Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 122-135).

Horses are at high risk to develop systemic inflammation due to the release of bacterial endotoxin from an inflamed gastrointestinal tract. Neutrophils are critical for mounting an immune response to bacterial endotoxins. Neutrophil activation following engagement of bacterial endotoxin expands their lifespan through suppression of their constitutive apoptosis. The prolonged lifespan of neutrophils propagates acute inflammation and delays the resolution of inflammation. Since equine neutrophil lifespan has not been well-studied, I investigated the occurrence of equine neutrophil apoptosis in vitro and in vivo. First, I investigated the effect of Escherichia coli lipopolysaccharide (LPS) treatment on the occurrence of equine neutrophil apoptosis in vitro. LPS treatment delayed in vitro equine neutrophil apoptosis in a dose-dependent manner at concentrations of 0.1-10 μg/ml through toll-like receptor (TLR)-4 signaling and down-regulation of the intrinsic pathway of apoptosis, specifically through reduced caspase-9 activity. Next, I found that ex vivo neutrophil apoptosis was delayed in two models of intestinal inflammation, jejunal ischemia and reperfusion (IR) and oligofructose-induced colitis, through down-regulation of both the intrinsic and extrinsic apoptosis pathways via reduced caspase-3, -8, and -9 activities. Pulmonary intravascular macrophages (PIMs) depletion with systemic gadolinium chloride (GC) prevented the prolongation of ex vivo neutrophil lifespan in horses undergoing jejunal IR through modulation of caspase-3, -8 and -9 activities. PIM depletion in IR horses resulted in an earlier and greater increase in tumor necrosis factor-alpha and a concomitant decrease in interleukin-10 to suggest an enhanced systemic pro-inflammatory response. I examined the effect of neutrophil concentration and co-incubation with aged, apoptotic neutrophils on the occurrence of neutrophil apoptosis in vitro. Neutrophil apoptosis was delayed with increasing concentrations of neutrophils in vitro, which may contribute to delayed neutrophil apoptosis in systemic inflammation. However, co-incubation with aged, apoptotic neutrophils did not alter in vitro neutrophil lifespan. Taken together, the data show that LPS delays equine neutrophils apoptosis in vitro in a TLR4-dependent manner through inhibition of caspase-9. Ex vivo neutrophil apoptosis was also delayed with systemic inflammation via down-regulation of caspase activity. A novel finding of this work was the reversal of delayed neutrophil apoptosis by depletion of PIMs in horses experiencing intestinal IR.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O ciclo vicioso de doenÃas entÃricas na infÃncia à um problema de saÃde pÃblica com consequÃncias graves e seus efeitos no desenvolvimento infantil nÃo estÃo totalmente elucidados. Orià e colaboradores, em 2005, demonstraram que crianÃas portadoras do gene APOE 4 com alta morbidade de diarreia apresentavam um melhor desempenho em testes cognitivos. O objetivo desse trabalho foi avaliar o papel protetor do gene APOE 4 em camundongos C57BL6J submetidos à desnutriÃÃo induzida por uma raÃÃo pobre em proteÃna (2%) e pela infecÃÃo intestinal induzida pelo Criptosporidium parvum. Utilizamos camundongos C57BL6J machos com peso mÃdio de 14 g, submetidos a um perÃodo de 14 dias de desnutriÃÃo e a 7 dias de infeÃÃo por C. parvum meio por meio da gavagem de 107 oocistos. Os animais foram separados segundo o genÃtipo: wildtype, APOE