京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18865号 / 医博第3976号 / 新制||医||1008(附属図書館) / 31816 / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 坂井 義治, 教授 福田 和彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The objective of this study was to evaluate developmental regulation of the expression of nutrient transporter and brushborder hydrolase genes in the small intestine of piglets. Seventy piglets from seven sows were killed at birth (d 0), during suckling (d 1, 3, 7, 14, 21) and postweaning (d 22, 24, 28, 35), and intestinal segments (duodenum, jejunum and ileum) were collected. The mRNA abundance was determined by Northern blot using specific cDNA probes for three disaccharidases (lactase-phlorizin hydrolase, LPH, sucrase-isomaltase, SI, and maltase-glucoamylase, MGA), three peptide hydrolases (aminopeptidase A, APA, aminopeptidase N, APN, and dipeptidyl peptidase IV, DPP IV), two sugar transporters (Na+-dependent glucose transporter 1, SGLT1, and facilitated glucose transporter 5, GLUT5), a peptide transporter (H+-dependent peptide transporter 1, PepT1), four amino acid transporters (excitatory amino acid carrier 1, EAAC1, Na+-dependent neutral amino acid transporter, ATB0, the light chain of a heterodimeric transport system b0,+ involved in the heteroexchange of cationic and neutral amino acids, b0,+AT, and Na+-independent large branched and aromatic neutral amino acid transporter 2, LAT2), and two iron transporters (divalent metal ion transporter 1, DMT1, and iron-regulated transporter 1, IREG1). Protein expression was quantified by Western blot using specific antibodies for LPH, SI, SGLT1, and PepT1. During suckling, the abundance of LPH, APA, APN, DPP IV, b0,+AT mRNA increased quadratically (P < 0.001) with age from birth to d 7 or 14 then remained unchanged or slightly declined with age to d 21. The mRNA abundance of SI increased and LAT2 decreased linearly (P < 0.001) with age, and the abundance of MGA and GLUT5 mRNA remained unchanged with age. There was an age x intestinal segment interaction (P < 0.001) for the abundance of EAAC1 and ATB0 mRNA. The abundance of EAAC1 mRNA increased from d 0 through 14 and remained stable to d 21 in the ileum, and it was low and slightly increased with age through d 21 in the duodenum and jejunum. The abundance of ATB0 mRNA generally increased from d 0 to 21 in the duodenum and ileum, and increased from d 0 to 7 and then decreased to d 21 in the jejunum. The abundance of SGLT1 and PepT1 mRNA was substantial at birth and transiently declined to d 1. The abundance of SGLT1 mRNA generally increased from d 1 to 21, and PepT1 mRNA abundance increased to d 3 and then plateaued through d 21. Postweaning, the mRNA abundance of all of these carbohydrate and protein assimilation related genes increased during the first day (3 d for ATB0) after weaning then declined to the levels at weaning in the jejunum and ileum, followed by a subsequent change pattern that varied among genes. During suckling, the mRNA abundance of LPH, SGLT1, and APA was greater in the duodenum and jejunum than the ileum (P < 0.001). The PepT1 and APN mRNA was evenly distributed among intestinal segments, and the expression of MGA, DPP IV, EAAC1, b0,+AT, ATB0, and LAT2 mRNA was generally greater in the jejunum and ileum than the duodenum or greatest in the ileum. Postweaning, the mRNA abundance of all of these carbohydrate and protein assimilation related genes examined was generally greater in the jejunum and ileum than the duodenum or highest in the ileum. From d 0 through 35, DMT1 and IREG1 mRNA was predominantly (P < 0.05) distributed in the duodenum, where the abundance of DMT1 and IREG1 mRNA increased with age during suckling, and then rapidly decreased after weaning. The protein expression of LPH and SI exhibited a similar developmental pattern as that for the mRNA abundance. Unlike the developmental regulation of their respective mRNA abundance, the protein expression of SGLT1 exhibited a general decline from suckling to postweaning. The protein expression of PepT1 gradually decreased with age from birth to d 35 in the duodenum, and initially declined from birth to the lowest value then slightly increased with age through d 21, followed by an increase to d 35 in the jejunum and ileum. In conclusion, the gene expression of these brushborder hydrolases and nutrient transporters was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages of life. These differences in ontogenetic regulation and the distribution may be related to the luminal substrate concentration as well as the nutrient categories, and the developmental regulation of these genes may occur not only at the transcriptional level but also at the posttranscriptional level. / Ph. D.

