This thesis investigated the number and function of natural killer T-cells (NK T-cells) as a function of age, in coeliac disease, Crohn’s disease and ulcerative colitis. NK T-cells are a newly appreciated class of immune cells that are able to regulate the activity of the broader T-cell population. NK T-cells have been implicated in animal models of autoimmune disease and in human autoimmune disease. A subset of NK cells express the T-cell receptor (TCR) and are termed NK T-cells. In humans a further small subset of NK T-cells express an invariant TCR α chain (Vα24Jα18) and contain the immunoregulatory cell population that is distinguished from classical T-cells by promptly producing interleukin-4 (IL-4). Invariant NK T-cells (iNK T-cells) have the surface phenotype of Vα24+ Vβ11+ T-cells and express CD161+ NK markers. They are CD4+ (single positive; SP) or CD4- (double negative; DN), CD1d restricted and are α-galactosylceramide (α-GalCer) reactive. NKT cells have been implicated in numerous autoimmune disorders. Early work showed a major deficiency of NKT cell numbers in nonobese diabetic (NOD) mice, a well-established model of spontaneous, autoimmune T-cell mediated insulin-dependent diabetes. Both the number of NKT cells and function, as assessed by IL-4 release following TCR ligation, are dramatically reduced in NOD mice. NK T-cells have been implicated in other models of autoimmunity such as, experimental allergic encephalomyelitis (EAE). They have since been investigated and shown to be deficient in a number of human autoimmune diseases including, systemic sclerosis (SSc), and systemic lupus erythematosus (SLE), multiple sclerosis, atopic asthma, atopic dermatitis, rheumatoid arthritis, type 1 diabetes mellitus and scleroderma. The basis of the work presented within this thesis originated from the deficiency of NK T-cells in models of autoimmune diseases and human autoimmune diseases. The initial aim of this thesis was to investigate the phenotype and function of Vα24+ NK T-cells in normal healthy control subjects and with respect to age. The original aim was to investigate whether NK cells, T-cells, NK T-like cells and invariant NK T-cells (iNK T-cells) are deficient in coeliac disease, Crohn’s disease and/or ulcerative colitis. Blood was collected for flow cytometry from normal control subjects, subjects with coeliac disease, Crohn’s disease and ulcerative colitis. The number of circulating NK cells, T-cells, NK T-like cells and iNK T-cells was assessed by three-colour flow cytometry. Intracellular cytokine production was measured after in vitro anti-CD3/ anti-CD28 antibodies, gluten fraction 3 and PMA:ionomycin stimulation. Vα24+ T-cells were quantified in ileocolonic biopsies by immunofluorescence and as mRNA by relative and real-time PCR (RT-PCR). The number of circulating Vα24+ T-cells and iNK T-cells decrease with age in normal healthy control subjects. Cytokine production was also affected by age. The work of this thesis has identified a subpopulation of otherwise normal healthy individuals whom have normal numbers of circulating Vα24+ T-cells, reduced numbers of circulating Vα24+ Vβ11+ T-cells and consequently iNK Tcells. Circulating CD161+ NK cells, Vα24+ T-cells and the SP subset of Vα24+ Tcells were reduced in coeliac disease. The low numbers of circulating Vα24+ T-cells was independent of diet. The number of circulating Vα24+ Vβ11+ Tcells were reduced in coeliac disease, and as a consequence, the number of circulating Vα24+ Vβ11+ α-GalCer/CD1d tetramer+ and Vα24+ 6B11+ iNK T-cells were reduced. The deficiency of Vα24+ T-cells was not confined to the blood, but observed within the intestinal mucosa. Intestinal Vα24 mRNA expression from subjects with coeliac disease was reduced compared to levels in normal subjects as assessed by relative and RT-PCR. Thus, Vα24+ T-cells were deficient in coeliac disease both systemically and mucosally. Cytokine production by Vα24+ T-cells, 6B11+ and Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro anti-CD3 stimulation was also impaired in subjects with coeliac disease. Circulating CD56+, CD57+, CD94+, CD161+ NK cells were reduced in Crohn’s disease and ulcerative colitis. Vα24+ T-cells and the SP subset of Vα24+ T-cells were reduced in Crohn’s disease but not in ulcerative colitis. Circulating Vα24+ Vβ11+ T-cells, Vα24+ Vβ11+ α-GalCer/CD1d tetramer+ and Vα24+ 6B11+ iNK T-cells were deficient in both Cohn’s disease and ulcerative colitis. The deficiency of Vα24+ T-cells was also observed within the intestinal mucosa. Intestinal Vα24 mRNA expression from Crohn’s disease and ulcerative colitis was reduced compared to levels in normal subjects as assessed by relative and RT-PCR. Cytokine production by Vα24+ T-cells, 6B11+ and Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro anti-CD3 stimulation