nocaute (ApoE Ko), APOE 3/3 (com gene APOE 3 humano) e APOE 4/4 (com gene APOE 4 humano). Os animais controles receberam PBS via gavagem. O peso dos animais foi monitorado diariamente. Os camundongos foram sacrificados em cÃmara de CO2 seguido de deslocamento cervical apÃs 14 dias do inÃcio do protocolo. Durante o perÃodo de pÃs-infecÃÃo foram coletadas as fezes dos animais infectados em dias alternados, para a realizaÃÃo do PCR quantitativo em tempo real (qPCR) para a anÃlise da quantidade de C. parvum liberada nas fezes. Amostras de Ãleo foram congeladas em nitrogÃnio lÃquido e armazenadas em freezer a -80ÂC para as anÃlises moleculares. Outras amostras foram fixadas em paraformaldeÃdo tamponado (4%) para processamento histolÃgico. Foram avaliados os parÃmetros morfomÃtricos de altura de vilo e profundidade de cripta nos segmentos ileais. Para a detecÃÃo de citocinas prÃinflamatÃrias de interesse (IL-1&#946;, IFN- &#947;, TNF-&#945; e IL-17), utilizou-se o ensaio multiplex (Luminex xMAP). Ainda por qPCR avaliou-se o transportador catiÃnico de aminoÃcidos (CAT-1), arginase 1, iNOS e TLR9. No peso encontramos uma maior adaptaÃÃo a perda de peso nos animais APOE 4 no 2o e 3o dias de desnutriÃÃo em comparaÃÃo a todos os grupos (p<0,05). No perÃodo de pÃs-infecÃÃo verificou-se diferenÃa significante no 2o dia (p<0,05) em comparaÃÃo a todos os grupos. Nas anÃlises morfomÃtricas, encontramos uma reduÃÃo na altura de vilos e profundidade de criptas nos animais APOE nocautes, jà nos animais APOE 4/4 ocorreu uma proteÃÃo contra esses danos em comparaÃÃo a todos os grupos (p<0,05). Os dados da anÃlise de liberaÃÃo de oocistos nas fezes evidenciaram um aumento do estado prÃ-inflamatÃrio e antiparasitÃrio nos animais APOE Ko e APOE 4/4, verificado por meio de uma reduÃÃo na quantidade de C. parvum liberado nas fezes de maneira significativa. Houve um aumento dos nÃveis intestinais das citocinas prÃ-inflamatÃrias IL-1&#946; (p<0,05) nos animais APOE Ko desnutridos e infectados em comparaÃÃo com APOE3/3 e APOE4/4, e altos nÃveis de IFN-&#947; (p<0,05) em comparaÃÃo com os controles selvagens e o grupo APOE Ko desnutrido controle. Os animais desnutridos controles APOE Ko tiveram aumento dos nÃveis intestinais de IL-17 (p<0,05) quando comparados aos animais APOE Ko desnutridos infectados. Dados de qPCR evidenciam que a presenÃa do genÃtipo APOE4 em camundongos aumenta os transcritos primÃrios de CAT-1 e arginase - 1 no Ãleo em relaÃÃo aos selvagens, APOE Ko e APOE3 (p<0,05) e que os animais nocautes aumentaram a expressÃo de iNOS em relaÃÃo aos outros grupos (p<0,05). Os animais APOE 4 desnutridos e infectados apresentaram uma expressÃo significativamente aumentada nos nÃveis de mRNA para TLR9 no Ãleo comparado com os APOE Ko igualmente desafiados (p<0,05). A partir dos nossos achados, podemos concluir que o animais com genÃtipo APOE 4 possuem uma aÃÃo prÃ-inflamatÃria controlada, o que favorece o combate ao C. parvum, visto que reduz a quantidade de DNA do parasita liberado nas fezes e melhora a taxa de crescimento de animais submetidos pela desnutriÃÃo/infecÃÃo, sugerindo que hospedeiros com genÃtipo APOE 4 possuem uma maior proteÃÃo contra as alteraÃÃes intestinais induzidas pela combinaÃÃo de C. parvum e desnutriÃÃo. / The vicious cycle of enteric infections and malnutrition during childhood is a major public health problem with devastating consequences and its effects are not fully elucidated. Oria and colleagues in 2005 showed that children with heavy diarrhea burdens when carrying the APOE 