Relationship between alcohol abuse upper gastro-intestinal haemorrhage and nutritional status during acute hospital admission

This thesis is concerned with the effect of alcohol consumption on the pathogenesis of bleeding from the upper gastrointestinal tract via nutritional pathways. Altered nutritional status is a frequently recognised clinical accompaniement of heavy alcohol consumption in hospitalized patients. Similarly, upper gastrointestinal bleeding is frequently accompanied by the presence of heavy alcohol consumption. Nevertheless, the clinical quantification of alcohol intake is often descriptive, so that a link between alcohol use and upper gastrointestinal haemorrhage via nutritional mechanisms has been only generally defined. In the literature review, the methods of defining alcohol use and abuse, using interview, biochemical and haematological techniques are noted. The relationship between alcohol abuse and nutrient imbalances is reviewed, especially in relation to possible effects on the gastrointestinal tract, appetite and eating habits. A further section reviews the relationship between alcohol use and anatomical lesions of the upper gastrointestinal tract likely to lead to bleeding. Following the chapter in which the methods used in this thesis are described. Chapter 4 seeks to describe the study population and its subgroups in this thesis in relation to interview, biochemical and haematological methods. Alcohol use is defined in relation to (1) a clinical classification of heavy or light drinking, based on a questionnaire administered in Casualty, (2) a quantified method of determining alcohol consumption during a subsequent ward dietetic assessment, (3) in relation to a biochemical definition (recent drinking and non-drinking), and a classification of (1) and (2) called, for the purposes of this thesis, 'alcohol abusers' and 'nonabusers'. Heavy, regular and recent drinkers and alcohol abusers tend to be male and younger than light, infrequent and nonrecent drinkers and nonabusers. Chapter 5 relates the nutritional status of those patients admitted acutely to hospital in relation to the groups defined in Chapter 4, Nutritional status is defined in terms of food intake, anthropometry, biochemical and haematological parameters. Different methods of defining alcohol use give rise to different patterns of nutritional impairment. Chapter 6 relates the nutritional status of those patients admitted acutely to hospital in relation to the presence or absence of an endoscopically defined site of upper gastrointestinal bleeding. A difference is seen between those bleeding from a Mailory-weiss tear and other sites of bleeding, similarly, biochemical differences in nutritional status emerge between those patients who presented in shock, and those who did not. Chapter 7 explores the relationships between biochemical markers of nutritional status and haemostatic variables in the groups of abusers/non-abusers, the various sites of primary bleeding/controls, and shock/non-shock. Serum copper appears to be related to altered haemostasis in a manner not apparently described elsewhere.

Effects of ovariectomy and estrogen on ischemia-reperfusion injury in hindlimbs of female rats

The effects of estrogen and ovariectomy on indexes of muscle damage after 2 h of complete hindlimb ischemia and 2 h of reperfusion were investigated in female Sprague-Dawley rats. The rats were assigned to one of three experimental groups: ovariectomized with a 17-estradiol pellet implant (OE), ovariectomized with a placebo pellet implant (OP), or control with intact ovaries (R). It was hypothesized that following ischemia-reperfusion (I/R), muscle damage indexes [serum creatine kinase (CK) activity, calpain-like activity, inflammatory cell infiltration, and markers of lipid peroxidation (thiobarbituric-reactive substances)] would be lower in the OE and R rats compared with the OP rats due to the protective effects of estrogen. Serum CK activity following I/R was greater (P < 0.01) in the R rats vs. OP rats and similar in the OP and OE rats. Calpain-like activity was greatest in the R rats (P < 0.01) and similar in the OP and OE rats. Neutrophil infiltration was assessed using the myeloperoxidase (MPO) assay and immunohistochemical staining for CD43-positive (CD43+) cells. MPO activity was lower (P < 0.05) in the OE rats compared with any other group and similar in the OP and R rats. The number of CD43+ cells was greater (P < 0.01) in the OP rats compared with the OE and R rats and similar in the OE and R rats. The OE rats had lower (P < 0.05) thiobarbituric-reactive substance content following I/R compared with the R and OP rats. Indexes of muscle damage were consistently attenuated in the OE rats but not in the R rats. A 10-fold difference in serum estrogen content may mediate this. Surprisingly, serum CK activity and muscle calpain-like activity were lower (P < 0.05) in the OP rats compared with the R rats. Increases in serum insulin-like growth factor-1 content (P < 0.05) due to ovariectomy were hypothesized to account for this finding. Thus both ovariectomy and estrogen supplementation have differential effects on indexes of I/R muscle damage.

