名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 原真由氏の博士論文として提出された

<p>Arbetet beskriver en analys av innehållet av membranbundna och periplasmiska c-cytokromer hos perkloratodlade GR-1 och jämförelser med c-cytokrominnehållet hos Ideonella dechloratans och andra kända c-cytokromer, samt med genomet för Dechloromonas aromatica. Den jämförande studien av c-cytokromer gjordes med syftet att undersöka en hypotes om att bakterierna använder olika vägar för elektronöverföring till det periplasmiska enzymet (per)kloratreduktas. Cellmembran från GR-1 renframställdes genom ultracentrifugering och periplasma preparerades fram med hjälp av osmotisk chock. Fraktionerna analyserades sedan med SDS-PAGE och peptider med kovalent bundet hem (c-cytokromer) detekterades med hjälp av en specifik färgreaktion. Även Touchdown PCR med degenererade primrar genomfördes på isolerat DNA från GR-1 i ett försök att finna en gen kodande för ett NapC/NirT-liknande protein. Slutligen kolmonoxidbubblades reducerade membran för att undersöka förekomsten av cbb3-typ oxidas.</p><p>Separation och infärgning av periplasmiska och membranbundna proteiner från GR-1 resulterade i sju respektive åtta peptidband med molekylvikter mellan 8-60 kDa. Inget som framkommit under arbetet talar emot hypotesen om att GR-1 och D. aromatica skulle använda ett NapC/NirT-liknande protein som elektronöverförare till det periplasmiska (per)kloratreduktaset. PCR-analysen resulterade i en produkt som troligtvis är en sekvens från en gen som kodar för ett NapC/NirT-liknande protein och två, eventuellt tre, kandidater för ett NapC/NirT-liknande protein hittades i membranet hos GR-1. Dessutom framkom att GR-1 troligtvis använder cbb3-typ oxidas som terminalt oxidas vid reduktion av syrgas under mikroaerofila förhållanden.</p><p>Vad gäller I. dechloratans och hypotesen om att denna bakterie använder ett lösligt cytokrom c för elektronöverföring till sitt kloratreduktas så har inget framkommit under arbetet som talar emot detta. Tre kandidater för lösliga cytokrom c-proteiner hittades. För teorin talar även att de försök som tidigare gjorts med att påvisa genen för ett NapC/NirT-liknande protein hos denna bakterie gett negativt resultat.</p> / <p>This work describes an analysis of membrane-anchored and periplasmic c-type cytochromes of perchlorate grown GR-1, and a comparison with the c-type cytochrome content of Ideonella dechloratans, other known c-type cytochromes and the genome of Dechloromonas aromatica. The aim of the comparison was to investigate a hypothesis that the bacteria use different routes for electron transfer to the periplasmic enzyme (per)chlorate reductase. Cell membrane from GR-1 was prepared through ultracentrifugation and periplasm was prepared through osmotic chock. The fractions were separated by SDS-PAGE and peptides containing covalently bound heme (c-type cytochromes) were detected by a specific staining reaction. In an attempt to probe a gene coding for a NapC/NirT-like protein Touchdown PCR was performed on isolated DNA from GR-1, using degenerate primers. Finally, reduced membranes were treated with carbon monoxide to investigate the presence of cbb3-type oxidase.</p><p>Separation and detection resulted in seven periplasmic peptides and eight membrane anchored peptides, all with molecular weights in a range of 8-60 kDa. Nothing has been revealed during this work that opposes the hypothesis of GR-1 and D. aromatica using a NapC/NirT-like protein as an electron carrier to their periplasmic (per)chlorate reductase. The PCR resulted in a product that most likely is a sequence from a gene coding for a NapC/NirT-like protein and two, maybe three, candidates for a NapC/NirT-like protein were also found in the membrane of GR-1. Analysis also revealed that GR-1 most likely makes use of a cbb3-type oxidase for reduction of oxygen during microaerofilic conditions.</p><p>Concerning I. dechloratans, nothing has been revealed during this work that opposes the hypothesis of this bacterium using a soluble cytochrome c as an electron carrier to its chlorate reductase. Three candidates for a soluble cytochrome c protein were found. The theory is also supported by the negative result from earlier attempts to probe a gene coding for a NapC/NirT-like protein in this bacterium.</p>

