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Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology.

Studying the cross talk between nonpathogenic organisms and their mammalian hosts represents an experimental challenge because these interactions are typically subtle and the microbial societies that associate with mammalian hosts are very complex and dynamic. A large, functionally stable, climax community of microbes is maintained in the murine and human gastrointestinal tracts. This open ecosystem exhibits not only regional differences in the composition of its microbiota but also regional differences in the differentiation programs of its epithelial cells and in the spatial distribution of its component immune cells. A key experimental strategy for determining whether "nonpathogenic" microorganisms actively create their own regional habitats in this ecosystem is to define cellular function in germ-free animals and then evaluate the effects of adding single or several microbial species. This review focuses on how gnotobiotics-the study of germ-free animals-has been and needs to be used to examine how the gastrointestinal ecosystem is created and maintained. Areas discussed include the generation of simplified ecosystems by using genetically manipulatable microbes and hosts to determine whether components of the microbiota actively regulate epithelial differentiation to create niches for themselves and for other organisms; the ways in which gnotobiology can help reveal collaborative interactions among the microbiota, epithelium, and mucosal immune system; and the ways in which gnotobiology is and will be useful for identifying host and microbial factors that define the continuum between nonpathogenic and pathogenic. A series of tests of microbial contributions to several pathologic states, using germ-free and ex-germ-free mice, are proposed.

Animals↗

Nitrosative stress in the ER: a new role for S-nitrosylation in neurodegenerative diseases.

S-Nitrosylation, the covalent addition of a nitrogen monoxide group to a cysteine thiol, has been shown to modify the function of a broad spectrum of mammalian, plant, and microbial proteins and thereby to convey the ubiquitous influence of nitric oxide on cellular signal transduction and host defense. Accumulating evidence indicates that dysregulated, diminished, or excessive S-nitrosylation may be implicated in a wide range of pathophysiological conditions. A recent study establishes a functional relationship between inhibitory S-nitrosylation of the redox enzyme protein disulfide isomerase (PDI), defects in regulation of protein folding within the endoplasmic reticulum (ER), and neurodegeneration. Further, an examination of human brains afflicted with Parkinson's or Alzheimer's disease supports a causal role for the S-nitrosylation of PDI and consequent ER stress in these prevalent neurodegenerative disorders.

Animals↗

[Progress in the effect of probiotics on cholesterol and its mechanism].

Because in vitro studies have shown that bacteria can remove cholesterol from culture media, much attention has been given to the cholesterol-lowering potential of probiotics--especially Lactobacillus and Bifidobacterium, which survive passage through the gastrointestinal tract and have beneficial effects on the host. Recent research development concerning the in vitro and in vivo experimental data were reviewed in this paper, including relationship between microbial growth and removal of cholesterol, impact of pH, function of bile salts, BSH activities, prebiotics, feeding trial on human beings and animals. Moreover, some hypotheses on mechanism of cholesterol removal were discussed, such as co-precipitation with bile salts, deconjugation of BSH to bile salts, incorporation of cholesterol into the cellular membrane and microbial assimilation of cholesterol. In addition, the future research directions and the prospect of the probiotics were also viewed.

Animals↗

Impaired regulatory T cell function in germ-free mice.

Regulatory T cells (Treg) are crucial for the maintenance of tolerance to auto-antigens and harmless exogenous antigens. Here, we studied the role of the commensal microbiota for the development and function of Treg. CD4+CD25+ T cells were obtained from peripheral lymph nodes (PLN) and mesenteric lymph nodes (MLN) of germ-free (GF) and conventional (conv) NMRI mice and tested for phenotype and functional suppressive capacity. CD4+CD25+ T cells from GF mice showed a lower relative gene expression of fork head box p3 gene (Foxp3) and were not as potent suppressors in vitro as CD4+CD25+ T cells from conv animals. Intracellular staining for Foxp3 and CTLA-4 revealed proportional and regional differences in putative Treg subsets between conv and GF mice. Fewer of the CD4+CD25+ T cells in GF MLN expressed Foxp3 and CTLA-4, while the expression of these markers was similar amongst the CD4+CD25+ T cells in PLN of conv and GF mice. The largest difference between conv and GF Treg was observed in the liver draining celiac lymph node, where GF mice had fewer putative Treg as compared to conv mice. We propose that the presence of a microbial flora favors the development of a fully functional Treg population.

Animals↗

Impaired proliferative response to B-lymphocyte activators in common variable immunodeficiency.