Influencing health through intestinal microbiota modulation and probiotics

Science has progressed fast in providing answers to probiotic health benefits to the consumers. This symposium also proves that progress is fast. However, several challenges still need to be solved and more effective strains and strain combinations discovered. This will pave the way from good probiotics to specific products for clearly identified target populations. The intestinal microbiota and its interaction(s) with probiotics challenges researchers to turn to the next new page to discover new approaches and treatment modalities that utilize probiotics as means of providing good nutrition with clear health benefits to all consumers.

Phospholipase activity in human mesenteric ischaemia and infarction

Currently, diagnostic tests for mesenteric ischaemia and infarction are inadequate due to poor sensitivity and specificity. In addition, many potential markers appear too late to be clinically useful. At present, definitive diagnosis can only be made at the time of surgery, which is not ideal as surgery is often to be avoided in critically ill and elderly patients. A clinically useful, minimally invasive test is likely to decrease the currently very high mortality rate and allow monitoring of 'at risk' patients during their hospital stay. A two-dimensional electrophoresis based proteomic approach was undertaken to assess plasma protein differences between patients with surgically confirmed bowel infarction and control Intensive Care patients. The major protein differences were found to be members or variants of acute phase proteins. Serum amyloid A showed the largest difference between the two patient groups, and this protein was investigated in greater depth. An analysis was performed to compare the diagnostic ability of several commonly used indicators of critical illness and bowel infarction with serum amyloid A and phospholipase A2. Although none of the variables were ideal for clinical use, plasma phospholipase A2 activity showed the best discriminatory power, as determined by Receiver Operating Characteristic curves. From a review of the literature, phospholipase AI (PLA2) appeared to be increased in the bowel as a result of ischaemia and infarction. In one patient, matched tissues were obtained, and PLA2 activity was found to be significantly higher in infarcted bowel tissue compared to ischaemic bowel tissue. PLA2 activity was significantly greater in bowel lumen than tissue, suggesting that the protein was being released, and may enter the circulation. PLA2 activity was increased in the plasma of bowel infarction patients compared with control patients, though the difference was not significant. The phospholipase activity exhibited a number of similarities to typical phospholipase A2 proteins, but also showed a number of inconsistent characteristics. For this reason, we wished to identify the protein responsible for the increased phospholipase activity in infarcted human bowel. The PLA2 activity in human bowel could not be abolished by immunoprecipitation of the PLA2 isoforms IIA (well described in bowel) and V (a closely related isoform). To investigate these proteins, a native urea protein gel devised for snake venom phospholipase A2 was modified for use with mammalian phospholipase AI. The modified gel was used to show that the protein with phospholipase activity from infarcted gut was different from normal gut PLA2 and type IIA PLA2. A number of extensions were devised for these native gels and were found to be useful both in this investigation and for venom investigations. Protein purification was undertaken to identify the protein responsible for the increased phospholipase activity in infarcted bowel. Protein was purified from infarcted human bowel using a number of techniques that exploited unusual characteristics of the protein. The purification techniques each retained the native activity of the protein and the purification could therefore be monitored with a phospholipid hydrolysis assay at each stage. The protein identified by mass spectrometry was an excellent match for cyclophilin B, an inflammatory protein that had previously been identified in rat bowel at the mRNA level (Hasel et al, 1991, Kainer & Doris, 2000). As the purification progress had been monitored throughout with a phospholipid hydrolysis assay, cyclophilin B was an unexpected identification, as it is not known to have phospholipase activity. Cyclophilin B was removed from the highly purified samples via immunoprecipitation and this process abolished all phospholipase activity. The addition of cyclosporin A, (the pharmaceutical ligand of cyclophilin B), did not effect the phospholipase activity. Cyclophilin B protein was found in normal and infarcted human bowel using Western blotting. Cyclophilin B protein also appeared to be present in the bowel lumen and plasma of several patients with bowel infarction, but not in control patients. Immunohistochemistry confirmed the ubiquitous nature of cyclophilin B that had been reported by other groups. This project has investigated the use of two dimensional gel electrophoresis based proteomics to identify proteins present in the plasma of patients with confirmed bowel infarction and control intensive care patients. The major protein classes observed were members of the acute phase proteins, which highlights the need for pre-fractionation of plasma to identify lower abundance, disease associated proteins. A series of potential plasma markers were compared using Receiver Operating Characteristic Curves. Although no ideal marker was clear from this analysis, phospholipase activity appeared to warrant further investigation. Phospholipase activity was investigated in human infarcted bowel. Protein purification identified cyclophilin B as a bowel protein that showed unusual phospholipid hydrolysing activity. Cyclophilin B is a ubiquitous protein in intestinal cell types in both normal and infarcted tissue. There appears to be release of cyclophilin B into bowel lumen and plasma under conditions of mesenteric ischaemia and infarction.

