliado por actimetria e por protocolo diário de atividades durante 10 dias consecutivos, abrangendo ao menos um ciclo de turnos completo. Amostras de saliva coletadas em momentos diferentes durante três dias de trabalho foram analisadas por ELISA para determinação das concentrações dos cinco biomarcadores. Todos os dados obtidos foram então submetidos às análises matemáticas e estatísticas mais adequadas a cada caso. Resultados. O trabalho noturno foi associado a uma diminuição na secreção da melatonina e a alterações em seu ritmo de secreção. Foram observadas também alterações no ritmo de secreção do cortisol e no padrão de variação diário das citocinas salivares TNF, IL-1 e IL-6. Nos trabalhadores diurnos, a privação parcial do sono foi associada a aumento significativo da concentração da IL-6 na saliva. Um maior grau de dessincronização crônica entre os trabalhadores noturnos estaria associado a um maior encurtamento do sono diurno em dias de trabalho, a uma grande variabilidade no ritmo circadiano de secreção do cortisol e a uma maior variabilidade no padrão de variação diário da produção da IL-1 salivar. Conclusões. Os resultados indicam que o trabalho noturno em turnos fixos pode vir a ocasionar prejuízos à saúde dos trabalhadores deste esquema de turnos, tornando-o desaconselhável desse ponto de vista. Diferentes graus de sincronização do sistema de temporização endógeno estariam associados a maior ou menor adaptação ao trabalho em turnos noturnos fixos, com possíveis reflexos, em médio e longo prazos, sobre a tolerância ao trabalho em turnos e os efeitos à saúde nesses trabalhadores. Turnos diurnos com um horário de início associados à privação parcial do sono também poderiam acarretar prejuízos futuros à saúde desses trabalhadores / Introduction. Despite its relevance for the economic development and widespread employment in modern societies, night work contributes to social distress and health injuries of workers. Aim. Assess the effects of permanent night work on sleep and selected hormonal and inflammatory biomarkers. Methods. This cross-sectional study in a sanitary metals industry in São Paulo, Brazil, included only male workers. Demographic, health and working conditions data were obtained for seventeen permanent night workers (shift hours: 21:00 to 6:00) and twenty one permanent day workers (shift hours: 7:00 to 17:00). They also answered questionnaires about fatigue, sleepiness, stress and morningness-eveningness. Activity/rest cycle was evaluated by actimetry and activity protocols for ten consecutive days, thus covering at least an entire shift work cycle. Saliva samples collected in different moments for three workdays were submitted to ELISA analysis for the assessment of the salivary levels of the five biomarkers. All data were further submitted to the appropriate mathematical and statistical analyzes. Results. Night work was associated with lower levels of salivary melatonin and with a disturbed rhythm of secretion. It was also associated with disturbances in the cortisol rhythm and in the daily variation pattern of salivary cytokines TNF, IL-1, and IL-6. Partial sleep deprivation was associated with significant elevated levels of salivary IL-6. Higher levels of chronic dessinchronization among night workers were associated with a shorter day sleep during workdays, great variability in the cortisol rhythm and to more variability in the daily variation pattern of salivary IL-1. Conclusions. Permanent night work might cause health injuries and should be avoided. Distinct levels of desynchronization seem to be associated with a better or worse adaptation to permanent night work, which could be later reflected in the tolerance to shift work and general health of night workers. An early morning start of day shifts is associated with partial sleep deprivation and could cause later health injuries; for this reason, they should also be avoided

