Intestinal Micro Research Articles

Effect of Dai-kenchu-to on Intestinal Micro Blood Flow

Effect of Dai-kenchu-to on Intestinal Micro Blood Flow

Conservation of mucosal associated invariant T (MAIT) cells and the MR1 restriction element in ruminants, and abundance of MAIT cells in spleen

MHC-related protein 1 (MR1) is a highly conserved MHC class I-like molecule. Human and murine mucosal associated invariant T (MAIT) cells are restricted by MR1 and express an invariant T cell receptor. Even though MR1 protein expression on the cell surface has not been demonstrated in vivo or ex vivo, it is assumed that MR1 presents a bacterial antigen from the intestinal lumen to MAIT cells because MAIT cells are present in the lamina propria and their expansion is dependent on the presence of intestinal micro flora. The existence of bovine MAIT cells and MR1 has been demonstrated recently although ovine MAIT cells and MR1 have not yet been described. We cloned bovine and ovine MR1 transcripts, including splice variants, and identified an anti human MR1 antibody that recognizes cells transfected with the bovine homolog. Using this antibody, no MR1 staining was detected using cells freshly isolated from blood, thymus, spleen, colon, ileum, and lymph node. MAIT cells are known to be enriched in the CD4/CD8 double negative peripheral blood T cell population, but their relative abundance in different tissues is not known. Comparison of the amount of MAIT cell-specific TCR transcript to the amount of constant α chain transcript revealed that numbers of MAIT cells are low in neonates and increase by 3-weeks of age. In 3-month old animals, MAIT cells are abundant in spleen and less so in ileum, peripheral blood, lymph node, colon, and thymus.

Inflammatory bowel disease in Asia

See article in J. Gastroenterol. Hepatol. 2010; 25: 453–468

Simultaneous determination of diosmin and diosmetin in human plasma by ion trap liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry: Application to a clinical pharmacokinetic study

Diosmetin (3′,5,7-trihydroxy-4′-methoxyflavone) is the aglycone of the flavonoid glycoside diosmin (3′,5,7-trihydroxy-4′-methoxyflavone-7-ramnoglucoside). Diosmin is hydrolyzed by enzymes of intestinal micro flora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the simultaneous determination of diosmin and diosmetin in human plasma was developed and validated. Plasma samples were incubated with β-glucuronidase/sulphatase. The analytes were isolated by liquid–liquid extraction with tert-butyl methyl ether at pH 2, and separated on a C 18 reversed-phase column using a mixture of methanol/1% formic acid (58:42, v/v) at a flow rate of 0.5 ml/min. APCI in the positive ion mode and multiple reaction monitoring (MRM) method was employed. The selected transitions for diosmin, diosmetin and the internal standard (7-ethoxycoumarin) at m/ z were: 609.0 → 463.0, 301.2 → 286.1 and 191, respectively. A good linearity was found in the range of 0.25–500 ng/ml ( R 2 > 0.992) for both compounds. The intra-batch assay precision (CV) for diosmin and diosmetin ranged from 1.5% to 11.2% and from 2.8% to 12.5%, respectively, and the inter-batch precision were from 5.2% to 11.5% and 8.5% to 9.8%, respectively. The accuracy was well within the acceptable range the accuracies (from −2.7% to 4.2% and −1.6% to 3.5% for diosmin and diosmetin, respectively). The mean recoveries of diosmin, diosmetin and the internal standard were 87.5%, 89.2% and 67.2%. Stability studies showed that diosmin and diosmetin were stable in different conditions. Finally, the method was successfully applied to the pharmacokinetic study of diosmin in healthy volunteers following a single oral administration (Daflon ®).

Immunomodulatory Effect of Probiotic Bacteria

Probiotics are usually defined as live microbial food ingredients beneficial to health which comprise of normal commensally bacteria as a part of the healthy human gut micro flora. The gut microflora is an important component of the gut defense barrier and have been shown to induce and maintain oral tolerance in experimental animal models. Functional foods, including probiotic bacteria, are an attractive medium for maintaining the steady nutritional state of the host with defective gut barrier functions. The gut-associated lymphoid tissue (GALT) embraces a crucial component of the total immunological capacity of the host in recognizing and selectively handling alien antigens for the initiation of immune responses. Normalization of increased intestinal permeability and altered gut micro ecology can ensure improvement of the function of the gut barrier. Probiotics do modify the composition of the gut microflora and, as a consequence, they have been shown to influence both intestinal and body functions. This review also discussed some patent related to the field.