Undiagnosed and potentially lethal parasite infections among immigrants and refugees in Australia

Background: Intestinal parasite infections are a major cause of ill health in many resource-poor countries. This study compares the types and rates of these infections and their risk factors in recently arrived and long-term immigrants in Australia.

Method: Cross-sectional surveys of 127 East African and 234 Cambodian immigrants and refugees were undertaken in 2000 and 2002, respectively, to assess the burden of intestinal parasites and collect demographic information. Serum samples were assessed for eosinophilia and Strongyloides stercoralis and Schistosoma antibodies, and feces examined for ova, cysts, and parasites.

Results: Intestinal parasites were identified in 77/117 fecal samples from East African and in 25/204 samples collected from Cambodian participants. Eleven percent (14/124) of East Africans and 42% (97/230) of Cambodians had positive or equivocal serology for S stercoralis. Schistosoma serology was positive or equivocal in 15% (19/124) of East African participants.

Conclusion: Potentially serious intestinal parasite infections are common among recent and longer term immigrants despite multiple visits to health care providers. Immigrants and refugees from high-risk countries would benefit from comprehensive health checks soon after resettlement.

Studies on dietary fibre : analysis, epidemiological and physiological aspects

This thesis involves an investigation in three areas; first, a study of an enzymatic-gravimetric method for the analysis of dietary fibre; second, a survey of dietary fibre intake in an area of a developing country, and finally, some observations on the functional aspects of gel-forming dietary fibre in the rat. A simple and rapid enzymatic-gravimetric assay for both soluble and insoluble dietary fibre has been critically investigated. Reference samples were also analysed by a more comprehensive, enzymatic gas chromatographic method to allow testing of the relative accuracy of the enzymatic-gravimetric method. The enzymatic-gravimetric method was found to be highly reproducible but gave a slightly higher value for total dietary fibre than the more comprehensive method. This discrepancy is probably due to the presence of small quantities of resistant starch and protein residue which are recovered in the enzymatic-gravimetric method. In the enzymatic-gas chromatographic method, protein residue is not measured, and resistant starch is estimated, but not counted as dietary fibre. The enzymatic-gravimetric method was applied to the analysis of foods commonly consumed in the Padang region of West Sumatra in Indonesia, in order to estimate dietary fibre intake in the region. Daily intakes of usual foods were estimated by use of a 24-hour recall procedure aided by food photographs to assist in the estimation of portion size. Samples of approximately 60 of the most commonly consumed foods were collected and analysed for dietary fibre. These appear to be the first data which report values for dietary fibre in Indonesion foods and they represent a significant improvement upon the existing data on crude fibre content. Knowledge of the amounts of foods usually consumed and their dietary fibre content allowed an estimation of usual intakes of dietary fibre. Fibre intake was found to be lower than in the developing countries of Africa and was comparable to intakes measured in the U.K. This is the first study to show that in this part of South East Asia, a developing country area using polished rice as a staple food, dietary fibre intakes are as low as in Western countries. Low intakes of fibre are believed to be related to the prevalence of a range of diseases and, in this study, preliminary data on the rates of non-infective, chronic diseases were collected from the two main hospitals in West Sumatra. Chronic, non-infectious diseases such as inguinal hernia, appendicitis, haemorrhoids, diabetes mellitus, hypertension and malignant neoplasms of the rectum are relatively frequent in West Sumatra. While no firm conclusions can be drawn from these data, they do show the possibility of a relationship between low intakes of dietary fibre and the prevalence of these diseases, and suggest that further investigation is necessary. Some observations were made of the effect of gel-forming dietary fibre on stomach emptying and intestinal transit rate in the rat. Xanthan gum was added to iso-osmotic solutions to produce increased viscosity and phenol sulphonphthalein (phenol red) was used as a non-absorbable marker. Gavage feeding of solutions with a range of viscosities was used to study the effect of viscosity on the rate of stomach emptying and intestinal transit. Increased viscosity was observed to slow gastro-intestinal transit and this provides one mechanism by which dietary fibre of the gel-forming type ray improve glucose tolerance.