The chicken yolk sac and small intestine are connected through the yolk stalk and share many biological similarities. During the embryonic stage, the extra-embryonic yolk sac helps the embryo to absorb nutrients primarily in the last two weeks of incubation. The chicken yolk sac physically moves yolk contents from the yolk sac to the small intestine at the end of embryogenesis. This is the time when the small intestine replaces the yolk sac in assimilating nutrients for the embryo and later for the posthatch chicken. Additionally, both chicken small intestinal epithelia and the yolk sac secrete beta defensins for promoting intestinal health. Since there are heterogeneous cell types along the mammalian intestinal villus, which are derived from the intestinal stem cells in the crypts, we investigated if cells of the chicken yolk sac and small intestine have the same ontogeny as mammalian intestinal epithelial cells. In this dissertation, we mainly focused on the spatial expression of nutrient transporters (PepT1 and SGLT1), intestinal stem cell markers (Lgr5 and Olfm4), and avian beta defensins in the chicken yolk sac and small intestine during the embryonic and early posthatch stages. RNAscope in situ hybridization was used to identify the distribution of cells expressing PepT1 mRNA in both the chicken yolk sac and small intestine. PepT1 mRNA was found to be expressed by epithelial cells in both the yolk sac and small intestine. In the yolk sac, PepT1 mRNA was uniformly distributed in each endodermal epithelial cell along the villus-like structure. The pattern of PepT1 mRNA expression observed in the chicken yolk sac during the last 10 days of incubation revealed that PepT1 mRNA was increased from e11 to e13, and decreased from e15 to day of hatch. The peak of PepT1 mRNA expression was between e13 and e15, when the yolk sac reaches maximum absorptive area and the growth of the chicken embryo is at its fastest rate. However, the expression of PepT1 mRNA in the intestine was only detected in columnar enterocytes along the villus and not in goblet cells or cells in the crypts. The immunofluorescence assay confirmed that PepT1 protein was located at the brush border membrane of the enterocytes and that protein expression of PepT1 was restricted to the intestinal epithelial cells from approximately the middle to the tip of the villus. In order to identify intestinal stem cells, we used the known mammalian stem cell markers, Lgr5 and Olfm4. Both Lgr5 and Olfm4 are specifically expressed by cells in the chicken intestinal crypts, suggesting that they can be used as biomarkers for chicken intestinal stem cells. Dual labelling of PepT1 and Olfm4 mRNA on the same chicken intestinal sample revealed that there was a gap between PepT1-expressing enterocytes and Olfm4-expressing intestinal stem cells. The cells in this gap were presumably transit amplifying (TA) cells. Additionally, we also found that the TA cell zone along the intestinal villus was reduced during chicken growth. This TA cell population could be clearly detected at day of hatch and d1 posthatch but not later. The expression of SGLT1 mRNA was localized to yolk sac endodermal epithelial cells and showed a sharp increase at the end of incubation. This increase of SGLT1 mRNA coincided with the increase in glucose in the yolk, indicating that the chicken embryo needs glucose as energy for hatching. The mRNA expression profiles of various avian beta defensins have been examined by qPCR and in situ hybridization to investigate the immune function of the yolk sac and small intestine. We found that AvBD10 mRNA showed the highest expression level in the yolk sac and was expressed predominantly in the yolk sac endodermal epithelial cells. Additionally, the expression of AvBD10 mRNA showed a development-specific pattern, which increased from e9 to e11, and decreased from e13 towards day of hatch. The expression patterns of AvBD1, 2, and 7 mRNA were similar to each other. These three genes were found to be expressed by chicken heterophils distributed in the yolk sac blood islands and small intestinal blood vessels. Only a subset of heterophils, which might be activated, were able to express AvBD1, 2, and 7 mRNA. In the intestine, the expression of AvBD10 mRNA was localized to cells along the villus at e19 and day of hatch, but later to only a few cells located above the intestinal crypts. In summary, the endodermal epithelial cells are responsible for the absorptive and immune functions of the chicken yolk sac. The yolk sac mesoderm is critical for embryonic hematopoiesis and innate immunity. The chicken small intestinal