4 gene had a better cognitive performance. The aim of this study was to evaluate the protective role of APOE 4 gene in C57BL6J mice challenged by malnutrition induced by a 2% protein diet and intestinal infection caused by Cryptosporidium parvum. We used male C57BL6J mice weighing in average 14g, challenged by malnutrition for a period of 14 days compound with 7 days of C. parvum infection through a single dose of 107 oocysts given by gavage. Study animals were separated according to their genotype, as following: wild-type, APOE knock-out, APOE 3/3 (carriers of the human APOE 3 gene) and APOE 4/4 (carriers of human APOE 4 gene). Control animals received PBS by gavage. Body weight of the animals was monitored daily. Mice were sacrificed in CO2 chamber with posterior cervical dislocation after 14 days from the beginning of the protocol. During the post-infection period, stools samples were collected from the infected mice every other day for real time quantitative PCR (qPCR) assays in order to quantify C. parvum oocysts released in the stools. Ileal samples were immediately frozen in liquid nitrogen and then stored in a freezer at -80ÂC for molecular analyses. Other samples were fixed in buffered paraformaldehyde (4%) for histological processing. Morphometric parameters were evaluated for villus height and crypt depth in the ileal segments. For detection of a proinflammatory cytokine panel (IL- 1&#946;, IFN-&#947;, TNF-&#945;, and IL-17), we used the multiplex assay (Luminex xMAP). In addition by qPCR, the cationic amino acid transporter (CAT-1), arginase 1, iNOS, and TLR9 were assessed. Regarding weight, we found a greater adaptation to weight loss in APOE 4 animals in the 2nd and 3rd days of malnutrition (p<0.05) and in the postinfection time there was a significant difference on the 2nd day (p<0.05) compared to all groups. In the morphometric analyses, we found villus blunting and crypt disorganization in APOE knockout mice. We found APOE 4 protection against these alterations compared to all groups (p<0.05). The C. parvum oocyst shedding data indicate an increase in the pro-inflammatory state and anti-parasitic effects seen in the APOE Ko and APOE 4/4 mice, as confirmed by a significant reduction of the C. parvum released in the stools. In addition, we found increased levels of the intestinal pro-inflammatory cytokine (IL-1&#946;) (p<0.05) in the APOE Ko when compared with APOE3/3 and APOE4/4, higher levels of IFN-&#947; (p<0.05) when compared with wild-type and undernourished APOE Ko controls. The APOE Ko undernourished mice have increased intestinal levels of IL-17 compared with APOE Ko undernourished infected mice. qPCR data demonstrate that the presence of the APOE4 genotype in mice increased the primary transcripts of CAT-1 and arginase 1 in comparison to wild types, APOE Ko, and APOE 3/3 (p<0.05). Furtermore, APOE knockout mice had higher iNOS expression in comparison to all groups (p<0.05). The APOE 4 mice showed significant increase in the expression of TLR9 mRNA in the ileum when compared to APOE Ko mice (p<0.05). Altogether we concluded that the APOE 4 carriers have a balanced pro-inflammatory response, benefiting the C. parvum control, as seen by reduction of the parasite DNA released in the stools, and by improvements in the growth rates in the mice challenged malnutrition/infection, suggesting that the hosts carrying the APOE4 genotype have a better protection against the intestinal alterations induced by the compound challenge of C. parvum infection and malnutrition.