Introdução: A toxicidade da cisplatina é bem compreendida nos sistemas renal, gastrointestinal, auditivo e nervoso, assim como na medula óssea. No entanto, os mecanismos causadores de infertilidade induzidos pela cisplatina são pouco compreendidos. Objetivo: Nosso objetivo foi verificar a participação do sistema calicreína-cininas e do estresse oxidativo na infertilidade induzida pela cisplatina, auxiliando no desenvolvimento de novas alternativas terapêuticas. Métodos: Os camundongos fêmeas C57BL/6 adultos (n=9) receberam dois ciclos de 2,5 mg/kg de cisplatina por via intraperitoneal durante cinco dias, com um período de recuperação de sete dias entre os ciclos. O grupo controle (n=9) recebeu solução de NaCl 0,9%. Foi feita a avaliação do ciclo estral e a contagem de folículos ovarianos. O marcador Ki67 foi avaliado por imunohistoquímica. Testes bioquímicos para calicreína plasmática, intersticial e glandular, tempo de tromboplastina parcial ativada (TTPa), óxido nítrico (NO), superóxido dismutase (SOD), glutationa reduzida (GSH), mieloperoxidase (MPO) e N-acetil glucosaminidase (NAG); e Western-blot para os receptores de bradicinina B1R e B2R também foram realizados Resultados: Após o protocolo de cisplatina, 100% das fêmeas do grupo controle mantiveram a ciclicidade estral versus 44,4% das fêmeas do grupo cisplatina. O grupo controle apresentou maior número de folículos antrais (p=0,011) e folículos viáveis totais (p=0,006). O grupo cisplatina apresentou maior número de folículos atrésicos (p=0,014). O marcador Ki67 demonstrou semelhantes taxas de proliferação celular entre os grupos. Os marcadores inflamatórios foram aumentados no grupo cisplatina, incluindo a geração de calicreína plasmática (p=0,003), a diminuição do TTPa (p=0,02), o aumento da atividade da calicreína intersticial (p=0,002) e glandular (p=0,008) e na expressão dos receptores B1R (p=0,001) e B2R (p=0,001), MPO (p=0,03) e NAG (p=0,04). Os marcadores de estresse oxidativo também foram aumentados no grupo cisplatina, com maior produção de NO (p=0,01) e diminuição na SOD (p=0,003) e na GSH (p=0,01). Conclusão: Todas as reações inflamatórias parecem ser ativadas pelo tratamento com cisplatina, exemplificadas pelo aumento da atividade da calicreína plasmática, intersticial e glandular, bem como a diminuição no TTPa e o aumento na expressão de B1R e B2R. A toxicidade mediada pela reação inflamatória da cisplatina é bem conhecida em seus efeitos colaterais, como ototoxicidade e nefrotoxicidade. Houve aumento da produção de NO nos ovários dos animais tratados, associado à indicação de menores concentrações de SOD e de GSH. Os desequilíbrios nos antioxidantes parecem contribuir para o estresse oxidativo ovariano. Quanto à MPO (neutrófilos) à NAG (macrófagos), a maior atividade de ambas no grupo cisplatina se explica pelo fato de que as células fagocíticas ativadas produzem grandes quantidades de espécies reativas de oxigênio, aumentando ainda mais o estresse oxidativo e a inflamação. O aumento da atividade do sistema calicreína-cininas e dos marcadores de estresse oxidativo no tecido ovariano propiciaram uma melhor compreensão da infertilidade induzida por cisplatina e indicam possíveis alternativas para proteção ovariana durante a quimioterapia, como inibidores do sistema calicreína-cinina e antioxidantes. / Background: Cisplatin toxicity is well understood in the renal, gastrointestinal, auditory and nervous systems, as well as in the bone marrow. However, the mechanisms causing infertility induced by cisplatin are poorly understood. Purpose: Our objective was to verify the participation of the kallikreinkinin system and oxidative stress in cisplatin-induced infertility, aiding in the development of new therapeutic alternatives. Methods: C57BL/6 adult (n=9) female mice received two 2.5 mg/kg intra-peritoneal cycles