Aspergilose Pulmonar Invasiva é uma doença fúngica oportunista, causada principalmente por Aspergillus fumigatus, que acomete pacientes imunodeprimidos. Para melhor compreensão dessa micose inicialmente estabelecemos em camundongos C57BL/6 um modelo experimental de depleção de neutrófilos por inoculação intraperitoneal de anticorpos anti-GR-1/Ly-6G, confirmado por contagem total e diferencial de leucócitos sanguíneos. A seguir, avaliamos a evolução da infecção pulmonar experimental utilizando duas amostras de A. fumigatus, caracterizadas previamente em fraca (amostra 699) e forte (amostra 1753) produtoras de elastase. Nenhum dos animais imunocompetentes e infectados evoluiu para o óbito, no período de 7 dias de observação. Os animais neutropênicos, infectados por ambas as amostras, apresentaram 100% de mortalidade após 5 dias, com curvas de sobrevivência praticamente sobrepostas, sugerindo que a maior contribuição para a virulência foi a condição imunológica e não a atividade de elastase da amostra fúngica. Para análise do comprometimento pulmonar, os animais foram sacrificados nos tempos 24, 48 e 72 horas pós-infecção. Durante a evolução da infecção experimental foi observada uma redução da carga fúngica nos pulmões dos animais, para ambas as amostras de A. fumigatus, mas não foi observada uma redução da carga fúngica, diferenciada e estatisticamente significativa, entre os grupos de animais neutropênicos e imunocompetentes. O padrão celular do infiltrado inflamatório observado nos pulmões dos animais neutropênicos, infectados por qualquer uma das amostras de A. fumigatus, mostrou predominância de células mononucleares, em infiltrados difusos, indícios de angioinvasão e invasão brônquica com ruptura de fibras elásticas em ambas as estruturas, além de exuberância de filamentação dos conídios para ambas as amostras fúngicas, desde os tempos iniciais da infecção experimental. O processo inflamatório observado nos pulmões dos animais imunocompetentes, infectados por ambas as amostras de fungos, foi constituído nos tempos iniciais por neutrófilos e se tornou exuberante após 72 horas, com predomínio de macrófagos. Foi observada integridade de vasos sanguíneos e discreta ruptura de parede brônquica no parênquima pulmonar. Para estes animais, salienta-se a ausência de transformação dos conídios de A. fumigatus em hifas para a amostra 699, em todos os períodos de observação. A contagem total de leucócitos no lavado broncoalveolar (LBA) foi significativamente maior, 72 horas pós-infecção, para os animais neutropênicos e imunocompetentes, infectados por ambas as amostras do fungo. A contagem diferencial revelou a presença de macrófagos e neutrófilos, com a primeira célula sempre em maior quantidade no LBA dos animais neutropênicos em comparação com os animais imunocompetentes, independentemente do período da infecção e da amostra fúngica infectante. Ao contrário, o número de neutrófilos foi sempre mais relevante nos animais imunocompetentes. Por microscopia eletrônica de transmissão foi observado que a interação do fungo (conídios ou hifas) com as células de defesa do LBA envolveu íntima adesão e fusão entre os componentes de superfície de ambas as células. A presença de hemoglobina no LBA foi oriunda de lesão alvéolo-capilar causada pelo crescimento e invasão provocados pelas amostras fúngicas ou por lesão determinada pela própria reação inflamatória. Concluímos que os neutrófilos são essenciais na defesa contra A. fumigatus, pois na ausência dessa população celular os fungos rapidamente invadem e lesam o parênquima pulmonar. No entanto, deve-se considerar que a simples presença do fungo em animais imunocompetentes induz a migração de neutrófilos para o sítio da infecção, os quais também causam dano tecidual. As amostras de A. fumigatus com perfis distintos de produção de elastase não