Amy Murphy

Changes in barrier function of a model intestinal epithelium by intraepithelial lymphocytes require new protein synthesis by epithelial cells.

BACKGROUND: Elements of the mucosal immune system may play an important part in regulating epithelial barrier function in the intestinal tract. Intraepithelial lymphocytes (IELs) represent a subtype of immunocyte which is strategically placed to regulate epithelial function at most mucosal sites. AIMS AND METHODS: An IEL derived cell line (SC1) was used to examine its effects on the model epithelium T84--a tumour derived cell line which retains the phenotype of colonic crypt cells. Transepithelial electrical resistance (TER) was used as a marker of epithelial integrity. RESULTS: Coculture of T84 cells with SC1 produced a significant fall in TER as did exposure of T84 monolayers to IEL derived supernatant. Recombinant interferon-gamma (rIFN gamma) also reduced TER in T84 monolayers. Cycloheximide prevented the effects of IEL supernatant and of rIFN gamma on TER. The fall in TER in response to rIFN gamma was attenuated by blocking antibodies, which did not alter the fall in resistance induced by IEL supernatant. Fractions of IEL supernatant, separated on the basis of size, evoked temporally distinct changes in TER. Ultrastructural studies support the hypothesis that the slow onset but severe fall in TER indicates catastrophic effects on the monolayer. The more rapid onset fall in TER was not associated with gross changes in monolayer morphology. Reduction of TER by IEL supernatant was not influenced by inhibitors of tyrosine phosphatase or of protein kinase C. Although herbimycin did reduce the rapid onset change in TER, the tyrosine kinase inhibitor genistein did not alter responses to IEL supernatant. CONCLUSIONS: Mucosal T cells may influence barrier function by a process involving new protein synthesis by epithelial cells. This model may have relevance in some inflammatory conditions of the gastrointestinal tract.

Revisiting Metchnikoff: Age-related alterations in microbiota-gut-brain axis in the mouse

Over the last decade, there has been increased interest in the role of the gut microbiome in health including brain health. This is by no means a new theory; Elie Metchnikoff proposed over a century ago that targeting the gut by consuming lactic acid bacteria such as those in yogurt, could improve or delay the onset of cognitive decline associated with ageing. However, there is limited information characterising the relationship between the behavioural and physiological sequelae of ageing and alterations in the gut microbiome. To this end, we assessed the behavioural, physiological and caecal microbiota profile of aged male mice. Older mice (20-21months old) exhibited deficits in spatial memory and increases in anxiety-like behaviours compared to younger mice (2-3months old). They also exhibited increased gut permeability, which was directly correlated with elevations in peripheral pro-inflammatory cytokines. Furthermore, stress exacerbated the gut permeability of aged mice. Examination of the caecal microbiota revealed significant increases in phylum TM7, family Porphyromonadaceae and genus Odoribacter of aged mice. This represents a shift of aged microbiota towards a profile previously associated with inflammatory disease, particularly gastrointestinal and liver disorders. Furthermore, Porphyromonadaceae, which has also been associated with cognitive decline and affective disorders, was directly correlated with anxiety-like behaviour in aged mice. These changes suggest that changes in the gut microbiota and associated increases in gut permeability and peripheral inflammation may be important mediators of the impairments in behavioural, affective and cognitive functions seen in ageing.

Expression of CD44 on rheumatoid synovial fluid lymphocytes.

OBJECTIVES--To investigate the involvement of the adhesion molecule CD44 in the homing of lymphocytes to synovial tissue, by examining the density of expression and molecular mass of CD44 on rheumatoid synovial fluid lymphocytes. METHODS--Twenty patients with rheumatoid arthritis were studied. Peripheral blood and synovial fluid lymphocytes were isolated by Ficoll-Hypaque sedimentation. CD44 expression was analysed by two colour flow cytometry of CD3 positive T lymphocytes with calculation of mean fluorescence intensity. Expression of activation markers M21C5, M2B3, interleukin (IL)-2 receptor and transferrin receptor was quantitated. In addition, CD44 molecular mass was examined by Western blot in six patients. RESULTS--CD44 expression was markedly increased on synovial fluid T lymphocytes of rheumatoid patients relative to peripheral blood lymphocytes from the same individuals. CD44 molecular mass on peripheral blood mononuclear cells was 88 kDa, but that on synovial fluid lymphocytes was only 83 kDa. CD44 expression correlated significantly with expression of activation markers M21C5, M2B3, and the IL-2 receptor. CONCLUSIONS--Alterations in density of expression or of the molecular mass of CD44 could contribute to local tissue injury, either directly by facilitating adhesion, or indirectly through effects on other adhesion molecules.