of cisplatin for five days, with a seven-day recovery period between cycles. The control group (n=9) received 0.9% NaCl solution. The ovarian follicles were counted with hematoxylin and eosin staining. Ki67 marker was evaluated by immunohistochemistry. Biochemical tests for plasma, interstitial and glandular kallikrein, activated partial thromboplastin time (aPTT), nitric oxide (NO), superoxide dismutase (SOD), reduced glutathione (GSH), myeloperoxidase (MPO) and N-acetyl glucosaminidase (NAG); and Western blotting for the bradykinin B1R and B2R receptors were also performed. Results: After cisplatin protocol, 100% of the females in the control group maintained estral cyclicity versus 44.4% of females in cisplatin group. The control group had a higher number of antral follicles (p=0.011) and total viable follicles (p=0.006). Cisplatin group had a higher number of atretic follicles (p=0.014). Ki67 marker demonstrated similar rates of cell proliferation between groups. Inflammatory markers were increased in cisplatin group, including plasma kallikrein generation (p=0.003), a decrease of aPTT (p=0.02), increased interstitial (p=0.002) and glandular (p=0.008) kallikrein, B1R (p=0.001) and B2R (p=0.001) expression, MPO (p=0.03) and NAG (p=0.04) Oxidative stress markers were also increased in cisplatin group, with higher NO production (p=0.01) and a decrease in SOD (p=0.003) and GSH (p=0.01). Conclusion: All inflammatory reactions appear to be activated by cisplatin treatment, exemplified by increased plasma, interstitial and glandular kallikrein activity, as well as the decrease in aPTT and increased expression of B1R and B2R. The toxicity mediated by cisplatin inflammatory reaction is well known in its side effects, such as ototoxicity and nephrotoxicity. There was an increase in NO production in the ovaries of treated animals, associated with the indication of lower concentrations of SOD and GSH. Imbalances in antioxidants appear to contribute to ovarian oxidative stress. Regarding MPO (neutrophils) and NAG (macrophages), the greater activity of both in cisplatin group is explained by the fact that activated phagocytic cells produce large amounts of reactive oxygen species, further increasing oxidative stress and inflammation. Increased activity of the kallikrein-kinin system and markers of oxidative stress in ovarian tissue provided a better understanding of cisplatin-induced infertility and indicate possible alternatives for ovarian protection during chemotherapy, such as inhibitors of the kallikrein-kinin system and antioxidants.

In the gastrointestinal tract, probiotics have been shown to promote host immunity and to regulate immune signaling pathways. This study used Caco-2 cell line to examine the effects of a Lactobacillus rhamnosus isolate from “amabere amaruranu” a Kenyan traditional cultured milk, on the production inflammatory cytokines in enterocytes. Live Lactobacillus rhamnosus (MRS6AN), its cytoplasmic fraction (CF), filtered spent broth (FSB) or heat inactivated FSB (HIB) were used as treatments on differentiated Caco-2 cell monolayer in transwells. Cytokine content in the cell lysates, apical and basolateral supernatants were determined using ELISA. Caco-2 cell lysate treatments showed significantly increased anti-inflammatory TGF-β (ng/ml) levels on average about 100x more compared to the increase in pro-inflammatory IL-8 (pg/ml) levels. These levels were significantly reduced after inhibition of NF-κB. In conclusion, live Lactobacillus rhamnosus, its CF, FSB or HIB seemed to modulate the production of inflammatory cytokines in enterocytes partly via the NF-κB signaling pathway.

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

African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

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