was impaired for subjects with Crohn’s disease and ulcerative colitis. In summary, Vα24+ T-cell number and function were affected by age. Further investigations are warranted to see if deficiency of this immunoregulatory population is associated with disease. The decrease and dysfunction in immunoregulatory cells, Vα24 T-cells and iNK T-cells could contribute to the pathogenesis of coeliac disease, Crohn’s disease and ulcerative colitis. Coeliac disease, Crohn’s disease and ulcerative colitis are polygenetic diseases in which environmental factors play a significant role in disease development and state. The reduced numbers of iNK T-cell along with their impaired function may only be two factors. Presumably, other factors are involved. Nevertheless, iNK T-cells offer a potential target for the therapeutic intervention of coeliac disease, ulcerative colitis and Crohn’s disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345088 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2008

Irritable Bowel Syndrome (IBS) is one of the most common functional disorders of the gastrointestinal tract. Visceral hypersensitivity is the most commonly reported symptom of IBS, yet is the least adequately treated. Mechanosensitive information from the colon is relayed to the CNS by extrinsic colonic primary afferent nerves which have their cell bodies within dorsal root ganglia (DRG). This thesis aims to identify the contribution of several putatively mechanosensitive ion channels (ASIC1, 2 and 3, TRPV4 and TRPA1) toward detection of mechanical stimuli in the colon. This involvement is assessed by both molecular and functional means. The abundance of each of these channels was assessed by comparing expression within whole DRG against that in specifically colonic DRG neurons using an in situ hybridization methodology developed as part of this PhD. The functional role TRPV4 and TRPA1 impart toward colonic mechanosensation was investigated by recording responses to mechanical stimuli from colonic primary afferent fibres and comparing the results from mice genetically modified to lack either TRPV4 or TRPA1 with those of their intact littermates. The results from these studies indicate expression patterns within whole DRG do not provide accurate representation of the organ of interest, with abundances of each of the channels investigated differing between colonic DRG cells and the whole DRG. In particular ASIC3 and TRPV4 are preferentially expressed in colonic DRG neurons, unlike ASIC2 and TRPA1. Further, TRPV4 is functionally restricted to detection of noxious mechanical stimuli in the colon, while expression of TRPA1 is more widespread and functionally less restricted. Each of these channels are each potential targets for the treatment of IBS as each affects specific aspects of colonic mechanotransduction. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1347202 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Sciences, 2008

This thesis investigated the number and function of natural killer T-cells (NK T-cells) as a function of age, in coeliac disease, Crohn’s disease and ulcerative colitis. NK T-cells are a newly appreciated class of immune cells that are able to regulate the activity of the broader T-cell population. NK T-cells have been implicated in animal models of autoimmune disease and in human autoimmune disease. A subset of NK cells express the T-cell receptor (TCR) and are termed NK T-cells. In humans a further small subset of NK T-cells express an invariant TCR α chain (Vα24Jα18) and contain the immunoregulatory cell population that is distinguished from classical T-cells by promptly producing interleukin-4 (IL-4). Invariant NK T-cells (iNK T-cells) have the surface phenotype of Vα24+ Vβ11+ T-cells and express CD161+ NK markers. They are CD4+ (single positive; SP) or CD4- (double negative; DN), CD1d restricted and are α-galactosylceramide (α-GalCer) reactive. NKT cells have been implicated in numerous autoimmune disorders. Early work showed a major deficiency of NKT cell numbers in nonobese diabetic (NOD) mice, a well-established model of spontaneous, autoimmune T-cell mediated insulin-dependent diabetes. Both the number of NKT cells and function, as assessed by IL-4 release following TCR ligation, are dramatically reduced in NOD mice. NK T-cells have been implicated in other models of autoimmunity such as, experimental allergic encephalomyelitis (EAE). They have since been investigated and shown to be deficient in a number of human autoimmune diseases including, systemic sclerosis (SSc), and systemic lupus erythematosus (SLE), multiple sclerosis, atopic asthma, atopic dermatitis, rheumatoid arthritis, type 1 diabetes mellitus and scleroderma. The basis of the work presented within this thesis originated from the deficiency of NK T-cells in models of