The type II secretion system and its ubiquitous lipoprotein substrate, SsIE are required for biofilm formation and virulence of enteropathogenic escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in infants in developing countries. We have identified a functional type II secretion system (T2SS) in EPEC that is homologous to the pathway responsible for the secretion of heat-labile enterotoxin by enterotoxigenic E. coli. The wild-type EPEC T2SS was able to secrete a heat-labile enterotoxin reporter, but an isogenic T2SS mutant could not. We showed that the major substrate of the T2SS in EPEC is SslE, an outer membrane lipoprotein (formerly known as YghJ), and that a functional T2SS is essential for biofilm formation by EPEC. T2SS and SslE mutants were arrested at the microcolony stage of biofilm formation, suggesting that the T2SS is involved in the development of mature biofilms and that SslE is a dominant effector of biofilm development. Moreover, the T2SS was required for virulence, as infection of rabbits with a rabbit-specific EPEC strain carrying a mutation in either the T2SS or SslE resulted in significantly reduced intestinal colonization and milder disease.

Effects of dietary selenium on post-ischemic expression of antioxidant mRNA

Cardiac ischemia reperfusion leads to oxidative stress and poor physiological recovery. Selenium deficiency down-regulates thioredoxin reductase (Txnrd) and glutathione peroxidase (Gpx) activity, impairing recovery from ischemia-reperfusion. Furthermore, selenium supplementation has been shown to be cardioprotective and lessens oxidative stress in reperfused rat hearts. In this study we have investigated the role of selenium in the mRNA expression of these, and related antioxidant proteins, post ischemia-reperfusion. Male rats were fed varying doses of selenium for five weeks. Hearts were isolated and perfused using the Langendorff method with 22.5 min of global ischemia and 45 min reperfusion. RNA was extracted for quantitative real-time PCR analysis of glutathione peroxidase (Gpx)-1 and 4, glutathione reductase (Gsr), thioredoxin peroxidase-2 (Prdx2), thioredoxin (Txn) and thioredoxin reductase (Txnrd)-1 and 2 gene expression. Selenium deficiency produced significant reductions in Gpx-1, Gpx-4, Prdx2, Txnrd-1 and Txnrd-2 expression. Conversely, selenium supplementation of 1000 μg/kg significantly up-regulated Gpx-1, Gpx-4, Txn, Txnrd-1 and Txnrd-2 transcription. Our results show selenium modulates the cardiac mRNA expression of thioredoxin and glutathione related enzymes post ischemia-reperfusion, and impacts on tolerance to ischemia-reperfusion.

Evidence for the existence of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) with and without abdominal discomfort (irritable bowel) syndrome.