The cell-mediated immune responses of 39 patients with common variable immunodeficiency (CVI) were studied in vitro, using Staphylococcus aureus and Escherichia coli prepared as whole cells and Candida albicans extract. These microbial activators wee found to require intact B-lymphocyte function for normal proliferative response. The patient group was observed to have significantly depressed lymphocyte responses compared wit those of controls studied in parallel (P less than 0.01). Negative lymphocyte response to one activator and strongly positive response to another were found in individual patients. Examination of patients' lymphocyte response to S. aureus and E. coli in association with serum IgG levels demonstrated that a rough correlation could be drawn, showing that patients with serum IgG less than 125 mg/dl had markedly lower (P less than 0.01) lymphocyte responses than those with serum IgG greater than 300 mg/dl. No similar correlation with phytohaemagglutinin activation was observed. Since depressed lymphocyte responses did not correlate with reduced B-cell number in these patients, intrinsic B-lymphocyte deficiency was indicated. These preparations of microbial activators are potentially useful tools in exploring lymphocyte subpopulation functions in primary immunodeficiency diseases.

Adolescent↗

The inflammatory reflex--introduction.

Sepsis is the third leading cause of death in the developed world. Despite recent advances in intensive care treatment and the discovery of antibiotics, sepsis remains associated with a high mortality rate. The pathogenesis of sepsis is characterized by an overwhelming systemic inflammatory response that is central to the development of lethal multiple organ failure. This volume of the Journal of Internal Medicine contains three reviews addressing novel aspects of a system we are only beginning to understand - the interactions between the immune and the nervous systems, the 'neuro-immune axis'. Tracey (Nature 2002; 420: 853) recently discovered that the nervous system, through the vagus nerve, can modulate circulating TNF-alpha levels induced by microbial invasion or tissue injury. This cholinergic anti-inflammatory pathway is mediated primarily by nicotinic acetylcholine receptors on tissue macrophages - the pathway leads to decreased production of proinflammatory cytokines. The author reports that treatment with the acetylcholine receptor agonist, nicotine, modulates this system and reduces mortality in 'established' sepsis. Watkins and Maier (J Intern Med 2005; 257: 139) illustrate that pathological pain (induced by inflammation) is not simply a strict neuronal phenomenon, but is a component of the immune response, and is modulated by peripheral immune cells and spinal cord glia cells. This may be of importance for future development of novel drugs for neuropathic pain as well as our understanding of increased risks for infections in anaesthetic skin areas. Blalock (J Immunol 1984; 132: 1067) elucidates the possibility that the immune system actually functions as the sixth sense, sensing microbes and microbial toxins that we cannot see, hear, taste, touch or smell. Activation of the sympathetic nervous system also has predominantly anti-inflammatory effects that are mediated through direct nerve to immune cell interaction or through the adrenal neuro-endocrine axis.

Autoimmunity↗

Microbial aspects of anaerobic BTEX degradation.

Combined with conventional methods, developments in both geochemical (delineation of redox processes) and molecular microbial methods (analysis of 16S rDNA genes and functional genes) have allowed us to study in details microorganisms and genes involved in the anaerobic degradation of benzene, toluene, ethylbenzene and xylene (BTEX) under specific redox conditions. This review summarizes recent research in this field. The potential for anaerobic BTEX degradation is widely spread. Specific groups of microorganisms appear to be involved in degradation under different redox conditions. Members of the Azoarcus/Thauera cluster perform BTEX degradation under denitrifying conditions, Geobacteraceae under Fe (III) reducing conditions and Desulfobacteriaceae under sulfate reducing conditions. The information so far obtained on biochemistry and molecular genetics of BTEX degradation indicates that each BTEX compound is funneled into the central benzyol-CoA pathway by a different peripheral pathway. The peripheral pathways of per BTEX compound show similarities among different physiological groups of microorganisms. We also describe how knowledge obtained on the microbial aspects of BTEX degradation can be used to enhance and monitor anaerobic BTEX degradation.

Bacteria, Anaerobic↗

Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix.

The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.

Animals↗

[Synthesis, function, and evolutionary origin of secondary metabolites produced by micro-organisms].

