Typescript (photocopy). / Digitized by Kansas Correctional Industries

E. coli O157:H7 is the most important serotype of enterohaemorrhagic E. coli (EHEC) which is of concern to public health worldwide. As a common cause of haemorrhagic colitis, EHEC infection can progress to life threatening sequelae including haemolytic uraemic syndrome (HUS) in humans. Human infection rates are higher in Scotland than found in the rest of the UK. Cattle are asymptomatic carriers of EHEC and are an important reservoir from which disease outbreaks can spread. The terminal rectum has been indicated as a site of E. coli O157:H7 colonisation in the bovine intestinal tract. This is the location of numerous lymphoid follicles which contain dendritic cells (DCs) which are professional antigen presenting cells and important directors of immune responses. DCs are likely to come into contact with EHEC and therefore could be key in this location for enabling EHEC to colonise the bovine host. The first aim of this project was to characterise dendritic cells within the bovine intestinal tract at various anatomical locations, including the terminal rectum, using immunohistochemistry techniques. Following this, work to extract and further phenotype dendritic cells from terminal rectal tissues was undertaken. Finally, a widely-used bovine dendritic cell model was employed to generate dendritic cells from circulating blood monocytes. This model was utilised to investigate the interactions of dendritic cells with EHEC strains compared with responses to bovine enterotoxigenic (ETEC) and bovine commensal E. coli strains. Early work identified that there are potentially numerous DCs within the bovine intestinal tissues and these cells were found in greater numbers at the terminal rectum. Protocols to extract and further characterise these cells were developed but proved inconsistent, with large variation between animals. Using the monocyte derived dendritic cells (moDCs), differences were observed between immunological responses to challenge with E. coli O157:H7 strains and bovine pathogenic or commensal E. coli strains. Cytokine production, cell surface molecule expression, cell phenotype and viability as well as intracellular bacterial counts were compared. The data presented here shows that the bovine moDCs respond differently to EHEC strains when compared with commensal or pathogenic E. coli in several key areas. This has important implications for the responses of the bovine host to various E. coli strains. This work also indicates that dendritic cells could be central to these responses and if studied further still, may hold the key to reducing the colonization and persistence of E. coli O157:H7 in cattle, and subsequent human disease outbreaks.

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

The aetiology of IBD is unknown, but available evidence suggests that an aberrant immune response towards the commensal microbial flora is responsible for intestinal inflammation in genetically susceptible individuals. Studies from animal models of intestinal inflammation have greatly advanced our understanding of the immunological basis of IBD. However, translation of results from animal research into human studies is essential in order to improve treatment options and patient quality of life. In this thesis we present the successful introduction of translational studies on human tissue in our laboratory. In particular, we evaluated the role of the IL23/IL17 pathway in the human immune response and its role in IBD. IL23-driven inflammation has been primarily linked to its activity on Th-17 cells; however, work from our laboratory has identified a novel population of IL23-responsive ILC, which are responsible for innate colitis in mice. Here we have analyzed the role of IL23-responsive innate cells in IBD. Our results show increased expression of Th-17 signature genes amongst intestinal CD3- cells in patients with IBD. Furthermore, we observed a marked and selective increase in IL17 producing CD56- ILC in the inflamed intestine of patients with CD. ILC may contribute to intestinal inflammation through secretion of cytokines, such as IL17A and IL17F, and recruitment of other inflammatory cells, representing a novel tissue-specific target for the treatment of IBD. In addition, we present here our preliminary data on the characterization of human intestinal and systemic DC populations. In particular, we aimed to evaluate if in the context of the intestinal microenvironment DC develop specific regulatory features, as observed in murine CD103+ DC. We show that human intestinal DC populations exhibit specific regulatory properties, such as expression of genes associated with TGF-β and RA activity. Furthermore, CD103+ DC are present in the human gut and are characterized by tolerogenic markers. Remarkably, patients with IBD have reduced frequencies of intestinal CD103+ DC, which display a more pro-inflammatory phenotype. Alteration in DC subset composition and functional activity may result in a distort balance between immune effector and regulatory responses, promoting the development of intestinal inflammation.