Τα τελευταία χρόνια, ανοσολογικές διεργασίες άσηπτης φλεγμονής έχουν αναγνωρισθεί ότι συμμετέχουν τόσο στην πρόκληση, όσο και στην (πρώιμη ή όψιμη) έκβαση των ισχαιμικών αγγειακών εγκεφαλικών επεισοδίων (Ι-ΑΕΕ). Στην παρούσα μελέτη ελέγχθηκε κατά πόσον η γενετική προδιάθεση, προς εντονότερη (ή ασθενέστερη) ανοσολογική απάντηση, θα μπορούσε να προβλέπει το οξύ Ι-ΑΕΕ, τη νευρολογική του εξέλιξη, την 6μηνη έκβασή του και την ανάπτυξη κοινών νευροψυχιατρικών επιπλοκών (κατάθλιψη, νοητική έκπτωση). Για το σκοπό αυτό αναλύθηκε η πιθανή προγνωστική αξία 5 σημειακών λειτουργικών πολυμορφισμών στα γονίδια φλεγμονωδών κυτταροκινών (IL4-589C>T, IL10-1082G>A, TNFa-308G>A, IL6-174G>C και IL12B 1188A>C) σε 145 διαδοχικούς ασθενείς με οξύ Ι-ΑΕΕ και ισάριθμους μάρτυρες. Επιπρόσθετα, η μελέτη αξιολόγησε το ρόλο της αντιφλεγμονώδους κυτταροκίνης αντιπονεκτίνης στην οξεία φάση του Ι-ΑΕΕ, καθώς και την πιθανή προβλεπτική της αξία στη νευρολογική του εξέλιξη, την πρόγνωση και την 6μηνη έκβασή του. Τα κύρια αποτελέσματα της μελέτης μπορούν να συνοψισθούν ως εξής: - Σε έναν ομογενή Eλληνικό πληθυσμό, οι λευτουργικοί πολυμορφισμοί IL4-589C>T, IL10-1082G>A, TNFa-308G>A, IL6-174G>C και IL12B 1188A>C δεν προβλέπουν την επέλευση ενός Ι-ΑΕΕ, την ηλικία έναρξης της νόσου ή την ανάπτυξη κατάθλιψης και νοητικής έκπτωσης όψιμα μετά το Ι-ΑΕΕ. - Ο IL4-589CT γονότυπος σχετίστηκε με την τάση της νόσου για υποτροπή. - Ο IL10-1082GG αρχέγονος γονότυπος προέβλεπε την πρώϊμη νευρολογική επιδείνωση και τη χειρότερη λειτουργική του έκβαση 1ου και 3ου μήνα. - Ο TNFa-308GG αρχέγονος γονότυπος συνέβαλλε ευεργετικά στην καλύτερη λειτουργική έκβαση κατά τον 6ο μήνα. - Ο IL12B 1188AC ετεροζυγώτης προέβλεπε σημαντικά τα ελαφρύτερα Ι-ΑΕΕ και στη χαμηλότερη θνητότητα από αυτά. - Τα αυξανόμενα επίπεδα αντιπονεκτίνης ορού σχετίζονται με μείωση των πιθανοτήτων για ένα Ι-ΑΕΕ, ανεξάρτητα από τους κλασσικούς παράγοντες κινδύνου. - Η αντιπονεκτίνη ορού βρίσκεται κατεσταλμένη στην οξεία φάση του Ι-ΑΕΕ, αλλά και 6 μήνες μετά. Τα χαμηλά της επίπεδα δεν αποτελούν αντίδραση οξείας φάσης, αλλά αντικατοπτρίζουν το σταθερό αντιφλεγμονώδη ρόλο της. - H αντιπονεκτίνης ορού οξείας φάσης μετά ένα Ι-ΑΕΕ δεν προβλέπει τη νευρολογική εξέλιξη και την 6μηνη έκβαση της νόσου στον άνθρωπο. Προτείνουμε ότι στο μέλλον η έγκαιρη ταυτοποίηση των γενετικά προδιατεθειμένων ατόμων για βαρύτερη νόσο και επιβαρυμένη έκβαση μετά ένα Ι-ΑΕΕ, θα επιτρέπει την αναγνώριση ομάδων ασθενών με τις μεγαλύτερες ανάγκες εντατικοποιημένης αποκατάστασης και βελτιστοποίησης των ιατρικών μας θεραπειών. Επιπρόσθετα, προτείνουμε ότι η αντιπονεκτίνη θα μπορούσε να αποτελεί ένα δυνητικό θεραπευτικό στόχο τροποποίησης του αγγειακού κινδύνου. / Immune processes of aseptic inflammation have been recently recognized as key participants into both the occurrence and (early or late) outcome of an ischemic stroke (IS). The current study tested whether the genetic predisposition for an enhanced (or suppressed) immunological response, could be predicting the occurrence, neurological evolution, 6-month outcome, and two common neuropsychiatric complications (depression, cognitive impairment) of an IS. For this purpose, 5 functional single nucleotide polymorphisms in the genes of inflammatory cytokines (IL4-589C>T, IL10-1082G>A, TNFa-308G>A, IL6-174G>C and IL12B 1188A>C) were analyzed for their putative prognostic value in 145 consecutive acute IS patients and in an equal-sized control group. Moreover, the present study evaluated the role of an anti-inflammatory cytokine, adiponectin, into the acute phase of an IS, as well as its possible predicting value in the neurological evolution, prognosis and 6-month outcome of the ischemic event. The main results of this study may be summarized as follows: - In a homogenous Greek population, the functional polymorphisms IL4-589C>T, IL10-1082G>A, TNFa-308G>A, IL6-174G>C and IL12B 1188A>C do not predict IS occurrence, age of disease onset, or the