autoimmune diseases and human autoimmune diseases. The initial aim of this thesis was to investigate the phenotype and function of Vα24+ NK T-cells in normal healthy control subjects and with respect to age. The original aim was to investigate whether NK cells, T-cells, NK T-like cells and invariant NK T-cells (iNK T-cells) are deficient in coeliac disease, Crohn’s disease and/or ulcerative colitis. Blood was collected for flow cytometry from normal control subjects, subjects with coeliac disease, Crohn’s disease and ulcerative colitis. The number of circulating NK cells, T-cells, NK T-like cells and iNK T-cells was assessed by three-colour flow cytometry. Intracellular cytokine production was measured after in vitro anti-CD3/ anti-CD28 antibodies, gluten fraction 3 and PMA:ionomycin stimulation. Vα24+ T-cells were quantified in ileocolonic biopsies by immunofluorescence and as mRNA by relative and real-time PCR (RT-PCR). The number of circulating Vα24+ T-cells and iNK T-cells decrease with age in normal healthy control subjects. Cytokine production was also affected by age. The work of this thesis has identified a subpopulation of otherwise normal healthy individuals whom have normal numbers of circulating Vα24+ T-cells, reduced numbers of circulating Vα24+ Vβ11+ T-cells and consequently iNK Tcells. Circulating CD161+ NK cells, Vα24+ T-cells and the SP subset of Vα24+ Tcells were reduced in coeliac disease. The low numbers of circulating Vα24+ T-cells was independent of diet. The number of circulating Vα24+ Vβ11+ Tcells were reduced in coeliac disease, and as a consequence, the number of circulating Vα24+ Vβ11+ α-GalCer/CD1d tetramer+ and Vα24+ 6B11+ iNK T-cells were reduced. The deficiency of Vα24+ T-cells was not confined to the blood, but observed within the intestinal mucosa. Intestinal Vα24 mRNA expression from subjects with coeliac disease was reduced compared to levels in normal subjects as assessed by relative and RT-PCR. Thus, Vα24+ T-cells were deficient in coeliac disease both systemically and mucosally. Cytokine production by Vα24+ T-cells, 6B11+ and Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro anti-CD3 stimulation was also impaired in subjects with coeliac disease. Circulating CD56+, CD57+, CD94+, CD161+ NK cells were reduced in Crohn’s disease and ulcerative colitis. Vα24+ T-cells and the SP subset of Vα24+ T-cells were reduced in Crohn’s disease but not in ulcerative colitis. Circulating Vα24+ Vβ11+ T-cells, Vα24+ Vβ11+ α-GalCer/CD1d tetramer+ and Vα24+ 6B11+ iNK T-cells were deficient in both Cohn’s disease and ulcerative colitis. The deficiency of Vα24+ T-cells was also observed within the intestinal mucosa. Intestinal Vα24 mRNA expression from Crohn’s disease and ulcerative colitis was reduced compared to levels in normal subjects as assessed by relative and RT-PCR. Cytokine production by Vα24+ T-cells, 6B11+ and Vα24+ α-GalCer/CD1d tetramer+ iNK T-cells after 4 h in vitro anti-CD3 stimulation was impaired for subjects with Crohn’s disease and ulcerative colitis. In summary, Vα24+ T-cell number and function were affected by age. Further investigations are warranted to see if deficiency of this immunoregulatory population is associated with disease. The decrease and dysfunction in immunoregulatory cells, Vα24 T-cells and iNK T-cells could contribute to the pathogenesis of coeliac disease, Crohn’s disease and ulcerative colitis. Coeliac disease, Crohn’s disease and ulcerative colitis are polygenetic diseases in which environmental factors play a significant role in disease development and state. The reduced numbers of iNK T-cell along with their impaired function may only be two factors. Presumably, other factors are involved. Nevertheless, iNK T-cells offer a potential target for the therapeutic intervention of coeliac disease, ulcerative colitis and Crohn’s disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345088 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2008

Abstract Nutritional status affects every aspect of a child?s health. Thorough nutritional assessment is hampered by the lack of a single comprehensive tool, which can cover all aspects of nutritional assessment. In three distinct studies, this thesis investigated the nutritional status of hospitalised children, children with coeliac disease and children with inflammatory bowel disease. Study 1 The objectives of this study were to assess prevalence of malnutrition and nutritional risk, and define demographic and anthropometric factors associated with nutritional risk among hospitalized children. In this cross sectional study, 157 hospitalised children were assessed for nutritional status using nutritional risk score (NRS) and anthropometric measurements. We found that 4.5%, 8.9%, 15.1% and 10.4% children were wasted, stunted, overweight and obese