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of secreted proteins, which include in addition to BDNF, nerve growth factor (NGF) and neurotrophin 3-6 (NT-3-6). BDNF mediates its functions by activating two cell surface receptors, pan-neurotrophin receptor (P75NTR) and tropomyosin-related kinase B (TrkB) and their downstream intracellular cascades. BDNF is best known for its role in neuronal survival, regulation of neuronal differentiation, migration and activity-dependent synaptic plasticity. However, BDNF is widely expressed in non-neuronal tissues as well. The localization and the function of BDNF in intestinal smooth muscle cells (SMCs) are not well defined. Thus, the main purpose of the present study was the identification and characterization of BDNF in intestinal SMCs. Using xviii biochemical and molecular techniques, we have demonstrated in this study that BDNF is synthesized and released in rabbit intestinal longitudinal SMCs cultures. Furthermore, gut neuropeptides, Pituitary Adenylate Cyclase Activating Peptide (PACAP) and substance P (SP) increased BDNF expression and release in SMCs cultures after 24 hrs and 48 hrs incubation. We have also shown that intracellular Ca2+ levels are essential for SP stimulation of BDNF expression and secretion. Lastly, we have demonstrated that exogenous BDNF enhanced carbachol (CCh)-induced contraction of isolated longitudinal muscle strips, and this was inhibited by preincubation with TrkB inhibitor K252a and PLC inhibitor U73122 sugesting that BDNF sensitize longitudinal SMCs to CCh by activating PLC pathway, which is normally absent in those muscle cells. These results provide new insight into the mechanisms of neurotrophin (BDNF) modulation of gut function, which may lead to new therapeutic avenues for treatment of gastrointestinal disorders, and explain some of the pathological changes associated with inflammation such as hypercontractility associated with gut infection or IBD.

Thesis advisor: Kenneth C. Williams / Elucidating the mechanisms through which viral infection and persistence in CNS occurs is critical to understanding the development and progression of neurological disease. To date, no study has demonstrated that monocyte traffic in HIV and SIV infection directly results in neuronal injury. The central hypothesis in this thesis is that continuous trafficking of monocytes into tissues is essential for pathogenesis with viral infection. In the dissertation work presented here, two studies addressed this hypothesis. In Chapter 2, experiments examining the role of peripheral monocyte activation in the development of neuroAIDS using the tetracycline antibiotic minocycline will be described. We hypothesized that decreased monocyte activation with minocycline treatment would play a neuroprotective role in the context of rapid SIV infection with a high incidence of SIV encephalitis (SIVE). We observed a reversal of neuronal injury within days of minocycline treatment that correlated with loss of monocyte activation. From these findings we concluded that decreased activation of monocytes results in lower CNS traffic. However this effect may have occurred due to lower plasma virus, decreased SIV infection of monocytes, or the ability of minocycline to cross the BBB and modulate changes within the CNS directly. In Chapter 3 of this thesis, we hypothesized that continuous traffic of activated monocytes from the periphery into the CNS is required for neuronal injury with AIDS, and that by effectively stopping monocyte accumulation, CNS pathology can be blocked or reversed. We also hypothesized that monocyte trafficking is necessary for the seeding of brain and small intestine with cell-associated virus. In order to test these hypotheses, we utilized the anti-α4 blocking antibody natalizumab (Tysabri; Biogen Idec), which selectively binds to the α4 subunit of α4β1 (VLA-4) and α4β7 integrins, preventing the interaction between α4 and its various ligands. To address the first hypothesis, natalizumab was administered after four weeks of infection once significant neuronal damage had already occurred. We found that preventing cell traffic with natalizumab is sufficient to stabilize neuronal injury and loss, demonstrating conclusively that stopping monocyte traffic stabilizes CNS disease. To address the second hypothesis, rhesus macaques were treated with natalizumab on the day of SIV infection. Natalizumab treatment completely blocked SIV infection in the brain, and virus traffic to the small intestine was significantly suppressed. Overall, these studies demonstrate that continuous traffic of monocytes is required for neuronal injury and the formation of CNS lesions, and that early trafficking of leukocytes is critical for seeding of the CNS and contributes to seeding of the small intestine with virus. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Styelid ascidians are diverse in developmental modes, varying from strictly sexual solitary species to highly integrated colonies. Circulatory stem cells (CSCs) accomplish fundamental roles in developmental processes of styelid ascidians. In the colonial styelids, CSCs enable budding and are capable of giving origin to the germline in certain species. The function of these cells have been tested experimentally in models within Styelidae. However, the understanding of coloniality as an evolutionary novelty requires reconstructing the possible ancestral CSCs characteristics in Styelidae. To address this issue, this work analyzes the possible developmental origin and the identity of putative CSCs among blood cell populations. The first chapter of this dissertation aimed to characterize and compare the hemocyte populations in two solitary styelids: Styela plicata and Styela canopus. In addition, the early development, the metamorphosis and the early maturation were compared in both species. After metamorphosis, S. canopus briefly develops a network of extracorporeal vessels with numerous terminal ampullae. These characters are usually associated to colonial ascidians, and were not found in S. plicata. With respect to the hemocyte populations, similar morphotypes were present in both species. However, S. canopus shows a lower frequency of vacuolated cells, which may be due to a reduced level of cytotoxicity in the tunic relative to S. plicata. These differences observed between S. canopus and S. plicata may be related to differences in the degrees of gregariousness or body size among the two species. In order to investigate possible approaches to distinguish and isolate CSC populations in a solitary styelid model, I used imaging flow cytometry. Putative CSCs were identified through measurement of morphological parameters and aldehyde dehydrogenase (ALDH) activity. The correlation between these parameters allowed to determine 2 gates enriched with particular cell types. A significant difference was found on the ALDH+ population within a gate of cells with low granularity, suggesting the presence of cells among circulatory hemocytes. To scrutinize the biogenesis of CSCs in S. plicata, I present a description of a candidate hematopoietic niche in this species. An exhaustive histological survey for hemoblast-like cells was performed, and complemented with immunohistochemistry with stem cell (piwi) and proliferation (pHH3) markers. The morphological and expression profiles of the intestine support the intestinal submucosa (IS) as a hematopoietic niche. At this region there are aggregations of cells with and undifferentiated morphological profile, corroborated by ultrastructural analysis. Furthermore, the IS holds high cellular proliferation and frequency of piwi+ cells. Ascidians are considered interesting models to investigate asexual reproduction and modular development. This study represents an advancement towards understanding the processes, cell populations and structures that may be related to facilitating the appearance of this evolutionary novelty / As ascídias da família Styelidae são diversas em modos de desenvolvimento, variando de espécies estritamente sexuais solitárias até colônias altamente integradas. As células-tronco circulatórias (CTCs) desempenham papéis fundamentais nos processos do desenvolvimento de ascídias styelídeas. Nas especies coloniais deste grupo, as CTCs permitem a brotação e são capazes de originar a linha germinativa em certas espécies. A função dessas células tem sido testada experimentalmente em modelos dentro de Styelidae. No entanto, a compreensão da colonialidade como uma novidade evolutiva requer reconstruir as características das possíveis CTCs ancestrais para Styelidae. Com o fim de abordar essa questão, este trabalho analisa a possível origem do desenvolvimento e a identidade de CSCs putativas entre populações de células sanguíneas de styelídeas solitárias. O