Spirulina in Health Care Management

Spirulina is a photosynthetic, filamentous, spiral-shaped and multicellular edible microbe. It is the nature's richest and most complete source of nutrition. Spirulina has a unique blend of nutrients that no single source can offer. The alga contains a wide spectrum of prophylactic and therapeutic nutrients that include B-complex vitamins, minerals, proteins, gamma-linolenic acid and the super anti-oxidants such as beta-carotene, vitamin E, trace elements and a number of unexplored bioactive compounds. Because of its apparent ability to stimulate whole human physiology, Spirulina exhibits therapeutic functions such as antioxidant, anti-bacterial, antiviral, anticancer, anti-inflammatory, anti-allergic and anti-diabetic and plethora of beneficial functions. Spirulina consumption appears to promote the growth of intestinal micro flora as well. The review discusses the potential of Spirulina in health care management.

A validated HPLC determination of the flavone aglycone diosmetin in human plasma.

Diosmetin, 3',5,7-trihydroxy-4'-methoxy flavone, is the aglycone of the flavonoid glycoside diosmin that occurs naturally in foods of plant origin. Diosmin exhibits antioxidant and anti-inflammatory activities, improves venous tone and it is used for the treatment of chronic venous insufficiency. Diosmin is hydrolyzed by enzymes of intestinal micro flora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the determination of diosmetin in human plasma was developed and validated. Diosmetin and the internal standard 7-ethoxycoumarin were isolated from plasma by liquid-liquid extraction and separated on a C8 reversed-phase column with methanol-water-acetic acid (55:43:2, v/v/v) as the mobile phase at 43 degrees C. Peaks were monitored at 344 nm. The method was linear in the 10-300 ng/mL concentration range (r > 0.999). Recovery for diosmetin and internal standard was greater than 89.7 and 86.8%, respectively. Intra-day and inter-day precision for diosmetin ranged from 1.6 to 4.6 and from 2.2 to 5.3%, respectively, and accuracy was better than 97.9%.

Human Isolates of Lactic Acid Bacteria Differentially Affect Maturation and Cytokine Production by Human Dendritic Cells

The intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut‐associated immune system. Evidence indicates that lactic acid bacteria (LAB), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. Established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. The objective of this study is to evaluate the immunomodulating properties of a range of LAB of human origin. As dendritic cells (DCs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro‐generated immature DCs serve as a suitable model for studying the immunomodulating effects of lab. Human immature DCs were generated in vitro from monocytes and exposed to lethally UV‐irradiated LAB. The effect of various species of LAB on DCs in direct contact was evaluated. Furthermore, the maturation pattern of DCs separated from the bacteria by an epithelial cell layer (CaCo‐2 cells), which should mimic the intestinal environment, was studied. Cytokine secretion (IL‐12, IL‐10 and TNF‐α) and upregulation of maturation surface markers on DCs (CD83 and CD86) was measured. Different LAB induced diverse cytokine responses. Some strains were strong IL‐12 and TNF‐α inducers and others weak. All strains induced IL‐10. Different LAB also differentially modulated expression of CD83 and CD86 on DCs. Although some variation in the response to LAB of DCs generated from different blood donors was observed, general differences in the effect of the various LAB was revealed. Experiments with the DC CaCo‐2 coculture system are ongoing. Different species of LAB differentially affect DC maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response.