refletiram em diferenças significantes para a mortalidade ou gênese das lesões pulmonares observadas em camundongos neutropênicos, sugerindo que embora a elastase contribua para a ruptura das fibras elásticas observadas no tecido pulmonar, outros fatores de virulência, como a morfogênese, podem assumir um papel mais relevante para a patogênese da API experimental. / Invasive Pulmonary Aspergillosis (IPA) is an opportunistic fungal disease, caused mainly by Aspergillus fumigatus, that affects immunocompromised patients. To better understand this mycoses, we originally established in C57BL/6 mice an experimental model of neutrophils depletion by intraperitoneal inoculation of antibodies anti GR-1/Ly-6G, confirmed by total and differential leukocyte counts from blood. Next, we evaluated the evolution of experimental pulmonary infection using two strains of A. fumigatus, previously characterized as weak (strain 699) and strong (strain 1753) elastase producers. None of immunocompetent infected mice died with 7 days of observation, while neutropenic mice, infected with both strains, showed 100% mortality after 5 days, with survival curves nearly overlap, suggesting that the major contribution to the virulence was the immune status instead of elastase activity of each fungal strain. For analysis of lung parenchyma, mice were sacrificed 24, 48 and 72 hours post-infection. During the course of experimental infection it was observed a reduction of fungal burden in the lungs, for both strains of A. fumigatus, but this reduction was not statistically significant between the infected groups (neutropenic and immunocompetent). The cellular pattern of the inflammatory infiltrate observed in lungs from neutropenic mice, infected with both strains of A. fumigatus, revealed a predominance of mononuclear cells, a diffuse pattern and clear evidences of angioinvasion, bronchial disruption with break of elastic fibers in both structures, besides exuberance of conidia filamentation for both fungal strains, since the early period of experimental infection. The inflammatory process observed in lungs from immunocompetent mice, infected with both fungal strains, was composed on early times by neutrophils and became exuberant after 72 hours, with predominance of macrophages. It was observed integrity of blood vessels and moderate bronchial wall disruption in lung parenchyma. A relevant observation was the lack of transformation of conidia in hyphae for 699 A. fumigatus strain, in all periods of observation. Total leukocytes count in bronchoalveolar lavage (BAL) was significantly higher at 72 hours post-infection for both groups infected with both strains. The differential count revealed the presence of macrophages and neutrophils, with the former always in greater percentage in BAL from neutropenic mice and the latter always more elevated in immunocompetent group. Analysis by transmission electron microscopy demonstrated that the interaction of fungal structures (conidia or hyphae) with the defense cells (neutrophlis or macrophages) of BAL involved an intimate adhesion and fusion between the surface components from both cells. The presence of hemoglobin in BAL was a result of alveolar injury caused by the fungal development and invasion, but also by injuries determined by the inflammatory process itself. We concluded that neutrophils have a critical role against A. fumigatus since the pathogen quickly invades and damages the lung parenchyma in its absence. However, we must consider that the mere presence of A. fumigatus in immunocompetent mice induces the neutrophils migration to the infection site, which can also cause a tissue injury. Strains of A. fumigatus with distinct patterns of elastase production did not reflect in significant differences in mortality or origin of pulmonary lesions observed in neutropenic mice, suggesting that although elastase contributes to elastic disruptions observed in pulmonary tissue, another virulence factors, such as morphogenesis, can assume a more relevant role for pathogenesis of experimental IPA.