development of late post-stroke depression and cognitive impairment. - The IL4-589CT mutant genotype was associated with relapsing IS. - The IL10-1082GG ancestral genotype significantly predicted early stroke pregression and worse functional outcome on months 1 and 3. - The TNFa-308GG ancestral genotype beneficially contributed into better 6-month functional outcome. - The IL12B 1188AC heterozygote significantly predicted milder strokes and decreased IS mortality. - Increasing serum adiponectin levels are associated with reduced odds for an IS, independently of common vascular risk factors. - Low serum adiponectin is present at the acute phase and 6 months after an IS. Suppressed adiponectin levels are not an effect of the acute-phase response, but reflect its solid anti-inflammatory role. - Acute post-stroke serum adiponectin does not predict neurological evolution and 6-month outcome of an IS in humans. We propose that early identification of genetically predisposed subjects, for a more severe disease and for a worse outcome, will allow in the future the recognition of those IS sufferers, who would most benefit from intensive rehabilitation programs, and optimization of our medical therapies. Moreover, we propose that adiponectin may represent a promising candidate for the therapeutic risk modification of common vascular complications.

Introdução: A toxicidade da cisplatina é bem compreendida nos sistemas renal, gastrointestinal, auditivo e nervoso, assim como na medula óssea. No entanto, os mecanismos causadores de infertilidade induzidos pela cisplatina são pouco compreendidos. Objetivo: Nosso objetivo foi verificar a participação do sistema calicreína-cininas e do estresse oxidativo na infertilidade induzida pela cisplatina, auxiliando no desenvolvimento de novas alternativas terapêuticas. Métodos: Os camundongos fêmeas C57BL/6 adultos (n=9) receberam dois ciclos de 2,5 mg/kg de cisplatina por via intraperitoneal durante cinco dias, com um período de recuperação de sete dias entre os ciclos. O grupo controle (n=9) recebeu solução de NaCl 0,9%. Foi feita a avaliação do ciclo estral e a contagem de folículos ovarianos. O marcador Ki67 foi avaliado por imunohistoquímica. Testes bioquímicos para calicreína plasmática, intersticial e glandular, tempo de tromboplastina parcial ativada (TTPa), óxido nítrico (NO), superóxido dismutase (SOD), glutationa reduzida (GSH), mieloperoxidase (MPO) e N-acetil glucosaminidase (NAG); e Western-blot para os receptores de bradicinina B1R e B2R também foram realizados Resultados: Após o protocolo de cisplatina, 100% das fêmeas do grupo controle mantiveram a ciclicidade estral versus 44,4% das fêmeas do grupo cisplatina. O grupo controle apresentou maior número de folículos antrais (p=0,011) e folículos viáveis totais (p=0,006). O grupo cisplatina apresentou maior número de folículos atrésicos (p=0,014). O marcador Ki67 demonstrou semelhantes taxas de proliferação celular entre os grupos. Os marcadores inflamatórios foram aumentados no grupo cisplatina, incluindo a geração de calicreína plasmática (p=0,003), a diminuição do TTPa (p=0,02), o aumento da atividade da calicreína intersticial (p=0,002) e glandular (p=0,008) e na expressão dos receptores B1R (p=0,001) e B2R (p=0,001), MPO (p=0,03) e NAG (p=0,04). Os marcadores de estresse oxidativo também foram aumentados no grupo cisplatina, com maior produção de NO (p=0,01) e diminuição na SOD (p=0,003) e na GSH (p=0,01). Conclusão: Todas as reações inflamatórias parecem ser ativadas pelo tratamento com cisplatina, exemplificadas pelo aumento da atividade da calicreína plasmática, intersticial e glandular, bem como a diminuição no TTPa e o aumento na expressão de B1R e B2R. A toxicidade mediada pela reação inflamatória da cisplatina é bem conhecida em seus efeitos colaterais, como ototoxicidade e nefrotoxicidade. Houve aumento da produção de NO