Axioms of mathe- matical immunology

Current wisdom describes the immune system as a defense against microbial pathogens. It is claimed that the virgin immune system has a capacity to produce antibodies against the entire antigenic universe. We assume, by contrast, that the responding capacity of the immune system is limited. Thus it cannot stand in readiness to deal with a practi- cally endless diversity and abundance of microbes. Axioms and theorems are suggested for a mathematician audience delineating how the immune system could use its limited resources economically. It is suggested that the task of the immune system is twofold: (i) It sustains homeostasis to preserve the genome by constant surveillance of the intracellular antigenic milieu. This is achieved by standardization of the T cell repertoire through a positive selection. The driving force of positive selection is immune cell survival. T cells must constantly seek contact with complementary MHC structures to survive. Such contact is based on molecular complementarity between immune cell receptors and MHC/self-peptide complexes. At the highest level of complementarity a local free energy minimum is achieved, thus a homeostatic system is created. Homeostatic interactions happen at intermediate afinity and are reversible. Alteration in the presented peptides typically decreases complementarity. That pushes the system away from the free energy minimum, which activates T cells. Complementarity is restored when cytotoxic T cells destroy altered (mutated/infected) host cells. (ii) B cells carry out an immune response to foreign proteins what requires a change in the genome. B cells raised under the antigenic in uence of the normal intestinal micro o- ra, self-proteins and alimentary antigens must go through a hypermutation process to be able to produce specific antibodies. It has a certain probability that hypermutation will successfully change the genome in some clones to switch from low afinity IgM antibody production to high afinity IgG production. Interactions (typically antibody antigen reac- tions) in an immune response happen at high afinity and are irreversible. High afinity clones will be selected, stimulated and enriched by the invading microbes. A complete account of the course of an infectious disease must also include a descrip- tion of the ecology of the immune response. It is therefore suggested that during prolonged interaction between host and infectious organism, carried on across many generations, the adaptive antibody population may facilitate the evolution of the natural antibody reper- toire, in accordance with the Baldwin effect in the evolution of instinct (see Appendix 6).

The Presence of a Cytochrome P450-like Protein in the Human Intestinal MicrofloraEubacterium aerofaciens

Eighteen human intestinal microbial strains were tested for the presence of cytochrome P450, an enzyme involved in detoxifying and toxifying various xenobiotics. The polymerase chain reaction (PCR) technique was used to amplify conserved regions within the gene. From the human intestinal micro ora Eubacterium aerofaciens , a DNA fragment of the expected size could be ampli. ed, for which a percentage homology of 20% was determined. Southern blot analysis, using Pseudomonas putida P450 CAM probes, showed reactions with probes at 45 and 55°C. The carbon monoxide (CO) difference spectrum produced a P450-like pro. le for E. aerofaciens , similar to P. putida P450 CAM. The presence of cytochrome P450 in human intestinal  ora may in uence the expression of hepatic cytochrome P450s. Keywords : human intestinal  ora, cytochrome P450, PCR, CO difference spectrum.

コーヒーマンナン由来マンノオリゴ糖の腸内細菌資化性

A mannooligosaccharide mixture was obtained by hydrolysis of spent coffee grounds. Furthermore, beta-1,4-D-mannobiose, beta-1,4-D-mannotriose, beta-1,4-D-mannotetraose, and beta-1,4-D-mannopentaose were fractionated by active carbon chromatography from this mixture. Each mannooligosaccharide were investigated for its effect on the growth of established enterobacterial strains. Regardless of the mannooligosaccharide molecular weight, all mannooligosaccharides were used by Bifidobacterium adolescentis, Lactobacillus acidophilus, and Lactobacillus gasseri. On the other hand, bad bacteria such as Clostridium perfringens and Escherichia coli that produce mutagenic substances could not use mannooligosaccharides. Therefore it could be expected that mannooligosaccharides had a potential to promote the improvement of healthful human intestinal micro flora as prebiotics.

Microbial Modulation of Host Intestinal Glycosylation Patterns

The host and its intestinal microora function together as a complex ecologic system in which there is a significant impact of the intestinal flora on the host. The structure and functioning of the gastrointestinal tract are dependent upon the presence of the resident and transient flora. The inuence of the microflora on the secretory pattern of the intestinal glycoconjugates is an emerging field of research. This review summarizes and examines the evidence that commensal intestinal bacteria can modulate the host intestinal glycosylation patterns. Changes in the glycosylation patterns might be associated with the induction and/or degradation of glycans, and are usually studied using either in vitro or in vivo models. In vitro microbial modulation of the intestinal glycosylation remains largely related to the degradation of glycoconjugates. On the other hand, most of the in vivo studies are associated with the induction of particular glycan structures. These two processes are associated but often analysed independently. Likewise, there are few studies involving the same bacterial species and for which both in vitro and in vivo approaches are used. On-going research should focus both the induction and degradation of glycoconjugates and the use of in vitro and in vivo models in a combined manner. The biological role of alteration in complex carbohydrate expression has yet to be determined. Due to their localisation at the cell surface in the gastrointestinal tract, glycans act as mediators of many host-microbial interactions, providing a wide range of binding sites for bacteria, pathogens and toxins. The knowledge of host receptors and their possible microbial modulation may lead to improved means of control and treatment of bacterial infections. Keywords: host-microbial interactions, intestinal glycosylation, in vivo , in vitro , commensal microflora.