Pilot translational study of dietary vitamin C supplementation in Barrett's esophagus

The transcription factor Nuclear factor kappa B (NF-kappaB) is central to the regulation of genes encoding for mediators of inflammation and carcinogenesis. In the esophagus, NF-kappaB is progressively activated from inflammation to Barretts metaplasia and adenocarcinoma. Vitamin C, an antioxidant, can inhibit NF-kappaB in in vitro models, and the aim of this study was to prospectively assess the effect of supplemental vitamin C on NF-kappaB and associated cytokines in patients with Barretts esophagus. Twenty-five patients with long-segment Barretts and specialized intestinal metaplasia received dietary vitamin C (1000 mg/day) orally for four weeks, and had pre- and post-vitamin C endoscopic biopsies. NF-kappaB activity (activated p50 and p65 subunits) of nuclear extracts was assessed using the Active Motif NF-kappaB assay, and cytokines and growth factors were measured using the Evidence Investigator biochip array. NF-kappaB and related pro-inflammatory cytokines and growth factors (IL-8, VEGF, IL-10) were activated in all Barretts tissue pre-treatment. Down-regulation in activated NF-kappaB and cytokines was observed in 8/25 (35%) patients. Dietary vitamin C supplementation may down-regulate pro-inflammatory markers in a subset of Barretts patients. Further studies with larger numbers of endpoints will be needed to further evaluate this effect.

Tyrosine phosphorylation in the human duodenum.

Many growth factor receptors including the epidermal growth factor receptor function through tyrosine kinase activity. The aim of this study was to examine the constitutive level of tyrosine phosphorylation in the normal duodenum and in the hyperproliferative coeliac duodenum. A flow cytometric assay was devised using monoclonal antibody to phosphorylated (but not native) tyrosine residues to determine the levels of tyrosine phosphorylation in both CD3 positive intraepithelial lymphocytes and CD3 negative epithelial cells obtained by EDTA treatment of endoscopically obtained duodenal biopsy specimens. In addition, immunohistochemistry was performed on 18 formalin fixed coeliac duodenal biopsy specimens and eight control specimens. Tyrosine phosphorylation could be detected by flow cytometry on duodenal enterocytes and this expression was up regulated by pretreatment with epidermal growth factor. Tyrosine phosphorylation decreased with progression from the villus to the crypt, however, and was virtually undetectable on crypt enterocytes. Immunohistochemistry of the coeliac duodenum showed virtually absent tyrosine phosphorylation in the crypt. Increased tyrosine phosphorylation was detected in the infiltrating T cells. In conclusion, tyrosine phosphorylation in the duodenum is confined to the non-proliferative villous epithelium and is virtually undetectable in the proliferative crypt compartment. These findings suggest that tyrosine kinase activity is not a significant factor in the regulation of crypt cell proliferation in the human duodenum either in normal subjects or in coeliac disease patients.

Dietary Supplementation with a Magnesium-Rich Marine Mineral Blend Enhances the Diversity of Gastrointestinal Microbiota

Accumulating evidence demonstrates that dietary supplementation with functional food ingredients play a role in systemic and brain health as well as in healthy ageing. Conversely, deficiencies in calcium and magnesium as a result of the increasing prevalence of a high fat/high sugar “Western diet” have been associated with health problems such as obesity, inflammatory bowel diseases, and cardiovascular diseases, as well as metabolic, immune, and psychiatric disorders. It is now recognized that modulating the diversity of gut microbiota, the population of intestinal bacteria, through dietary intervention can significantly impact upon gut health as well as systemic and brain health. In the current study, we show that supplementation with a seaweed and seawater-derived functional food ingredient rich in bioactive calcium and magnesium (0.1% supplementation) as well as 70 other trace elements, significantly enhanced the gut microbial diversity in adult male rats. Given the significant impact of gut microbiota on health, these results position this marine multi-mineral blend (MMB) as a promising digestive-health promoting functional food ingredient.

Chapter 5 – Probiotics as Curators of a Healthy Gut Microbiota: Delivering the Solution

Abstract In recent years, the influence of the gut microbiota on many aspects of human health has become clear. Accumulating evidence suggests that perturbations of the gut microbiota have consequences that extend beyond the gastrointestinal tract and have been associated with the development of several immunological, metabolic, and mental disorders. Consequently, the use of probiotics to modulate the gut microbiota has gained significant interest. In this review we describe a typical healthy gut microbiota composition in infants and adults and the factors known to cause disturbances. Strategies to restore the gut microbiota using probiotics are discussed, with examples of food- and nonfood-based probiotic carriers. The potential of probiotic therapy for various conditions is evident; however, further research is necessary to develop successful probiotic treatments focused on modulating the gut microbiota to maintain health or alleviate disease.