primeiro capítulo desta dissertação teve como objetivo caracterizar e comparar as populações de hemócitos em dois espécies solitárias: Styela plicata e Styela canopos. Além disso, o desenvolvimento inicial, a metamorfose e a maturação do juvenil foram comparados em ambas as espécies. Após a metamorfose, S. canopus desenvolve brevemente uma rede de vasos extracorpóreos com numerosas ampolas terminais. Esses caracteres são geralmente associados a ascídias coloniais e não foram encontrados em S. plicata. Com relação às populações de hemócitos, morfotipos semelhantes estavam presentes em ambas as espécies. No entanto, o S. canopos apresenta menor frequência de células vacuoladas, o que pode ser devido a um nível reduzido de citotoxicidade na túnica em relação a S. plicata. Essas diferenças observadas entre S. canopos e S. plicata podem estar relacionadas a diferenças nos graus de gregariedade ou tamanho corporal entre as duas espécies. A fim de investigar possíveis abordagens para distinguir e isolar populações de CTCs em um modelo de styelídeo solitário, usei citometria de fluxo com adquisição de imagem. As CTCs putativas foram identificadas através da medição de parâmetros morfológicos e da atividade da aldeído desidrogenase (ALDH). A correlação entre estes parâmetros permitiu determinar 2 gates enriquecidos com tipos celuláres particulares. Uma diferença significativa foi encontrada na população ALDH+ dentro de um gate de células com baixa granularidade, sugerindo a presença de células-tronco circulatórias. Para examinar a biogênese das CTCs em S. plicata, foi realizada uma descrição de um nicho hematopoiético candidato nesta espécie. Um exame histológico exaustivo para células semelhantes a hemoblastos foi realizado e complementado com imunohistoquímica com marcadores de células-tronco (piwi) e proliferação (pHH3). Os perfis morfológicos e de expressão do intestino sustentam a submucosa intestinal (SI) como nicho hematopoiético. Nesta região há agregações de células com morfolia indiferenciada, corroborada pela análise ultraestrutural. Além disso, a SI mantém alta proliferação celular e freqüência de células piwi+. As ascídias são consideradas modelos interessantes para investigar a reprodução assexuada e o desenvolvimento modular. Este estudo representa um avanço na compreensão dos processos, populações celulares e estruturas que podem estar relacionadas a facilitar o surgimento desta novidade evolutiva

As doenças do trato digestório nos equinos apresentam altas taxas de morbidade e mortalidade, com diferentes etiologias. Em alguns casos, o emprego da biopsia intestinal se faz necessário para auxílio no diagnóstico dessas enfermidades. No entanto, as técnicas convencionais podem trazer riscos aos pacientes, por serem invasivas, ou não serem elucidativas por apresentarem limitações de acesso a determinados segmentos. O presente estudo teve como objetivo validar uma técnica de biopsia intestinal, intracorpórea, assistida por videolaparoscopia, ainda não descrita na literatura, para coleta de fragmentos de mucosa de jejuno e cólon menor de equinos, que sejam considerados adequados para avaliação histológica. Para tanto, foram utilizados seis equinos machos, da raça Puro Sangue Árabe, com idade de dois anos, sem histórico prévio de doenças do trato digestório, com peso médio de 267 kg. Todos os animais foram submetidos ao mesmo procedimento laparoscópico, instituindo-se apenas jejum alimentar prévio de oito horas. Os equinos foram acompanhados com exame físico e de ultrassonografia abdominal, desde o dia precedente às laparoscopias, até o 15º dia do período pós-operatório, bem como avaliados por meio de hemograma, provas de funções hepática e renal, e análise do líquido peritoneal nos dias 0, 1, 2, 3, 5, 7, 10, 14, 21 e 30. O tempo cirúrgico foi cronometrado, sendo registrado o tempo total, iniciado na criação do primeiro portal de acesso e finalizado ao término da sutura de pele, e os tempos parciais para biopsia de jejuno e cólon menor separadamente, com início na apreensão do segmento intestinal e término quando constatada a polimerização da cola cirúrgica sobre o orifício de acesso da agulha. De cada segmento obtiveram-se dez fragmentos, e posteriormente submetidos à análise histológica. Atribuiu-se escore para cada um deles, sendo considerado 0 fragmentos com qualidade ruim; 1 para qualidade boa e 2 para qualidade ótima. Por sua vez, os