The dense and diverse community of microorganisms inhabiting the gastrointestinal tract of ruminant animals plays critical roles in the metabolism and absorption of nutrients, and gut associated immune function. Understanding microbial colonization in the small intestine of new born ruminants is a vital first step toward manipulating gut function through interventions during early life to produce long-term positive effects on host productivity and health. Yet the knowledge of microbiota colonization and its induced metabolites of small intestine during early life is still limited. In the present study, we examined the microbiota and metabolome in the jejunum and ileum of neonatal sika deer (Cervus nippon) from birth to weaning at days 1, 42, and 70. The microbial data showed that diversity and richness were increased with age, but a highly individual variation was observed at day 1. Principal coordinate analysis revealed significant differences in microbial community composition across three time points in the jejunum and ileum. The abundance of Halomonas spp., Lactobacillus spp., Escherichia-Shigella, and Bacteroides spp. tended to be decreased, while the proportion of Intestinibacter spp., Cellulosilyticum spp., Turicibacter spp., Clostridium sensu stricto 1 and Romboutsia spp. was significantly increased with age. For metabolome, metabolites separated from each other across the three time points in both jejunum and ileum. Moreover, the amounts of methionine, threonine, and putrescine were increased, while the amounts of myristic acid and pentadecanoic acid were decreased with age, respectively. The present study demonstrated that microbiota colonization and the metabolome becomes more developed in the small intestine with age. This may shed new light on the microbiota-metabolome-immune interaction during development.

No description available.

The mammalian small intestine is a major site (second in total activity only to the liver) for the synthesis and secretion of plasma apolipoproteins, and contributes significantly to overall whole-body lipid dynamics. A prominent feature of the small intestine is its exposure to periodic loads of meals often containing dramatically varying amounts or types of food components, including lipids such as tri-acylglycerols, cholesterol and cholesteryl esters. Since the trans-epithelial transport of most of these latter materials requires the elaboration of particles partially covered by apolipoproteins, the regulation of the biosynthesis or, more correctly, the availability of these proteins is an important and as yet little-understood problem. Previous studies have been conducted on systems which, for one or the other reason, have not permitted the following questions to be satisfactorily or coherently answered: Does the ingestion of fat-containing meals, either acutely or chronically, increase the rate of biosynthesis of intestinal apolipoproteins such as apo B-48, and is this the principal method of matching the "demand" with the supply of this "packaging material" needed for fat transport across the intestinal epithelial cells? Alternatively, does the maintenance of a large steady-state intracellular pool in the face of variations in intracellular apolipoprotein degradation, controlled by acute or chronic lipid ingestion, produce the required "match" between supply and demand for these proteins (as has recently been suggested in studies on liver cells)? An in vitro system was therefore devised whereby sheets of intestinal epithelial cells (enterocytes) were freshly isolated from the jejuna of adult male Syrian golden hamsters and incubated for several hours in a medium supporting steady-state protein synthesis, in a manner which was assumed to be similar to the activity just before the killing of the donor animals. (Hamsters appear on various grounds to be a better small-animal model of human lipoprotein metabolism than the more commonly studied rats). The isolated epithelial cell sheets produced primary apolipoprotein products that could be extracted from the cells or detected in the incubation media, free from the subsequent modifications that they are known to undergo in vivo. Hamsters maintained on a low-fat chow were either studied as such or subjected to a variety of dietary treatments designed to maximize (over short or long time periods) intracellular apolipoprotein requirements for the "packaging" of tri-acylglycerol-rich lipoproteins, especially chylomicrons: acute bolus administration of lipid into the gut; overnight feeding of fat-enriched food; and chronic (six week) fat feeding. Using specific antisera and immuno-precipitation techniques, apo B-48 and two other principal intestinal apolipoproteins were shown to be synthesized in the steady state by intestinal cell sheets derived from control animals and from those subjected to acute or chronic fat-containing diets. Secretion took place, however, only when prior fat exposure of the donor intestines had occurred. Pulse-chase labelling was used to compare the rates of apolipoprotein synthesis, degradation and secretion in the same cell sheet preparations. The rates of apolipoprotein B-48 synthesis did not vary significantly under conditions of low or high trans-epithelial lipid flux, supporting findings derived from in vivo experimental systems. In contrast with data from other systems, however, the biosynthesis of apolipoprotein A-IV was not reproducibly increased on fat challenge. The rates of apo B-48 degradation varied significantly and were markedly reduced under conditions of fat feeding. The experiments permit a choice between the two alternatives mentioned above: Ingestion of fatty foods, either acutely or over long periods of time, does not increase the rates of biosynthesis of apolipoproteins such as apo B-48; but variations in the rate of intracellular degradation of this and probably other apolipoproteins allows the intestinal cells to match their requirements for lipid-transporting molecules to the demands of any given situation, relying in each case on a large steady-state intracellular pool maintained by "constitutive" biosynthesis. Importantly, there seems also to be a specific, possibly related effect of fat feeding on the secretion of lipoproteins into the intestinal extracellular fluid. These conclusions coincide with those obtained by other workers from studies of apolipoprotein B dynamics in isolated hepatocytes and in the hepatoma-derived liver cell line, Hep G2. The mechanisms underlying these phenomena are as yet unresolved.