epithelial cells are derived from the intestinal stem cells in the crypts. These epithelial cells have different cell types, which are functioning to absorb nutrients and secrete antimicrobial peptides. / Ph. D. / The chicken yolk sac and small intestine are connected to each other and share many biological similarities. Both chicken small intestinal and yolk sac epithelia play critical roles for nutrient absorption and immune defense. In this dissertation, the mRNA for nutrient transporters such as the peptide transporter, PepT1 and the sodium-glucose co-transporter, SGLT1 were found to be expressed by absorptive epithelial cells in both the yolk sac and small intestine. Additionally, both intestinal and yolk sac epithelial cells expressed avian beta defensins (AvBDs), which are important chicken host defense peptides. In the small intestine, there are a number of differentiated cell types that originate from stem cells in the crypt that express the known mammalian stem cell markers, Olfm4 and Lgr5 mRNA. However, in the chicken yolk sac, only the stem cell marker Lgr5 mRNA was expressed by endothelial cells. In summary, the yolk sac epithelial cells are responsible for the absorptive and immune functions for the embryonic stage. The chicken small intestinal epithelial cells are derived from the intestinal stem cells in the crypts. These epithelial cells have different cell types, which function to absorb nutrients and secrete antimicrobial peptides.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

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.

¡iAbstract¡j Lipopolysaccharide (LPS) is the toxic chemical component of the cell wall in all gram-negative bacteria which can stimulate immune cells, including macrophages and white blood cell, to release cytokines such as interleukin-1£], interleukin-6 and tumor necrosis factor-£\. These pro-inflammatory mediators induce systemic acute inflammation and multiple organs dysfuction syndrome in sepsis. Plasma leakage from microvasculature is a hallmark of inflammation. Previous studies have demonstrated that other inflammatory agents, such as capsaicin, substance P and histamine could cause the acute formation of numerous endothelial gaps in the venules that result in extensive plasma leakage in the inflammatory tissues of the whole respiratory tract and a part of digestive tract in a few minutes. Mammalian intestines have many goblet cells that synthesize mucus and discharge it into the intestinal lumen. The mucus film that covers the surface epithelium facing the lumen of digestive system, is an immune defense that can prevent gastrointestinal epithelium from chemical and physical damage and act as a lubricant. Changes in goblet cell function and number are involved in microbial infection, inflammatory syndromes and immune factors. This study was aimed to investigate: (1) The degree of Plasma leakage and goblet cell hypersecretion in the small intestine of rats after an intravenous injection of a high dose of LPS (15 mg/kg), and (2) The involvement of vagus nerve and cholinergic receptors in plasma leakage and goblet cell secretion. For the study of plasma extravasation in small intestine during endotoxema, India ink was used as the tracer to mark the inflamed leaky microvessels. Rats were perfusion-fixed through the aorta, and endothelial gaps between endothelial cells of blood vessels were made visible with silver staining. The methacrylate sections of the ileum 3 £gm in thickness were stained with Alcian blue and periodic acid-schiff reagent to detect glycoproteins of goblet cells. Our results showed that LPS not only caused an increase in plasma leakage but also triggered degranulation of many goblet cells in villi and crypts. Numerous gaps were found in postcapillary venules and collecting venules, and plasma extravasation was observed in the serosa and tunica muscularis rat small intestine after LPS. Extensive plasma extravasation occurred in earier phases (5-30 min). However, numerous goblet cells started to discharge mucus granules 30 min after LPS treatment. A large amount of extracellular mucus was accumulated between intestinal villi 1 hour after LPS stimulation. Pretreatment with atropine, the muscarinic receptor antagonist, significantly inhibited goblet cell secretion. The inhibitory effect of pretreatment with atropine or bilateral cervical vagotomy on LPS-induced plasma leakage was not consistent. It is concluded that the plasma leakage and goblet cell hypersecretion induced by endotoxin shock was time-dependent and was associated with activation of muscarinic cholinergic receptors.