considerados viáveis foram somente os que se enquadraram nos escores 1 e 2. Amostras avaliadas como adequadas 11 apresentaram no mínimo 50% dos fragmentos viáveis. A média do tempo total de procedimento foi de 66,50 minutos (± 7,87), enquanto a média do tempo parcial para biopsia de jejuno foi de 14,2 minutos (± 4,3) e a de cólon menor 12,7 minutos (± 5,0). Clinicamente, os animais apresentaram desconforto abdominal nas primeiras 48 horas. Os exames ultrassonográficos do abdômen não revelaram alterações condizentes com peritonite ao longo de todo experimento. Os parâmetros laboratoriais apresentaram apenas características inflamatórias, sendo que o líquido peritoneal permaneceu alterado até o 21º de pós-operatório, havendo normalização de todos os seus valores no 30º dia do estudo. Na inspeção laparoscópica de dois equinos (E2, E4) foi identificada aderência de porção de omento no diafragma. Nas avaliações histológicas de jejuno, uma amostra (E5) de seis foi considerada inadequada, com 5/12 fragmentos viáveis, e em cólon menor, duas (E1, E2) de seis, foram inadequadas, com 4/9 e 5/10 fragmentos viáveis respectivamente. A nova técnica de biopsia intestinal possibilitou a coleta de amostras adequadas de mucosa para análise histológica, de forma segura para os animais, uma vez que as alterações clínicas e laboratoriais foram aquelas relacionadas ao processo inflamatório, compatível com procedimentos laparoscópicos na espécie / Gastrointestinal diseases in horses result in high rates of morbidity and mortality, with different aetiologies. In some cases, an intestinal biopsy is needed to aid in the diagnosis of such diseases. However, the conventional techniques can pose risks to patients for being invasive or for not being elucidating due to having limitations in accessing certain segments. The objective of this study was to validate an intestinal biopsy technique, intracorporeal, assisted by laparoscopy, which has not yet been described in the literature, to collect mucosal fragments from the jejuno and small colon, which might be considered suitable for histological assessment. For such, six male horses were used, Arabian breed, with two years of age, without any records of abdominal diseases, weighing 267 kg in average. All horses were subjected to the same laparoscopic procedure, fasting for eight hours previously to the procedure. All horses were monitored through physical examination and abdominal ultrasonography, from the day previous to laparoscopy, until the 15th postoperative day, as well as hemogram, tests of liver and kidney functions, and analysis of the peritoneal fluid in days 0, 1, 2, 3, 5, 7, 10, 14, 21 and 30. The total laparoscopic procedure time was registered, starting at the moment of the first incision and ending at the moment of the skin closure. The partial times for the jejunal biopsy and small colon biopsy were recorded as well, starting at the grasping of the intestinal segment and ending at the moment of polymerization of the surgical adhesive on the needle access site. From each segment, ten fragments were collected and later subjected to histological analysis. A score was assigned for each one of them, being scored \"0\" fragments of poor quality; \"1\" fragments of good quality and \"2\" fragments of optimal quality. The samples considered viable were only the ones which scored 1 and 2. The samples deemed as adequate showed at least 50% of it fragments to be viable. The average of the surgery total time was of 66,50 minutes (± 7.87), whereas the average of the jejunal biopsy was of 14.2 minutes (± 4.3) and the small colon biopsy time was of 12.7 minutes ( ± 5.0). Clinically, the animals showed mild abdominal 13 discomfort in the first 48 hours. Ultrasonographic examination of the abdomen did not reveal any alterations consistent with peritonitis throughout the entire experiment period. Laboratory parameters presented inflammatory characteristics, and the peritoneal fluid remained altered until the 21th postoperative day, with normalization of all its values on the 30th day of the study. During the laparoscopic inspection of two horses (E2, E4) was identified partial omental adhesion with the diaphragm. In the jejunal histological evaluations, one sample (E5) of six was considered inadequate, with 5/12 viable fragments, and as for the small colon, two (E1, E2) of six were inadequate, with 4/9 and 5/10 viable fragments respectively. The new technique proposed allowed a safe collection of adequate mucosal samples for histological analysis, since clinical and laboratory abnormalities identified were related to the inflammatory process associated to the laparoscopic techniques in horses