respectively. However, with the NRS, 47.8% of the children were at high nutritional risk. These children at high risk had lower weight for age (p=0.02), lower BMI percentiles for age (p=0.001) and longer hospitalization (p=0.001) than children at no risk. Study 2 The objectives of this study were to determine nutritional parameters in children with coeliac disease. Twenty-five children with coeliac disease and an equal number of age and gender matched controls were enrolled and anthropometric measurements, BIA and leptin levels were analysed in all. No significant differences were found between the children with coeliac disease and controls in these parameters. BMI percentile correlated with leptin levels in children with coeliac disease. Study 3 The objectives of this study were to determine anthropometric parameters and leptin levels in children with IBD and ascertain if BMI correlates with leptin levels in these children. Thirty children with IBD and 60 age and gender matched controls were enrolled. Anthropometric measurements and leptin levels were analysed and compared with controls. IBD children had significantly low weight for age (p=0.002), BMI percentiles (p=0.001) and leptin levels (p=0.009) compared to controls. There was a correlation between BMI and leptin levels in IBD children. In conclusion, this thesis has shown that one quarter of hospitalized children were overweight or obese, and further, that half of the hospitalised children were at high risk of nutritional deterioration and these children had longer hospital stay than children at no risk. Children with coeliac disease had similar anthropometric measurements, body compartments and leptin levels to controls. However, children with IBD had lower anthropometric measurements and leptin levels, indicating under-nutrition. Nutritional assessment should be a mandatory part of clinical management with nutritional status assessed by various tools including NRS, anthropometry, BIA and leptin levels.

Abstract Nutritional status affects every aspect of a child?s health. Thorough nutritional assessment is hampered by the lack of a single comprehensive tool, which can cover all aspects of nutritional assessment. In three distinct studies, this thesis investigated the nutritional status of hospitalised children, children with coeliac disease and children with inflammatory bowel disease. Study 1 The objectives of this study were to assess prevalence of malnutrition and nutritional risk, and define demographic and anthropometric factors associated with nutritional risk among hospitalized children. In this cross sectional study, 157 hospitalised children were assessed for nutritional status using nutritional risk score (NRS) and anthropometric measurements. We found that 4.5%, 8.9%, 15.1% and 10.4% children were wasted, stunted, overweight and obese respectively. However, with the NRS, 47.8% of the children were at high nutritional risk. These children at high risk had lower weight for age (p=0.02), lower BMI percentiles for age (p=0.001) and longer hospitalization (p=0.001) than children at no risk. Study 2 The objectives of this study were to determine nutritional parameters in children with coeliac disease. Twenty-five children with coeliac disease and an equal number of age and gender matched controls were enrolled and anthropometric measurements, BIA and leptin levels were analysed in all. No significant differences were found between the children with coeliac disease and controls in these parameters. BMI percentile correlated with leptin levels in children with coeliac disease. Study 3 The objectives of this study were to determine anthropometric parameters and leptin levels in children with IBD and ascertain if BMI correlates with leptin levels in these children. Thirty children with IBD and 60 age and gender matched controls were enrolled. Anthropometric measurements and leptin levels were analysed and compared with controls. IBD children had significantly low weight for age (p=0.002), BMI percentiles (p=0.001) and leptin levels (p=0.009) compared to controls. There was a correlation between BMI and leptin levels in IBD children. In conclusion, this thesis has shown that one quarter of hospitalized children were overweight or obese, and further, that half of the hospitalised children were at high risk of nutritional deterioration and these children had longer hospital stay than children at no risk. Children with coeliac disease had similar anthropometric measurements, body compartments and leptin levels to controls. However, children with IBD had lower anthropometric measurements and leptin levels, indicating under-nutrition. Nutritional assessment should be a mandatory part of clinical management with nutritional status assessed by various tools including NRS, anthropometry, BIA and leptin levels.