Rat exon microarrays were utilized to detect changes in mRNA expression and alternative splicing in the liver, duodenum, jejunum, and ileum of the lactating rat when compared to age-matched virgin controls. Analysis of data at the level of gene expression revealed differential expression of genes involved in cholesterol biosynthesis in each tissue examined, suggesting increased Sterol Response Element Binding Protein activity. We also detected decreased mRNA from components of the T-cell signaling pathway in the jejunum and ileum. We characterized expression of solute carrier and adenosine triphosphate binding cassette proteins. In addition to characterizing genes by pathway, we have also grouped genes based on their pattern of expression to identify important genes. Amongst genes upregulated in all tissues was Slc39a4, which is a critical transporter in the absorption of zinc in enterocytes. Alternative splicing analysis detected a substantial amount of alternative splicing in the ileum compared to other tissues. In addition, in the liver Abcg8, a protein that functions as a heterodimer to export cholesterol in the bile, shows differential splicing in the liver, but not in other tissues. We also detected differential expression of Ugt1a6 in the liver based on usage of an alternative first exon, which is consistent with altered protein levels observed previously. Differential splicing also appears to occur in Ace2 in the ileum, which could have consequences on the renin-angiotensin pathway.

The effect of intestinal microbiota and dietary supplementation of mannan oligosaccharides (MOS) on mucosal architecture and digestive physiology in broiler chicks was examined. In experiment 1, pre-sterilized eggs (Ross x Ross 308) were placed in three HEPA (high efficiency particulate air)-filtered isolator units at day 19 of incubation. Germ-free chicks in one isolator were conventionalized by exposure to cecal contents from a laying hen. Bacterial contamination occurred in one germ-free isolator such that these birds were monoassociated by a bacterium within the Acinetobacter spp. resulting in 3 categories of microbial status including germ-free (GF, n=10), conventionalized (CV, n=19) and monoassociated (Mono, n=13) birds. Dietary treatments assigned to each isolator consisted of a negative control (NC, 0 g/kg of MOS in the basal diet) and MOS (2 g/kg of MOS in the diet) resulting in a 2X3 factorial treatment arrangement. At 7 d of age, body weight was recorded and birds were killed to permit collection of visceral organs, intestinal tissues and cecal contents. Body weight, relative length of small intestinal segments and relative bursa weight were significantly increased in CV birds. These birds also had increased crypt depth and lamina propria area. Dietary MOS increased villus height and villus surface area in CV birds compared with GF and Mono birds. Transcripts for all housekeeping genes tested in ileal tissue were increased by MOS such that transcripts were normalized to unit mass of total RNA. In comparison to birds fed the NC diet, MOS significantly increased the abundance of proliferative cell nuclear antigen (PCNA), toll-like receptor (TLR)-4, avian β-defensin (GAL)-6, interleukin (IL)-8, peptide transporter 1 (PepT1) and sodium-dependent glucose transporter (SGLT)-1 transcripts in ileum per unit total RNA. However, the effect of microbial status on selected gene expression profiles was surprisingly limited. A second experiment was conducted to confirm the conventionalization protocol produced a complex microbiota similar to conventionally reared birds. Twenty day-old broiler chicks (Ross x Ross 308) were assigned to one of two wire-floored battery cages provided the NC and MOS diets ad libitum and killed at 7 d of age. Terminal restriction fragment length polymorphism (TRFLP) analysis demonstrated that microbial diversity indices (Richness, Evenness, Shannon, and Simpson) were greater in conventionalized gnotobiotic birds compared to the conventionally reared birds confirming a successful conventionalization strategy in the gnotobiotic trial. These studies demonstrate that under good hygienic conditions, CV chicks thrive similar to GF animals. Based on responses to MOS observed in GF birds, evidence indicates that MOS, independent of changes in microbial composition, directly modifies host response parameters including innate immune activation, digestive and absorptive function.