nos ovários dos animais tratados, associado à indicação de menores concentrações de SOD e de GSH. Os desequilíbrios nos antioxidantes parecem contribuir para o estresse oxidativo ovariano. Quanto à MPO (neutrófilos) à NAG (macrófagos), a maior atividade de ambas no grupo cisplatina se explica pelo fato de que as células fagocíticas ativadas produzem grandes quantidades de espécies reativas de oxigênio, aumentando ainda mais o estresse oxidativo e a inflamação. O aumento da atividade do sistema calicreína-cininas e dos marcadores de estresse oxidativo no tecido ovariano propiciaram uma melhor compreensão da infertilidade induzida por cisplatina e indicam possíveis alternativas para proteção ovariana durante a quimioterapia, como inibidores do sistema calicreína-cinina e antioxidantes. / Background: Cisplatin toxicity is well understood in the renal, gastrointestinal, auditory and nervous systems, as well as in the bone marrow. However, the mechanisms causing infertility induced by cisplatin are poorly understood. Purpose: Our objective was to verify the participation of the kallikreinkinin system and oxidative stress in cisplatin-induced infertility, aiding in the development of new therapeutic alternatives. Methods: C57BL/6 adult (n=9) female mice received two 2.5 mg/kg intra-peritoneal cycles of cisplatin for five days, with a seven-day recovery period between cycles. The control group (n=9) received 0.9% NaCl solution. The ovarian follicles were counted with hematoxylin and eosin staining. Ki67 marker was evaluated by immunohistochemistry. Biochemical tests for plasma, interstitial and glandular kallikrein, activated partial thromboplastin time (aPTT), nitric oxide (NO), superoxide dismutase (SOD), reduced glutathione (GSH), myeloperoxidase (MPO) and N-acetyl glucosaminidase (NAG); and Western blotting for the bradykinin B1R and B2R receptors were also performed. Results: After cisplatin protocol, 100% of the females in the control group maintained estral cyclicity versus 44.4% of females in cisplatin group. The control group had a higher number of antral follicles (p=0.011) and total viable follicles (p=0.006). Cisplatin group had a higher number of atretic follicles (p=0.014). Ki67 marker demonstrated similar rates of cell proliferation between groups. Inflammatory markers were increased in cisplatin group, including plasma kallikrein generation (p=0.003), a decrease of aPTT (p=0.02), increased interstitial (p=0.002) and glandular (p=0.008) kallikrein, B1R (p=0.001) and B2R (p=0.001) expression, MPO (p=0.03) and NAG (p=0.04) Oxidative stress markers were also increased in cisplatin group, with higher NO production (p=0.01) and a decrease in SOD (p=0.003) and GSH (p=0.01). Conclusion: All inflammatory reactions appear to be activated by cisplatin treatment, exemplified by increased plasma, interstitial and glandular kallikrein activity, as well as the decrease in aPTT and increased expression of B1R and B2R. The toxicity mediated by cisplatin inflammatory reaction is well known in its side effects, such as ototoxicity and nephrotoxicity. There was an increase in NO production in the ovaries of treated animals, associated with the indication of lower concentrations of SOD and GSH. Imbalances in antioxidants appear to contribute to ovarian oxidative stress. Regarding MPO (neutrophils) and NAG (macrophages), the greater activity of both in cisplatin group is explained by the fact that activated phagocytic cells produce large amounts of reactive oxygen species, further increasing oxidative stress and inflammation. Increased activity of the kallikrein-kinin system and markers of oxidative stress in ovarian tissue provided a better understanding of cisplatin-induced infertility and indicate possible alternatives for ovarian protection during chemotherapy, such as inhibitors of the kallikrein-kinin system and antioxidants.