Development and Function of Intestinal B and T Cells

The gastrointestinal immune system has evolved under the dual evolutionary pressure of preventing bacterial invasion along its extensive epithelial surfaces, while permitting absorption of nutrients. Accordingly, the development, selection and function of mucosal immune cells differ from comparable characteristics of lymphoid cells in other peripheral tissues. In the normal state, intestinal immune responses are aiming at exclusion of antigens – that is, preventing translocation of intestinal microorganisms and dangerous immunogenic substances such as bacterial toxins into the lamina propria. Furthermore, presentation of soluble food proteins and components of the autologous intestinal microora to mucosal T and B cells, apparently results in immunological downregulation as well as active suppression of systemic immune responses, a phenomenon termed mucosal or ‘oral’ tolerance. The normal microbial flora has a major impact on the pool size, composition, antigen recognition repertoire, and effector function of mucosal immune cells. Local antibody responses are characterized by differentiation of B lymphocytes to lamina propria plasma cells which produce polymeric immunoglobulins, a process strictly regulated by mucosal T cells. Polymeric immunoglobulins (e.g., dimeric IgA and pentameric IgM) are actively transported by a specific receptor across the epithelial barrier into the gut lumen where they can bind bacteria and soluble antigens, thereby preventing them from adhering to, and penetrating, the mucosal surface. A major population of intestinal T cells is located in the surface epithelium, thus being strategically situated at the interface between the gut lumen and the interior of the body. However, their precise role in microbial defence has not yet been established. It is remarkable how the immune system normally maintains the balance between exclusion of (but coexistence with) the commensal gut bacteria, while protecting against (and eventually eliminating) intestinal pathogens. This immune balance appears to be tightly regulated by intestinal T cells. Recent findings in various genetically manipulated mouse models suggest that a dysregulated T-cell response against the normal intestinal microflora may lead to progressive inflammatory disease in the gut. This adverse development appears to reflect abrogation of oral tolerance and may be a central mechanism in the pathogenesis of inflammatory bowel disease. Also the development of atopic allergy appears to be influenced by immunoregulatory mechanisms on which the intestinal microflora exerts a substantial impact in terms of both mucosal barrier maintenance, oral tolerance and a balanced cytokine profile. Keywords: intestinal microflora, secretory immunity, oral tolerance, intraepithelial lymphocytes, gut disease.

Modulation of Intestinal Microflora by Specific Dietary Components

This review focuses on the specific effects of nutrients on the colonic microora with special emphasis on how human milk and its components influence the intestinal microora in early infancy compared with formula-fed infants. Some proteins such as lactoferrin and lysozyme and some casein and whey peptides, as well as non-protein nitrogen derivatives, particularly nucleotides, influence the growth of bifidobacteria. In addition, β -lactose and milk oligossaccharides, together with milk gangliosides, seem to affect the intestinal colonisation of the infant and the establishment of a particular microflora. Specific effects of protein and nitrogen derivatives, carbohydrates, lipids and some minerals on colonic microorganisms and how this affects health are also considered. There is an increasing interest in how non-absorbable carbohydrates affect the colonic microecology and how they may have a role in the maintenance of human health. However, there are only few data on how other nutrients, namely non-protein nitrogen derivatives, lipids, macrominerals, microminerals, and other non-nutrient components of foods may affect individual colonic microorganisms. Further research in these areas is highly recommended.

Influence of Dietary Compounds on Intestinal Immunity

This review focuses on the specific effects of nutrients on the intestinal immune system. The intestinal immune system possesses a number of special characteristics different from those of the systemic immune system, which are summarised in this report. Although some of these characteristics are well known, there is a big gap regarding the specific mechanisms that regulate immunity at intestinal level with respect to the systemic immunity. Because of that, during the last years, there is an increasing interest in elucidating the inner mechanisms that regulate the intestinal immune function, as well as how specific nutrients influence that function. We have reviewed this topic with special emphasis on how human milk and its components influence the development of the intestinal immunity in early infancy compared with formula-fed infants. Interactions between nutrients and the intestinal microora have also been reviewed. Some micronutrients, which are present in human milk and in most of foods, are capable of influencing immune functioning at very low concentrations. The specific action of these micronutrients on some parameters of the intestinal immunity as well as the potential mechanisms of action promoted by them have been considered more in detail. However, few data exist on how other specific nutrients, namely protein and non-protein compounds, lipids, carbohydrates, and others, such as mineral and vitamins, influence the intestinal immunity. Further research in these areas is highly recommended.