Made available in DSpace on 2014-06-11T19:27:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2003Bitstream added on 2014-06-13T18:31:10Z : No. of bitstreams: 1 cerqueira_bf_dr_botfmvz.pdf: 1499209 bytes, checksum: 7ff8a218fa7c9053994488fe2ecdb205 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A oclusão e a reperfusão das artérias esplâncnicas provoca alterações locais e sistêmicas derivadas principalmente da liberação de substâncias citotóxicas e da interação entre neutrófilos e células endoteliais. Buscando-se estudar os efeitos da pentoxifilina e da n-acetilcisteína em lesões provocadas em um modelo de isquemia e reperfusão (I/R), 60 ratos foram divididos em 6 grupos: SAL/CONT (salina/controle): animais submetidos à cirurgia, mas não à I/R, tratados com solução fisiológica; SAL/ISQ (salina/isquemia): animais submetidos à oclusão da artéria celíaca (AC), artéria mesentérica cranial (AMCr) e artéria mesentérica caudal (AMCa) durante 30 minutos, seguidos de 120 minutos de reperfusão, tratados com solução fisiológica; PTX/CONT: animais submetidos à cirurgia, mas não à I/R, tratados com pentoxifilina; PTX/ISQ: animais submetidos à I/R, tratados com pentoxifilina; NAC/CONT: animais submetidos à cirurgia, mas não à I/R, tratados com n-acetilcisteína; NAC/ISQ: animais submetidos à I/R, tratados com n-acetilcisteína. Os parâmetros avaliados foram pressão arterial média (PAM) carótida, pressão venosa jugular, fluxo sangüíneo na aorta e na AMCr, temperatura esofágica, hematócrito, hemogasometria, histologia intestinal, dosagem de espécies reativas ao ácido tiobarbitúrico (TBARS) em duodeno, jejuno e íleo e de malondialdeído (MDA) plasmático e do íleo. Nos grupos submetidos à I/R, a PAM teve queda significativa após 120 minutos de reperfusão; a pressão venosa não mostrou alterações no decorrer do experimento; houve queda do fluxo sangüíneo na aorta após os 30 minutos de isquemia, com queda mais acentuada ao final do período de reperfusão; o fluxo sangüíneo na AMCr diminuiu significativamente após 120 minutos de reperfusão, porém, em menor grau no grupo NAC/ISQ; a freqüência... . / Splanchnic artery occlusion and reperfusion result in local and remote tissue destruction caused by toxic factors released into the circulation and by neutrophil-endothelial cell interactions. In order to evaluate the effects of pentoxifylline (PTX) and n-acetilcysteine (NAC) on ischemia/reperfusion model (I/R), sixty rats were allocated into six groups: 1) SAL/CONT: Sham operation + saline; 2) SALI/ISQ: 30 min celiac, cranial and caudal mesenteric arteries occlusion + 120 min reperfusion + saline; 3) PTX/CONT: Sham operation + PTX (50 mg/kg); 4) PTX/ISQ: I/R + PTX; 5) NAC/SAL: Sham operation + NAC (430 mg/kg) and 6) NAC/ISQ: I/R + NAC. In all groups above mean arterial blood pressure (MABP), vein pressure, aorta and cranial mesenteric artery blood flow, heart rate, esophagus temperature, hematocrit, blood gas determination, histology, thiobarbituric acid reaction species (TBARS) and malondialdehyde (MDA) levels were determined. In I/R groups, MABP decreased significantly 120 minutes after reperfusion (p<0,05); as no difference in vein pressure were observed. Aorta blood flow decreased 30 minutes after ischemia, decreasing dramatically following restoration of blood flow. Cranial mesenteric artery blood flow decreased significantly after reperfusion, although the fall in NAC/ISQ group was attenuated. During the assays, heart rate was reduced, temperature decreased progressively and metabolic acidosis took place. Pentoxiffyline prevented histopathologic signs of injury in duodenum, jejunum and ileum, as well as n-acetilcysteine prevented ileum histopathologic signs of injury. I/R caused TBARS increase in NAC group jejunum and saline group ileum, but no difference was observed in plasmatic and ileal MDA. The present study allowed concluding that PTX was more efficient in preventing histophatologic injury than NAC, but neither PTX nor NAC... (Complete abstract, click electronic address below).

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第18319号 / 農博第2044号 / 新制||農||1021(附属図書館) / 学位論文||H26||N4826(農学部図書室) / 31177 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 伏木 亨, 教授 金本 龍平 / 学位規則第4条第1項該当