Abstract Nutritional status affects every aspect of a child?s health. Thorough nutritional assessment is hampered by the lack of a single comprehensive tool, which can cover all aspects of nutritional assessment. In three distinct studies, this thesis investigated the nutritional status of hospitalised children, children with coeliac disease and children with inflammatory bowel disease. Study 1 The objectives of this study were to assess prevalence of malnutrition and nutritional risk, and define demographic and anthropometric factors associated with nutritional risk among hospitalized children. In this cross sectional study, 157 hospitalised children were assessed for nutritional status using nutritional risk score (NRS) and anthropometric measurements. We found that 4.5%, 8.9%, 15.1% and 10.4% children were wasted, stunted, overweight and obese respectively. However, with the NRS, 47.8% of the children were at high nutritional risk. These children at high risk had lower weight for age (p=0.02), lower BMI percentiles for age (p=0.001) and longer hospitalization (p=0.001) than children at no risk. Study 2 The objectives of this study were to determine nutritional parameters in children with coeliac disease. Twenty-five children with coeliac disease and an equal number of age and gender matched controls were enrolled and anthropometric measurements, BIA and leptin levels were analysed in all. No significant differences were found between the children with coeliac disease and controls in these parameters. BMI percentile correlated with leptin levels in children with coeliac disease. Study 3 The objectives of this study were to determine anthropometric parameters and leptin levels in children with IBD and ascertain if BMI correlates with leptin levels in these children. Thirty children with IBD and 60 age and gender matched controls were enrolled. Anthropometric measurements and leptin levels were analysed and compared with controls. IBD children had significantly low weight for age (p=0.002), BMI percentiles (p=0.001) and leptin levels (p=0.009) compared to controls. There was a correlation between BMI and leptin levels in IBD children. In conclusion, this thesis has shown that one quarter of hospitalized children were overweight or obese, and further, that half of the hospitalised children were at high risk of nutritional deterioration and these children had longer hospital stay than children at no risk. Children with coeliac disease had similar anthropometric measurements, body compartments and leptin levels to controls. However, children with IBD had lower anthropometric measurements and leptin levels, indicating under-nutrition. Nutritional assessment should be a mandatory part of clinical management with nutritional status assessed by various tools including NRS, anthropometry, BIA and leptin levels.

A review of the literature revealed that diet plays an important role in serious human noninfectious large bowel diseases including cancer and inflammatory bowel diseases. Dietary protein (especially as red and processed meats) has been implicated as a positive risk factor for colorectal cancer while starch which is not digested in the small intestine (resistant starch, RS) appears to be protective. The series of experiments described in this thesis were aimed to determine the effects of dietary proteins and RS on indices of colon health in an animal model, the laboratory rat. Genetic damage is a prerequisite for carcinogenesis and this was assessed by a specific assay (the comet assay) which gives a measure of DNA strand breaks. Loss of mucus barrier function is thought to contribute to inflammatory bowel disease by permitting bacterial translocation and this was measured optically using a microscope micrometer. Other biomarkers were measured as described below. There were four major experiments. 1. Effects of dietary red meat and casein on colonic DNA damage and interaction with resistant starch Previous studies had shown that higher dietary protein (as casein) induced genetic damage in rat colonocytes and that RS (fed as a high amylose maize starch) was protective. This study was aimed at establishing whether a high protein diet fed as cooked red meat had similar effects and whether RS was protective. Rats were fed diets containing either 15 % or 25% casein or 25% barbecued lean red beef, each with or without 48% high amylose maize starch (as a source of RS) for 4 weeks. As expected, high dietary casein caused a 2-fold increase in colonic single-strand DNA breaks compared with a low casein diet and reduced the thickness of the colonic mucus layer by 41%. High levels of cooked meat caused 26% more DNA damage than the high casein diet but reduced mucus thickness to a similar degree as casein. Addition of RS to the diet abolished the increase in DNA damage and the loss of colonic mucus thickness induced by either high protein diet. It is thought that RS promotes large bowel health through the SCFA produced by the large bowel bacteria. One acid in particular (butyrate) has been associated particularly with promotion of normal large bowel function and protection against disease. In keeping with this hypothesis, caecal and faecal short chain fatty acid pools (including those of butyrate) were increased by inclusion of RS in the diet. DNA damage is an early step in the initiation of cancer and these findings agree with the population data which suggest that total dietary protein and red meat promote risk of colorectal cancer. However, inclusion of resistant starch in the diet could significantly reduce that risk. 2. Differential effects of dietary whey, soy and casein on colonic DNA damage and interaction with resistant starch The preceding experiments showed that high levels of animal-derived proteins increased colonocyte genetic damage and loss of the mucus barrier in rats. This second experiment was designed to determine whether diets high in different types of dairy protein (casein or whey) or a plant protein