Made available in DSpace on 2016-12-06T17:06:59Z (GMT). No. of bitstreams: 1 Alexandra Amin Lineburger.pdf: 1015080 bytes, checksum: 1d7fd8b623689b7bd6fd0e3efda64391 (MD5) Previous issue date: 2014-05-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Heart failure (HF) is a disease with systemic involvement, because in addition to cardiac involvement it causes cardiorenal, hemodynamic and neurohormonal dysfunctions contributing to aggravate the catabolism and exercise intolerance and impacting on quality of life. In this context, the high prevalence of hypotestosteronemia is associated with increased cardiovascular events and mortality in men with HF. Regular physical exercise, especially high intensity, and testosterone supplementation are therapeutic options in the management of the disease due to the reduction of risk factors such as dyslipidemia and low-grade inflammation. The aim of this study was to analyze the effects of high intensity exercise and testosterone supplementation therapy (TST) on lipid and inflammatory profile of men with HF. The study is characterized as a randomized controlled trial, with 17 men participants of cardiopulmonary rehabilitation program, HF functional class II and III (NYHA) with ejection fraction <45% and testosterone levels <400ng/dl, with 8 patients undergoing high intensity exercise (EAI) and 9 patients who received hormonal supplementation with testosterone undecanoate (Nebido 1000mg) and performed high-intensity exercise (EAIT group). The physical exercise protocol was carried out for 12 weeks and testosterone supplementation was applied in the first and sixth week. Lipid profile (LDL-c, HDL-c, triglycerides and total cholesterol) and plasma levels of inflammatory mediators (TNF-&#945; and CRP) were analyzed before and after the intervention. The results showed significant increase of 26.7% from the level of HDL-C (p = 0.04) in the EAI group. In comparing the two groups, there were statistically significant differences for the variations in the levels of triglycerides (p = 0.03). Both the EAI group as EAIT decreased plasma levels of TNF-&#945;, respectively 47.4% (p = 0.02) and 43.7% (p = 0.04) with no statistically significant difference between groups. Based on these results it is suggested that high intensity exercise positively influence lipid and inflammatory profile without additional effects associated with supplementation of testosterone therapy. / A insuficiência cardíaca (IC) é uma doença de acometimento sistêmico, pois além do comprometimento cardíaco causa disfunções cardiorrenais, hemodinâmicas e neurohormonais que contribuem para agravar o catabolismo e a intolerância ao exercício impactando na qualidade de vida. Neste contexto, a alta prevalência de hipotestosteronemia está associada ao aumento de eventos cardiovasculares e mortalidade em homens com IC. A prática regular de exercícios físicos, especialmente o de alta intensidade, e a suplementação de testosterona são opções terapêuticas no manejo da doença, devido à redução de fatores de risco como a dislipidemia e a inflamação de baixo grau. O objetivo do presente estudo foi analisar os efeitos do exercício de alta intensidade e terapia de suplementação de testosterona (TST) no perfil lipídico e inflamatório de homens com IC. O estudo é caracterizado como ensaio clínico controlado randomizado, com 17 homens participantes de um programa de reabilitação cardiopulmonar, portadores de IC classe funcional II e III (NYHA), com frequência de ejeção < 45 % e níveis de testosterona < 400 ng/dl, sendo 8 pacientes submetidos a exercício de alta intensidade (EAI) e 9 pacientes que receberam suplementação hormonal com undecanoato de testosterona (Nebido 1000 mg) e realizaram exercício de alta intensidade (grupo EAIT). O protocolo de exercícios físicos foi realizado por 12 semanas e a suplementação de testosterona foi aplicada na primeira e na sexta semana. Foi analisado o perfil lipídico (LDL-c, HDL-c, triglicerídeos e colesterol total) e os níveis plasmáticos de mediadores inflamatórios (TNF-&#945; e PCR) antes e após a intervenção. Os resultados mostraram aumento significativo de 26,7% do nível de HDL-c (p=0,04) no grupo EAI. Na comparação entre os dois grupos, houve diferença estatística para as variações dos níveis de triglicerídeos (p = 0,03). Tanto o grupo EAI quanto o EAIT apresentaram redução nos níveis plasmáticos de TNF- &#945;, respectivamente de 47,7% (p=0,02) e 43,7% (p=0,04) sem diferença estatística entre os grupos. Com base nestes resultados sugere-se que o exercício físico de alta intensidade influenciou positivamente o perfil lipídico e inflamatório, sem efeitos adicionais quando associado à terapia de suplementação de testosterona.