To study the mRNA found in sheep omasal epithelium encoding for a peptide transport protein(s), a 446-bp cDNA fragment was cloned from sheep omasal epithelium RNA. The predicted amino acid sequence of this fragment was 85.8, 90.5, and 90.5 percent identical to rabbit, human, and rat PepT1, respectively. The fragment was radiolabeled for use as a probe to study the distribution of the mRNA in various tissues. Total RNA was extracted and mRNA was isolated from the epithelium of gastrointestinal segments and other tissues as indicated. Northern blot analysis was conducted using the radiolabeled probe. In sheep (5) and lactating Holstein cows (3), hybridization was observed with mRNA from the omasum, rumen, duodenum, jejunum, and ileum. The estimated size of mRNA was 2.8 kb. No hybridization was observed with mRNA from the abomasum, cecum, colon, liver, kidney, and semitendinosus and longissimus muscles of either species or the mammary gland of the dairy cows. In pigs (6), the probe hybridized with mRNA from the duodenum, jejunum, and ileum. There was no hybridization with mRNA from the stomach, large intestine, liver, kidney, and semitendinosus and longissimus muscles. Two bands, 3.5 and 2.9 kb were observed with northern blot analysis, indicating two RNA transcripts that may result from alternative mRNA processing. In both Leghorns (15) and broilers (20), the strongest hybridization was found in the duodenum while the jejunum and ileum showed faint bands. The size of mRNA in chickens was 1.9 kb. Other tissues, including the crop, proventriculus, gizzard, ceca, liver, kidney, and muscles showed no hybridization to the probe. In conclusion, mRNA for a peptide transport protein(s) is present in the small intestine of all animals examined and the omasal and ruminal epithelium of sheep and dairy cows. The size of the mRNA varied among species. / Master of Science

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

Cold exposure impacts intestinal remodelling and metabolism. Signaling by glucagon-like peptide 1 (GLP-1), GLP-2 and glucose-dependent insulinotropic polypeptide (GIP) are tightly linked to nutrient intake and absorption. However, these peptide hormones' necessity to mediate gut adaptation and metabolic alternations during cold exposure has been incompletely explored. We hypothesize that GLP-1, GIP, and GLP-2 are released in proportion to required energy needs during cold exposure to enable efficient nutrient absorption and gut adaptation and subsequently impact nutrient handling. We evaluated morphological changes in the intestinal in wildtype, Glp1r-/-Glp2r-/- and Glp1r-/-Gipr-/- mice exposed to chronic cold or thermoneutral conditions for four weeks. Food intake and gut hormone secretion were significantly increased in all mice housed at 4-6 ̊C compared to those housed at thermoneutrality. Concomitantly, we observed increased remodeling measured by crypt to villus height (increased villi length) and intestinal circumference (increased circumference) in cold-exposed wildtype and Glp1r-/-Gipr-/- mice housed. In contrast, intestinal morphology in Glp1r-/-Glp2r-/- mice was unchanged in response to cold. Associated with these morphometric changes, we observed significant increases in fasting concentrations of GLP-1. These data suggest that GLP-1 and GLP-2 are key signaling molecules secreted from the gut in response to chronic cold exposure to enable intestinal remodeling.