Panel Discussion I: Microbiological Endpoints and Public Health Concern

Dr. Cerniglia: We heard the results of studies using both an in vitro and an in vivo models for evaluating the impact of antimicrobial residues on the human intestinal micro flora. Various microbiological endpoints were measured in these models and the panelists will discuss the public health impact of these endpoints. We hope to agree on what should be measured, what is relevant, and what is the magnitude of change that it is really indicative of a human adverse health effect.

Interactions of constitutive nitric oxide with PAF and thromboxane on rat intestinal vascular integrity in acute endotoxaemia

1. The involvement of endogenous platelet activating factor (PAF) and thromboxane A2 in the acute microvascular damage in the ileum and colon induced by the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) following endotoxin administration was investigated in the rat over a 1 h period. 2. Administration of L-NAME (1-10 mg kg-1, s.c.) concurrently with E. coli lipopolysaccharide (LPS; 3 mg kg-1, i.v.) dose-dependently increased vascular permeability in the ileum and colon, as determined by the leakage of radiolabelled albumin, and caused macroscopic mucosal damage in the ileum determined 1 h later. Neither LPS administration nor L-NAME (5 mg kg-1) alone affected resting vascular permeability. 3. Infusion of phenylephrine (10 micrograms kg-1 min-1, i.v. for 1 h) caused an elevation in blood pressure similar to that found following L-NAME administration (5 mg kg-1, i.v. or s.c.), but did not increase intestinal vascular permeability, when administered with LPS (3 mg kg-1, i.v.). 4. The increased vascular permeability in the ileum and colon and macroscopic damage in the ileum, induced by L-NAME (5 mg kg-1, s.c.) and LPS (3 mg kg-1, i.v.) was dose-dependently inhibited following s.c. pretreatment (15 min before challenge) with the thromboxane synthase inhibitors, OKY 1581 (5-25 mg kg-1) or 1-benzyl-imidazole (1-50 mg kg-1), or with the thromboxane receptor antagonist, BM 13177 (0.2-2 mg kg-1). 5. Pretreatment with the cyclo-oxygenase inhibitor, indomethacin (2-5 mg kg-', s.c., 15 min before challenge) reduced the microvascular injury in the ileum and colon and macroscopic lesions in the ileum,observed after the concurrent administration of L-NAME and LPS.6. Pretreatment (15 min) with the PAF-receptor antagonists, WEB 2086 (0.5-1 mg kg-', s.c.) or BN52021 (2.5-10 mg kg-', s.c.) likewise attenuated this intestinal vascular injury.7. Combined administration of low doses of l-benzyl-imidazole (1 mg kg-') with WEB 2086(0.5 mg kg-')15 min before L-NAME and LPS challenge, abolished this vascular damage and macroscopic injury.8. These results suggest that PAF and thromboxane A2 are released acutely following challenge with a low dose of endotoxin. However, these mediators do not appear to injure the intestinal micro vascular bed unless NO synthase is concurrently inhibited. Such findings support the protective role of constitutively-formed NO, counteracting the injurious vascular actions of cytotoxic mediators released under pathological conditions.

Antibiotic Associated Hypoprothrombinemia

DOI: http://dx.doi.org/10.5915/17-2_3-12755 Vitamin K is an important substance in synthesis of several clotting factors, most important of which is prothrombin. Many antibiotics have effects on intestinal micro organisms which are necessary for synthesis of Vitamin K and thus formation of essential clotting factors is interfered with. This antibiotic associated hypoprothrombinemia can lead to hemorrhage. Clinical set up for such a happening is generally easily recognized. Management includes parenteral administration of Vitamin K, plasma transfusion, or Prothrombin Complex in serious hemorrhagic conditions. Antibiotic associated hypoprothrombinemia can be prevented by prophylactic use of Vitamin K in situations likely to result in bleeding, e.g. post-operative conditions.