isolate (soy) had similar adverse effects on colonic DNA and mucus barrier function and whether inclusion of RS in the diet was protective. Adult male Sprague Dawley rats were fed a diet containing 15 % or 25 % casein, whey or soy protein, each with or without 48 % high amylose maize starch for 4 weeks. In confirmation of the earlier studies, higher levels of dietary casein increased colonocyte DNA damage significantly. However, whey did not increase genetic damage. Colonic DNA damage was highest for soy when fed at both 15% and 25% protein in the absence of RS. Inclusion of RS in the diet attenuated colonocyte DNA damage due to higher dietary protein in all three groups. The colonic mucus barrier was thinner in rats fed higher dietary protein but the effect was reversed by feeding RS. Caecal total SCFA and butyrate pools were low in rats fed the digestible starch and were higher in rats fed RS. However, there was no relationship between caecal or faecal SCFA and genetic damage or mucus thickness. Caecal and colonic tissue weight and colon length were higher in rats fed RS, consistent with greater SCFA supply. These data confirm that higher dietary protein of animal (casein) or plant (soy) origin increases genetic damage and loss of the mucus barrier indicating that this is an effect of protein and not its source. These findings accord with the epidemiological data which link dietary protein to greater risk of colorectal cancer and inflammatory bowel disease. However, the data show also that dietary proteins differ in their specific actions on genetic damage and mucus thickness. Further, the data from the feeding of whey suggest that not all proteins are equivalent in their capacity to provoke adverse changes in colonic integrity. While the data show that RS raised large bowel and faecal SCFA, they indicate their levels were not related directly to these biomarkers. 3. Dose response effects of resistant starch on protein induced colonic DNA damage The accumulated data linking greater protein intakes to adverse changes in the colon were obtained at dietary levels which were not unreasonable in terms of animal or human consumption. However, the dietary level of RS which were fed were relatively high (48% by weight) so this study was conducted to determine its effectiveness at lower levels of dietary inclusion. It was also important to ascertain whether there was a dose-response relationship between RS intake and the observed effects. One of the mechanisms proposed for the induction of colorectal cancer by high dietary protein intakes is oxidative damage to DNA. In this experiment, this was done by assaying with endonuclease III. Adult male rats were fed a diet containing 25% casein with 0%, 10%, 20%, 30% or 40% high amylose maize starch for 4 weeks. As in the preceding studies comet tail moment was greatest and the mucus barrier thinnest in rats fed 0% RS. DNA damage was reduced and the mucus barrier thickened in a logarithmic dose-dependent manner by RS. There was no significant difference between dietary groups associated with oxidative DNA damage as measured by endonuclease III. Caecal and faecal short chain fatty acid (SCFA) pools rose with the increased level of dietary RS. DNA damage of colonocytes correlated negatively with caecal SCFA but the strongest correlation was with caecal butyrate, which is consistent with the proposed role of this SCFA in promoting a normal cell phenotype. The data show that RS prevents protein induced colonic DNA damage in a dose-dependent manner. Inclusion of 10% high amylose maize starch was found to be sufficient to oppose colonocyte DNA damage, and to increase caecal and faecal SCFA pools. Intakes of this order are not unreasonable in terms of human consumption of RS. 4. Dose response effects of red and white meat on colonic DNA damage and interaction with resistant starch The accumulated evidence from large prospective human studies links diet to colorectal cancer risk strongly. The evidence from the animal studies described in this thesis that dietary protein induces colonocyte genetic damage supports a role for high protein intakes in increasing risk. Recently, several large epidemiological studies and a meta-analysis of prospective studies have found that consumption of dietary red or processed meats, but not white (poultry) meat, is associated with increased risk of colorectal cancer. This is consistent with the data from the preceding studies that specific proteins affected colonic integrity differentially. A large prospective European study (European Prospective Investigation into Cancer and Nutrition) has reported that dietary fibre was protective. The findings reported in this thesis that RS opposes the effects of high dietary protein accord with that conclusion. This study aimed to compare the effects of cooked red (beef) or white (chicken) meat on DNA damage and mucus barrier thickness in rats. The study was designed to determine whether the relationship between the intakes of these meats was dose-dependent. Double-strand DNA breaks are thought to relate more closely to carcinogenesis than single-strand breaks so both were measured. Adult male Sprague-Dawley rats were fed a diet containing 15%, 25% or 35% cooked beef or cooked chicken each with or without 20% high amylose maize starch for four weeks. Both red and white meat increased colonic DNA damage dose-dependently. However, both single and double strand breaks were significantly greater when the rats were fed the red meat diets compared to those fed the white meat. Colonocyte DNA damage was reduced by the consumption of RS while large bowel SCFA were increased. The findings of this study are consistent with the epidemiological data which show that red meat consumption is associated with greater risk of colorectal cancer but that white meat is not. Summary The data reported in this thesis support the findings of prospective population studies that high dietary protein, red meat in particular, appears to be harmful to the health of the large bowel. However, the data demonstrate also that different protein types have differential effects on the integrity of the colonocyte DNA. Furthermore, the addition of RS to the diet protects against protein-induced colonic DNA damage and maintenance of the colonic mucus barrier, apparently through increased SCFA production by colonic fermentation. The results of these experiments indicate a strong potential for RS to be effective in maintenance of large bowel integrity in the face of high dietary protein. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1292858 / Thesis(Ph.D.)-- School of Molecular and Biomedical Science, 2007.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The primary aim of this thesis was to identify probiotics and/or prebiotics with the potential to reduce the severity of experimental colitis. The specific aims were to i) screen a range of candidate pro biotic strains for capacity to reduce symptoms of DSS-colitis, ii) characterize the effects of DSS within the small intestine, iii) assess, in vitro, the effect of probiotics on intestinal epithelial cell integrity, iv) assess the potential for the prebiotic, fructooligosaccharide, to reduce the severity of DSS-colitis alone, and in synbiotic combination with a probiotic strain. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1280844 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2007

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The primary aim of this thesis was to identify probiotics and/or prebiotics with the potential to reduce the severity of experimental colitis. The specific aims were to i) screen a range of candidate pro biotic strains for capacity to reduce symptoms of DSS-colitis, ii) characterize the effects of DSS within the small intestine, iii) assess, in vitro, the effect of probiotics on intestinal epithelial cell integrity, iv) assess the potential for the prebiotic, fructooligosaccharide, to reduce the severity of DSS-colitis alone, and in synbiotic combination with a probiotic strain. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1280844 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 2007

A review of the literature revealed that diet plays an important role in serious human noninfectious large bowel diseases including cancer and inflammatory bowel diseases. Dietary protein (especially as red and processed meats) has been implicated as a positive risk factor for colorectal cancer while starch which is not digested in the small intestine (resistant starch, RS) appears to be protective. The series of experiments described in this thesis were aimed to determine the effects of dietary proteins and RS on indices of colon health in an animal model, the laboratory rat. Genetic damage is a prerequisite for carcinogenesis and this was assessed by a specific assay (the comet assay) which gives a measure of DNA strand breaks. Loss of mucus barrier function is thought to contribute to inflammatory bowel disease by permitting bacterial translocation and this was measured optically using a microscope micrometer. Other biomarkers were measured as described below. There were four major experiments. 1. Effects of dietary red meat and casein on colonic DNA damage and interaction with resistant starch Previous studies had shown that higher dietary protein (as casein) induced genetic damage in rat colonocytes and that RS (fed as a high amylose maize starch) was protective. This study was aimed at establishing whether a high protein diet fed as cooked red meat had similar effects and whether RS was protective. Rats were fed diets containing either 15 % or 25% casein or 25% barbecued lean red beef, each with or without 48% high amylose maize starch (as a source of RS) for 4 weeks. As expected, high dietary casein caused a 2-fold increase in colonic single-strand DNA breaks compared with a low casein diet and reduced the thickness of the colonic mucus layer by 41%. High levels of cooked meat caused 26% more DNA damage than the high casein diet but reduced mucus thickness to a similar degree as casein. Addition of RS to the diet abolished the increase in DNA damage and the loss of colonic mucus thickness induced by either high protein diet. It is thought that RS promotes large bowel health through the SCFA produced by the large bowel bacteria. One acid in particular (butyrate) has been associated particularly with promotion of normal large bowel function and protection against disease. In keeping with this hypothesis, caecal and faecal short chain fatty acid pools (including those of butyrate) were increased by inclusion of RS in the diet. DNA damage is an early step in the initiation of cancer and these findings agree with the population data which suggest that total dietary protein and red meat promote risk of colorectal cancer. However, inclusion of resistant starch in the diet could significantly reduce that risk. 2. Differential effects of dietary whey, soy and casein on colonic DNA damage and interaction with resistant starch The preceding experiments showed that high levels of animal-derived proteins increased colonocyte genetic damage and loss of the mucus barrier in rats. This second experiment was designed to determine whether diets high in different types of dairy protein (casein or whey) or a plant protein isolate (soy) had similar adverse effects on colonic DNA and mucus barrier function and whether inclusion of RS in the diet was protective. Adult male Sprague Dawley rats were fed a diet containing 15 % or 25 % casein, whey or soy protein, each with or without 48 % high amylose maize starch for 4 weeks. In confirmation of the earlier studies, higher levels of dietary casein increased colonocyte DNA damage significantly. However, whey did not increase genetic damage. Colonic DNA damage was highest for soy when fed at both 15% and 25% protein in the absence of RS. Inclusion of RS in the diet attenuated colonocyte DNA damage due to higher dietary protein in all