Aspergilose Pulmonar Invasiva é uma doença fúngica oportunista, causada principalmente por Aspergillus fumigatus, que acomete pacientes imunodeprimidos. Para melhor compreensão dessa micose inicialmente estabelecemos em camundongos C57BL/6 um modelo experimental de depleção de neutrófilos por inoculação intraperitoneal de anticorpos anti-GR-1/Ly-6G, confirmado por contagem total e diferencial de leucócitos sanguíneos. A seguir, avaliamos a evolução da infecção pulmonar experimental utilizando duas amostras de A. fumigatus, caracterizadas previamente em fraca (amostra 699) e forte (amostra 1753) produtoras de elastase. Nenhum dos animais imunocompetentes e infectados evoluiu para o óbito, no período de 7 dias de observação. Os animais neutropênicos, infectados por ambas as amostras, apresentaram 100% de mortalidade após 5 dias, com curvas de sobrevivência praticamente sobrepostas, sugerindo que a maior contribuição para a virulência foi a condição imunológica e não a atividade de elastase da amostra fúngica. Para análise do comprometimento pulmonar, os animais foram sacrificados nos tempos 24, 48 e 72 horas pós-infecção. Durante a evolução da infecção experimental foi observada uma redução da carga fúngica nos pulmões dos animais, para ambas as amostras de A. fumigatus, mas não foi observada uma redução da carga fúngica, diferenciada e estatisticamente significativa, entre os grupos de animais neutropênicos e imunocompetentes. O padrão celular do infiltrado inflamatório observado nos pulmões dos animais neutropênicos, infectados por qualquer uma das amostras de A. fumigatus, mostrou predominância de células mononucleares, em infiltrados difusos, indícios de angioinvasão e invasão brônquica com ruptura de fibras elásticas em ambas as estruturas, além de exuberância de filamentação dos conídios para ambas as amostras fúngicas, desde os tempos iniciais da infecção experimental. O processo inflamatório observado nos pulmões dos animais imunocompetentes, infectados por ambas as amostras de fungos, foi constituído nos tempos iniciais por neutrófilos e se tornou exuberante após 72 horas, com predomínio de macrófagos. Foi observada integridade de vasos sanguíneos e discreta ruptura de parede brônquica no parênquima pulmonar. Para estes animais, salienta-se a ausência de transformação dos conídios de A. fumigatus em hifas para a amostra 699, em todos os períodos de observação. A contagem total de leucócitos no lavado broncoalveolar (LBA) foi significativamente maior, 72 horas pós-infecção, para os animais neutropênicos e imunocompetentes, infectados por ambas as amostras do fungo. A contagem diferencial revelou a presença de macrófagos e neutrófilos, com a primeira célula sempre em maior quantidade no LBA dos animais neutropênicos em comparação com os animais imunocompetentes, independentemente do período da infecção e da amostra fúngica infectante. Ao contrário, o número de neutrófilos foi sempre mais relevante nos animais imunocompetentes. Por microscopia eletrônica de transmissão foi observado que a interação do fungo (conídios ou hifas) com as células de defesa do LBA envolveu íntima adesão e fusão entre os componentes de superfície de ambas as células. A presença de hemoglobina no LBA foi oriunda de lesão alvéolo-capilar causada pelo crescimento e invasão provocados pelas amostras fúngicas ou por lesão determinada pela própria reação inflamatória. Concluímos que os neutrófilos são essenciais na defesa contra A. fumigatus, pois na ausência dessa população celular os fungos rapidamente invadem e lesam o parênquima pulmonar. No entanto, deve-se considerar que a simples presença do fungo em animais imunocompetentes induz a migração de neutrófilos para o sítio da infecção, os quais também causam dano tecidual. As amostras de A. fumigatus com perfis distintos de produção de elastase não refletiram em diferenças significantes para a mortalidade ou gênese das lesões pulmonares observadas em camundongos neutropênicos, sugerindo que embora a elastase contribua para a ruptura das fibras elásticas observadas no tecido pulmonar, outros fatores de virulência, como a morfogênese, podem assumir um papel mais relevante para a patogênese da API experimental. / Invasive Pulmonary Aspergillosis (IPA) is an opportunistic fungal disease, caused mainly by Aspergillus fumigatus, that affects immunocompromised patients. To better understand this mycoses, we originally established in C57BL/6 mice an experimental model of neutrophils depletion by intraperitoneal inoculation of antibodies anti GR-1/Ly-6G, confirmed by total and differential leukocyte counts from blood. Next, we evaluated the evolution of