Os diversos estudos com a suplementação de glutamina em situações de estresse fisiológico demonstram o essencial papel deste aminoácido no metabolismo. Agudamente, a suplementação com glutamina aumenta a glutaminemia. Cronicamente, verifica-se maior concentração de glutamina em tecidos, tais como o muscular e o hepático. Entretanto, não é conhecido se esses efeitos são decorrentes diretos da suplementação oral com glutamina ou da redução de sua captação a partir da membrana basolateral de enterócitos. O estudo da cinética na absorção de glutamina traz informações relacionadas à proporção da concentração de glutamina absorvida e a retida no tecido intestinal, de acordo com as doses utilizadas, e aponta quais são as alterações decorrentes da sepse induzida pela endotoxemia. O objetivo deste trabalho foi investigar a cinética de absorção de glutamina em camundongos submetidos à endotoxemia, o que não fora previamente investigado em outros trabalhos. Para avaliar a cinética da absorção intestinal de glutamina foi realizada eversão intestinal em camundongos machos, o que permite a coleta dos líquidos das camadas mucosa e serosa com maior precisão. Foram utilizadas doses de 10, 20, 40 e 50 mM de L-glutamina associada a [14C]-glutamina, nos tempos 0, 5, 10, 20, 30, 40, 60, 90 e 120 minutos. Os resultados obtidos demonstraram que em alta concentração de glutamina (50 mM) ocorre maior absorção em relação à retenção tecidual e esta é realizada por transporte ativo de aminoácidos. Os animais que foram submetidos à endotoxemia por LPS (5mg/kg) apresentaram alterações estruturais no tecido intestinal, detectadas pela histologia. Neste grupo, a retenção tecidual de glutamina foi significativamente maior do que no grupo controle, sobretudo na presença de glicose. Conclui-se que a cinética na absorção de glutamina é dose e tempo dependente em animais saudáveis e que, em condições de endotoxemia, ocorre maior retenção de glutamina no tecido intestinal na presença de glicose. Sugere-se que a via da hexosamina está envolvida; no entanto, mais estudos são necessários para esclarecer tais mecanismos. / The various studies of glutamine supplementation in stressful situations demonstrate the physiological role of this essential amino acid in metabolism. Acutely, supplementation with glutamine improves glutaminemia. Chronically, there is a greater concentration of glutamine in tissues such as muscle and liver. However, it is not known whether these effects are direct result of glutamine oral supplementation or reduced uptake within the basolateral membrane of enterocytes. The kinetic study of the absorption of glutamine provides information related to the ratio of concentration of glutamine absorbed and retained in the intestinal tissue, according to the doses used, and points out the changes resulting from sepsis induced by endotoxemia. The objective of this study was to investigate the kinetics of glutamine uptake in mice subjected to endotoxemia. To evaluate the kinetics of intestinal absorption of glutamine intestinal eversion was performed in male mice, allowing to collect the liquid layers of mucosa and serosa with greater precision. The doses used were 10, 20, 40 and 50 mM L-glutamine associated with [14C]-glutamine at 0, 5, 10, 20, 30, 40, 60, 90 and 120 minutes. The results showed that high concentrations of glutamine (50 mM) a higher absorption occurs in relation to tissue retention and this is accomplished by active transport of amino acids. The animals subjected to endotoxemia by LPS (5mg/kg) showed structural changes in the intestinal tissue, detected by histology. In this group, the tissue retention of glutamine was significantly higher than in the control group, especially in the presence of glucose. It is concluded that the kinetics of glutamine uptake is dose and time dependent in healthy animals, and in conditions of endotoxemia, there is greater retention of glutamine in intestinal tissue in the presence of glucose. It is suggested that the hexosamine pathway is involved; however, more studies are needed to clarify these mechanisms.

Typescript. / Digitized by Kansas Correctional Industries