three groups. The colonic mucus barrier was thinner in rats fed higher dietary protein but the effect was reversed by feeding RS. Caecal total SCFA and butyrate pools were low in rats fed the digestible starch and were higher in rats fed RS. However, there was no relationship between caecal or faecal SCFA and genetic damage or mucus thickness. Caecal and colonic tissue weight and colon length were higher in rats fed RS, consistent with greater SCFA supply. These data confirm that higher dietary protein of animal (casein) or plant (soy) origin increases genetic damage and loss of the mucus barrier indicating that this is an effect of protein and not its source. These findings accord with the epidemiological data which link dietary protein to greater risk of colorectal cancer and inflammatory bowel disease. However, the data show also that dietary proteins differ in their specific actions on genetic damage and mucus thickness. Further, the data from the feeding of whey suggest that not all proteins are equivalent in their capacity to provoke adverse changes in colonic integrity. While the data show that RS raised large bowel and faecal SCFA, they indicate their levels were not related directly to these biomarkers. 3. Dose response effects of resistant starch on protein induced colonic DNA damage The accumulated data linking greater protein intakes to adverse changes in the colon were obtained at dietary levels which were not unreasonable in terms of animal or human consumption. However, the dietary level of RS which were fed were relatively high (48% by weight) so this study was conducted to determine its effectiveness at lower levels of dietary inclusion. It was also important to ascertain whether there was a dose-response relationship between RS intake and the observed effects. One of the mechanisms proposed for the induction of colorectal cancer by high dietary protein intakes is oxidative damage to DNA. In this experiment, this was done by assaying with endonuclease III. Adult male rats were fed a diet containing 25% casein with 0%, 10%, 20%, 30% or 40% high amylose maize starch for 4 weeks. As in the preceding studies comet tail moment was greatest and the mucus barrier thinnest in rats fed 0% RS. DNA damage was reduced and the mucus barrier thickened in a logarithmic dose-dependent manner by RS. There was no significant difference between dietary groups associated with oxidative DNA damage as measured by endonuclease III. Caecal and faecal short chain fatty acid (SCFA) pools rose with the increased level of dietary RS. DNA damage of colonocytes correlated negatively with caecal SCFA but the strongest correlation was with caecal butyrate, which is consistent with the proposed role of this SCFA in promoting a normal cell phenotype. The data show that RS prevents protein induced colonic DNA damage in a dose-dependent manner. Inclusion of 10% high amylose maize starch was found to be sufficient to oppose colonocyte DNA damage, and to increase caecal and faecal SCFA pools. Intakes of this order are not unreasonable in terms of human consumption of RS. 4. Dose response effects of red and white meat on colonic DNA damage and interaction with resistant starch The accumulated evidence from large prospective human studies links diet to colorectal cancer risk strongly. The evidence from the animal studies described in this thesis that dietary protein induces colonocyte genetic damage supports a role for high protein intakes in increasing risk. Recently, several large epidemiological studies and a meta-analysis of prospective studies have found that consumption of dietary red or processed meats, but not white (poultry) meat, is associated with increased risk of colorectal cancer. This is consistent with the data from the preceding studies that specific proteins affected colonic integrity differentially. A large prospective European study (European Prospective Investigation into Cancer and Nutrition) has reported that dietary fibre was protective. The findings reported in this thesis that RS opposes the effects of high dietary protein accord with that conclusion. This study aimed to compare the effects of cooked red (beef) or white (chicken) meat on DNA damage and mucus barrier thickness in rats. The study was designed to determine whether the relationship between the intakes of these meats was dose-dependent. Double-strand DNA breaks are thought to relate more closely to carcinogenesis than single-strand breaks so both were measured. Adult male Sprague-Dawley rats were fed a diet containing 15%, 25% or 35% cooked beef or cooked chicken each with or without 20% high amylose maize starch for four weeks. Both red and white meat increased colonic DNA damage dose-dependently. However, both single and double strand breaks were significantly greater when the rats were fed the red meat diets compared to those fed the white meat. Colonocyte DNA damage was reduced by the consumption of RS while large bowel SCFA were increased. The findings of this study are consistent with the epidemiological data which show that red meat consumption is associated with greater risk of colorectal cancer but that white meat is not. Summary The data reported in this thesis support the findings of prospective population studies that high dietary protein, red meat in particular, appears to be harmful to the health of the large bowel. However, the data demonstrate also that different protein types have differential effects on the integrity of the colonocyte DNA. Furthermore, the addition of RS to the diet protects against protein-induced colonic DNA damage and maintenance of the colonic mucus barrier, apparently through increased SCFA production by colonic fermentation. The results of these experiments indicate a strong potential for RS to be effective in maintenance of large bowel integrity in the face of high dietary protein. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1292858 / Thesis(Ph.D.)-- School of Molecular and Biomedical Science, 2007.