experimental pulmonary infection using two strains of A. fumigatus, previously characterized as weak (strain 699) and strong (strain 1753) elastase producers. None of immunocompetent infected mice died with 7 days of observation, while neutropenic mice, infected with both strains, showed 100% mortality after 5 days, with survival curves nearly overlap, suggesting that the major contribution to the virulence was the immune status instead of elastase activity of each fungal strain. For analysis of lung parenchyma, mice were sacrificed 24, 48 and 72 hours post-infection. During the course of experimental infection it was observed a reduction of fungal burden in the lungs, for both strains of A. fumigatus, but this reduction was not statistically significant between the infected groups (neutropenic and immunocompetent). The cellular pattern of the inflammatory infiltrate observed in lungs from neutropenic mice, infected with both strains of A. fumigatus, revealed a predominance of mononuclear cells, a diffuse pattern and clear evidences of angioinvasion, bronchial disruption with break of elastic fibers in both structures, besides exuberance of conidia filamentation for both fungal strains, since the early period of experimental infection. The inflammatory process observed in lungs from immunocompetent mice, infected with both fungal strains, was composed on early times by neutrophils and became exuberant after 72 hours, with predominance of macrophages. It was observed integrity of blood vessels and moderate bronchial wall disruption in lung parenchyma. A relevant observation was the lack of transformation of conidia in hyphae for 699 A. fumigatus strain, in all periods of observation. Total leukocytes count in bronchoalveolar lavage (BAL) was significantly higher at 72 hours post-infection for both groups infected with both strains. The differential count revealed the presence of macrophages and neutrophils, with the former always in greater percentage in BAL from neutropenic mice and the latter always more elevated in immunocompetent group. Analysis by transmission electron microscopy demonstrated that the interaction of fungal structures (conidia or hyphae) with the defense cells (neutrophlis or macrophages) of BAL involved an intimate adhesion and fusion between the surface components from both cells. The presence of hemoglobin in BAL was a result of alveolar injury caused by the fungal development and invasion, but also by injuries determined by the inflammatory process itself. We concluded that neutrophils have a critical role against A. fumigatus since the pathogen quickly invades and damages the lung parenchyma in its absence. However, we must consider that the mere presence of A. fumigatus in immunocompetent mice induces the neutrophils migration to the infection site, which can also cause a tissue injury. Strains of A. fumigatus with distinct patterns of elastase production did not reflect in significant differences in mortality or origin of pulmonary lesions observed in neutropenic mice, suggesting that although elastase contributes to elastic disruptions observed in pulmonary tissue, another virulence factors, such as morphogenesis, can assume a more relevant role for pathogenesis of experimental IPA.

Arbetet beskriver en analys av innehållet av membranbundna och periplasmiska c-cytokromer hos perkloratodlade GR-1 och jämförelser med c-cytokrominnehållet hos Ideonella dechloratans och andra kända c-cytokromer, samt med genomet för Dechloromonas aromatica. Den jämförande studien av c-cytokromer gjordes med syftet att undersöka en hypotes om att bakterierna använder olika vägar för elektronöverföring till det periplasmiska enzymet (per)kloratreduktas. Cellmembran från GR-1 renframställdes genom ultracentrifugering och periplasma preparerades fram med hjälp av osmotisk chock. Fraktionerna analyserades sedan med SDS-PAGE och peptider med kovalent bundet hem (c-cytokromer) detekterades med hjälp av en specifik färgreaktion. Även Touchdown PCR med degenererade primrar genomfördes på isolerat DNA från GR-1 i ett försök att finna en gen kodande för ett NapC/NirT-liknande protein. Slutligen kolmonoxidbubblades reducerade membran för att undersöka förekomsten av cbb3-typ oxidas. Separation och infärgning av periplasmiska och membranbundna proteiner från GR-1 resulterade i sju respektive åtta peptidband med molekylvikter mellan 8-60 kDa. Inget som framkommit under arbetet talar emot hypotesen om att GR-1 och D. aromatica skulle använda ett NapC/NirT-liknande protein som elektronöverförare till det periplasmiska (per)kloratreduktaset. PCR-analysen resulterade i en produkt som