There is evidence that Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is accompanied by gastro-intestinal symptoms; and IgA and IgM responses directed against lipopolysaccharides (LPS) of commensal bacteria, indicating bacterial translocation.

The gut microbiota and inflammatory noncommunicable diseases: associations and potentials for gut microbiota therapies

Rapid environmental transition and modern lifestyles are likely driving changes in the biodiversity of the human gut microbiota. With clear effects on physiologic, immunologic, and metabolic processes in human health, aberrations in the gut microbiome and intestinal homeostasis have the capacity for multisystem effects. Changes in microbial composition are implicated in the increasing propensity for a broad range of inflammatory diseases, such as allergic disease, asthma, inflammatory bowel disease (IBD), obesity, and associated noncommunicable diseases (NCDs). There are also suggestive implications for neurodevelopment and mental health. These diverse multisystem influences have sparked interest in strategies that might favorably modulate the gut microbiota to reduce the risk of many NCDs. For example, specific prebiotics promote favorable intestinal colonization, and their fermented products have anti-inflammatory properties. Specific probiotics also have immunomodulatory and metabolic effects. However, when evaluated in clinical trials, the effects are variable, preliminary, or limited in magnitude. Fecal microbiota transplantation is another emerging therapy that regulates inflammation in experimental models. In human subjects it has been successfully used in cases of Clostridium difficile infection and IBD, although controlled trials are lacking for IBD. Here we discuss relationships between gut colonization and inflammatory NCDs and gut microbiota modulation strategies for their treatment and prevention. (J Allergy Clin Immunol 2015;135:3-13.)

Alteration of copper physiology in mice overexpressing the human menkes protein ATP7A

The Menkes protein (ATP7A) is defective in the Cu deficiency disorder Menkes disease and is an important contributor to the maintenance of physiological Cu homeostasis. To investigate more fully the role of ATP7A, transgenic mice expressing the human Menkes gene ATP7A from chicken beta-actin composite promoter (CAG) were produced. The transgenic mice expressed ATP7A in lung, heart, liver, kidney, small intestine, and brain but displayed no overt phenotype resulting from expression of the human protein. Immunohistochemical analysis revealed that ATP7A was found primarily in the cardiac muscle, smooth muscle of the lung, distal tubules of the kidney, intestinal enterocytes, and patches of hepatocytes, as well as in the hippocampus, cerebellum, and choroid plexus of the brain. In 60-day- and 300-day-old mice, Cu concentrations were reduced in most tissues, consistent with ATP7A playing a role in Cu efflux. The reduction in Cu was most pronounced in the hearts of older T22#2 females (24%), T22#2 males (18%), and T25#5 females (23%), as well as in the brains of 60-day-old T22#2 females and males (23% and 30%, respectively).

Drug-herb interactions: eliminating toxicity with hard drug design.

By searching the literatures, it was found that a total of 32 drugs interacting with herbal medicines in humans. These drugs mainly include anticoagulants (warfarin, aspirin and phenprocoumon), sedatives and antidepressants (midazolam, alprazolam and amitriptyline), oral contraceptives, anti-HIV agents (indinavir, ritonavir and saquinavir), cardiovascular drug (digoxin), immunosuppressants (cyclosporine and tacrolimus) and anticancer drugs (imatinib and irinotecan). Most of them are substrates for cytochrome P450s (CYPs) and/or P-glycoprotein (PgP) and many of which have narrow therapeutic indices. However, several drugs including acetaminophen, carbamazepine, mycophenolic acid, and pravastatin did not interact with herbs. Both pharmacokinetic (e.g. induction of hepatic CYPs and intestinal PgP) and/or pharmacodynamic mechanisms (e.g. synergistic or antagonistic interaction on the same drug target) may be involved in drug-herb interactions, leading of altered drug clearance, response and toxicity. Toxicity arising from drug-herb interactions may be minor, moderate, or even fatal, depending on a number of factors associated with the patients, herbs and drugs. Predicting drug-herb interactions, timely identification of drugs that interact with herbs, and therapeutic drug monitoring may minimize toxic drug-herb interactions. It is likely to predict pharmacokinetic herb-drug interactions by following the pharmacokinetic principles and using proper models that are used for predicting drug-drug interactions. Identification of drugs that interact with herbs can be incorporated into the early stages of drug development. A fourth approach for circumventing toxicity arising from drug-herb interactions is proper design of drugs with minimal potential for herbal interaction. So-called ”hard drugs” that are not metabolized by CYPs and not transported by PgP are believed not to interact with herbs due to their unique pharmacokinetic properties. More studies are needed and new approached are required to minimize toxicity arising from drug-herb interactions.