troligtvis är en sekvens från en gen som kodar för ett NapC/NirT-liknande protein och två, eventuellt tre, kandidater för ett NapC/NirT-liknande protein hittades i membranet hos GR-1. Dessutom framkom att GR-1 troligtvis använder cbb3-typ oxidas som terminalt oxidas vid reduktion av syrgas under mikroaerofila förhållanden. Vad gäller I. dechloratans och hypotesen om att denna bakterie använder ett lösligt cytokrom c för elektronöverföring till sitt kloratreduktas så har inget framkommit under arbetet som talar emot detta. Tre kandidater för lösliga cytokrom c-proteiner hittades. För teorin talar även att de försök som tidigare gjorts med att påvisa genen för ett NapC/NirT-liknande protein hos denna bakterie gett negativt resultat. / This work describes an analysis of membrane-anchored and periplasmic c-type cytochromes of perchlorate grown GR-1, and a comparison with the c-type cytochrome content of Ideonella dechloratans, other known c-type cytochromes and the genome of Dechloromonas aromatica. The aim of the comparison was to investigate a hypothesis that the bacteria use different routes for electron transfer to the periplasmic enzyme (per)chlorate reductase. Cell membrane from GR-1 was prepared through ultracentrifugation and periplasm was prepared through osmotic chock. The fractions were separated by SDS-PAGE and peptides containing covalently bound heme (c-type cytochromes) were detected by a specific staining reaction. In an attempt to probe a gene coding for a NapC/NirT-like protein Touchdown PCR was performed on isolated DNA from GR-1, using degenerate primers. Finally, reduced membranes were treated with carbon monoxide to investigate the presence of cbb3-type oxidase. Separation and detection resulted in seven periplasmic peptides and eight membrane anchored peptides, all with molecular weights in a range of 8-60 kDa. Nothing has been revealed during this work that opposes the hypothesis of GR-1 and D. aromatica using a NapC/NirT-like protein as an electron carrier to their periplasmic (per)chlorate reductase. The PCR resulted in a product that most likely is a sequence from a gene coding for a NapC/NirT-like protein and two, maybe three, candidates for a NapC/NirT-like protein were also found in the membrane of GR-1. Analysis also revealed that GR-1 most likely makes use of a cbb3-type oxidase for reduction of oxygen during microaerofilic conditions. Concerning I. dechloratans, nothing has been revealed during this work that opposes the hypothesis of this bacterium using a soluble cytochrome c as an electron carrier to its chlorate reductase. Three candidates for a soluble cytochrome c protein were found. The theory is also supported by the negative result from earlier attempts to probe a gene coding for a NapC/NirT-like protein in this bacterium.

Preterm birth remains a major challenge in obstetrics. It complicates up to 13% of all pregnancies and accounts for approximately 80% of neonatal mortality and morbidity. Bacterial Vaginosis (BV) is associated with a 1.4-fold increased risk of preterm birth. Due to ineffectiveness of antibiotics in preventing preterm labour, probiotics have been proposed to serve as an alternative for treatment of BV and prevention of preterm birth. The objectives of this thesis were to determine 1) the effect of Lactobacillus rhamnosus GR-1 (L. rhamnosus GR-1) supernatant on cytokine profile and prostaglandin (PG)-regulating enzyme expression in lipopolysaccharide (LPS)-stimulated human chorion and placental trophoblast cells from human placentae, 2) the potential signaling pathways through which lactobacilli act and 3) the potential role of immune and placental trophoblast cells in initiating a response to LPS and L. rhamnosus GR-1 treatments. Primary cultures of human placental trophoblast cells were pre-treated with lactobacilli supernatant and then with LPS. In addition, immune cells were removed from cell suspensions using a magnetic purification technique to determine their role in modulating cytokine levels. The expression of pro- and anti-inflammatory cytokines and prostaglandin-regulating enzymes was then determined. We found sex-specific differences in the ability of LPS to increase the output of TNF-α, IL-10, and PTGS2. We also showed that L. rhamnosus GR-1 is able to act through the JAK/STAT and MAPK pathways to increase IL-10 and G-CSF, and independently down-regulates PTGS2 and TNF-α and up-regulates PGDH. The increase in G-CSF and PGDH were only observed in women carrying a female fetus. L. rhamnosus GR-1 may serve as an alternative to antibiotics in preventing some infection/inflammation-mediated cases of preterm birth.