'Iron-saturated' lactoferrin is a potent natural adjuvant for augmenting cancer chemotherapy

Bovine lactoferrin (bLf), an iron-containing natural defence protein found in bodily secretions, has been reported to inhibit carcinogenesis and the growth of tumours. Here, we investigated whether natural bLf and iron-saturated forms of bLf differ in their ability to augment cancer chemotherapy. bLf was supplemented into the diet of C57BL/6 mice that were subsequently challenged subcutaneously with tumour cells, and treated by chemotherapy. Chemotherapy eradicated large (0.6 cm diameter) EL-4 lymphomas in mice that had been fed iron-saturated bLf (here designated Lf(+)) for 6 weeks prior to chemotherapy, but surprisingly not in mice that were fed lesser iron-saturated forms of bLf, including apo-bLf (4% iron saturated), natural bLf (approximately 15% iron saturated) and 50% iron-saturated bLf. Lf(+)-fed mice bearing either EL-4, Lewis lung carcinoma or B16 melanoma tumours completely rejected their tumours within 3 weeks following a single injection of either paclitaxel, doxorubicin, epirubicin or fluorouracil, whereas mice fed the control diet were resistant to chemotherapy. Lf(+) had to be fed to mice for more than 2 weeks prior to chemotherapy to be wholly effective in eradicating tumours from all mice, suggesting that it acts as a competence factor. It significantly reduced tumour vascularity and blood flow, and increased antitumour cytotoxicity, tumour apoptosis and the infiltration of tumours by leukocytes. Lf(+) bound to the intestinal epithelium and was preferentially taken up within Peyer's patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumour, including TNF, IFN-gamma, as well as nitric oxide that have been reported to sensitize tumours to chemotherapy. Importantly, it restored both red and white peripheral blood cell numbers depleted by chemotherapy, potentially fortifying the mice against cancer. In summary, bLf is a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy, but needs to be saturated with iron to be effective.

Benchmarking ventricular arrhythmias in the mouse—revisiting the ‘Lambeth Conventions’ 20 years on

The isolated Langendorff-mode perfused heart has become a valuable experimental model, used extensively to examine cardiac function, pathophysiology and pharmacology. For the clinical cardiologist an ECG is often a simple practicality, however in experimental circumstances, particularly with ex vivo murine hearts it is not always possible to obtain an ECG due to experimental recording constraints. However, the mechanical record of ventricular contractile function can be highly informative in relation to electrical state. It is difficult though to achieve consistency in these evaluations of arrhythmia as a validated common reference framework is lacking. In 1988, a group of investigators developed the ‘Lambeth Conventions’—a standardised reference for the definition and classification of arrhythmias in animal experimental models of ischaemia, infarction and reperfusion in vivo. Now, two decades later it is timely to revisit the Lambeth Conventions, and to update the guidelines in the context of the marked increase in murine heart study in experimental cardiac pathophysiology. Here we describe an adjunct to the Lambeth Conventions for the reporting of ventricular arrhythmias post-ischaemia in ex vivo mouse hearts when ECG recordings are not employed. Of seven discrete and identifiable patterns of mechanical dysrhythmia observed in reperfusion, five could be classified using conventional ECG terminology: ventricular premature beat, bigeminy, trigeminy, ventricular tachycardia and ventricular fibrillation. Two additional rhythm variations detected from the pressure record are described (potentiated contraction and alternans).