Preterm birth remains a major challenge in obstetrics. It complicates up to 13% of all pregnancies and accounts for approximately 80% of neonatal mortality and morbidity. Bacterial Vaginosis (BV) is associated with a 1.4-fold increased risk of preterm birth. Due to ineffectiveness of antibiotics in preventing preterm labour, probiotics have been proposed to serve as an alternative for treatment of BV and prevention of preterm birth. The objectives of this thesis were to determine 1) the effect of Lactobacillus rhamnosus GR-1 (L. rhamnosus GR-1) supernatant on cytokine profile and prostaglandin (PG)-regulating enzyme expression in lipopolysaccharide (LPS)-stimulated human chorion and placental trophoblast cells from human placentae, 2) the potential signaling pathways through which lactobacilli act and 3) the potential role of immune and placental trophoblast cells in initiating a response to LPS and L. rhamnosus GR-1 treatments. Primary cultures of human placental trophoblast cells were pre-treated with lactobacilli supernatant and then with LPS. In addition, immune cells were removed from cell suspensions using a magnetic purification technique to determine their role in modulating cytokine levels. The expression of pro- and anti-inflammatory cytokines and prostaglandin-regulating enzymes was then determined. We found sex-specific differences in the ability of LPS to increase the output of TNF-α, IL-10, and PTGS2. We also showed that L. rhamnosus GR-1 is able to act through the JAK/STAT and MAPK pathways to increase IL-10 and G-CSF, and independently down-regulates PTGS2 and TNF-α and up-regulates PGDH. The increase in G-CSF and PGDH were only observed in women carrying a female fetus. L. rhamnosus GR-1 may serve as an alternative to antibiotics in preventing some infection/inflammation-mediated cases of preterm birth.

Investigation of tumor growth has traditionally been studied focusing only on the cancer cells. However, tumors consist of a complex tissue organization where heterotypic signaling occurs between different cell types. The cross-talk between tumor cells and other surrounding cell types may ultimately prove to be as important for the tumor cell behavior as the internal signaling cascades in the tumor cell itself.Myeloid cells, such as granulocytes and monocytes, and thrombocytes play an important role in the tumor tissue, as a tumor can be compared to a wound healing process without the normal regulation mechanisms. Platelets are thought to facilitate tumor cell extravasation by binding to the tumor cell and recruiting myeloid cells that secrete factors aiding tumor migration through the endothelial cells. Studying the content of granules and vesicles of the platelets and myeloid cells can provide important knowledge about how the tumor interactions are mediated and which key proteins that controls these processes.Serglycin is an intracellular proteoglycan that attaches chains of negatively charged glycosaminoglycans. It is thought to have a function in retaining and storing proteins in hematipoietic cells. In this project the impact of the loss of serglycin on platelets and myeloid cells was investigated, using a spontaneous insulinoma serglycin knockout mouse model. The results suggests that serglycin does not affect the amount of neutrophil granulocytes and monocytes in peripheral blood, nor does it seem to affect the amount of platelets sequestered to the tumor tissue. A co-staining for platelets and MMP9 positive granulocytes was also performed in order to assess if granulocyte-platelet interactions in the tumor were affected by loss of serglycin. Interactions between these cells were observed in both genotypes. Von Willebrand factor levels in the tumor tissue also remained unchanged upon loss of serglycin. However, preliminary experiments indicated that serglycin seems to play a role in the intracellular amounts of vimentin and VEGFB in undifferentiated primary bone marrow derived monocytes.