The microbial secondary metabolites are compounds with a wide range of chemical structures, produced mainly by actinomycetales and some fungi, usually in the late growth phase. Although a high proportion of this metabolites are antibiotics, there are also examples with pigment, herbicide and surfactant properties. Its function has been correlated to bacterial pathogenicity and cellular differentiation, however, properties dealing with chelation, hormonal and antitumor activities as well as nutritional reserve have been also reported. As in other examples of cellular compounds, the secondary metabolites are produced from low molecular weight precursors. For this purpose, specific biosynthetic pathways are utilized and regulated by processes which generally affect either the activity or the synthesis of the enzymes involved in it. Considering the secondary metabolites apparently are dispensable compounds, there are difficulties to explain their existence from an evolutionary point of view. Explanations to their existence have gone from laboratory artifacts to those conferring them an adaptative value in the past. It seems that they were maintained due to selective advantages to the producer microorganisms and probably, their sometimes complex biosynthetic pathways, have emerged from primary metabolites and evolved later independently by random mutation, amplification and genetic transfer.

Adaptation, Physiological↗

Use of a lux-marked rhizobacterium as a biosensor to assess changes in rhizosphere C flow due to pollutant stress.

The flow of carbon from plant roots into soil supports a range of microbial processes and is therefore critical to ecosystem function and health. Pollution-induced stress, which influences rhizosphere C flow is of considerable potential importance, and therefore needs to be evaluated. This paper reports on a method, based on reporter gene technology, for quantifying pollutant effects on rhizosphere C flow. The method uses the lux-marked rhizobacterium Pseudomonas fluorescens, where bioluminescence output of this biosensor is directly correlated with the metabolic activity and reports on C flow in root exudate. Plantago lanceolata was treated with paraquat (representing a model pollutant stress) in a simple microcosm system. The lux-biosensor response correlated closely with C concentrations in the exudate and demonstrated that the pollutant stress increased the C flow from the plantago roots, 24 h after application of the herbicide. The lux-reporter system therefore potentially offers a technique for use in assessing the impact of pollutant stress on rhizosphere C flow through the soil microbial biomass.

Carbon↗

Sensor molecules in intestinal innate immunity against bacterial infections.

PURPOSE OF REVIEW: Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-containing proteins are innate immune sensors for microbial signature molecules. This review highlights new insights into the functions of these sensors in intestinal physiology. RECENT FINDINGS: TLRs are membrane bound and survey the extracellular space for microbe-derived molecules, while NOD-containing proteins are cytoplasmic and detect microbial molecules in the cytoplasm. Most microbial sensors recognize components of the bacterial cell wall and its appendages. For example, TLR4 detects lipopolysaccharide in the Gram-negative bacterial cell wall. TLR5 recognizes flagellin, a component of bacterial flagella required for motility. NOD1 recognizes diaminopimelic acid-containing dipeptide or tripeptide motifs in the Gram-positive bacterial cell wall, while NOD2 detects muramyl dipeptide, a ubiquitous cell wall peptidoglycan motif. These sensors are important for host defense against gastrointestinal pathogens. Thus, TLR4 is required for Salmonella eradication, NOD1 contributes to controlling Helicobacter pylori infection, and NOD2 is involved in mucosal defense against Listeria monocytogenes. These sensors also regulate mucosal inflammation independent of pathogen infections. SUMMARY: Toll-like receptors and nucleotide-binding oligomerization domain-containing proteins not only play critical roles in host defense against known gastrointestinal bacterial pathogens, but also contribute to mucosal homeostasis in the apparent absence of such pathogens.

Animals↗

Concurrent stimulation of diflufenican biodegradation and changes in the active microbiome in gravel revealed by Total RNA.

The use of slowly degraded pesticides poses a particular problem when these are applied to urban areas such as gravel paths. The urban gravel provides an environment very different from agricultural soils; i.e., it is both lower in carbon and microbial activity. We, therefore, endeavored to stimulate the degradation of the pesticide diflufenican added to urban gravel microcosms amended with dry alfalfa to increase microbial activity. In the present study, alfalfa addition significantly increased the formation of diflufenican's primary metabolite, 2-[3-(trifluoromethyl)phenoxy]nicotinic acid (AE-B), indicating stimulated biotransformation. The concurrent changes of the active microbial communities within the gravel were explored using shotgun metatranscriptomic sequencing of ribosomal RNA and messenger RNA. Although bacterial taxa remained dominant (87.0%-98.5% relative abundance), the alfalfa treatment led to a 4-5-fold increase in eukaryotic groups, including fungi and microbial grazers. Several microbial taxa potentially involved in the degradation of complex carbon compounds and aromatic pollutants-including Bacteroidetes, Verrucomicrobia, Sordariomycetes, Mortierellales, Tremellales, Sphingopyxis, and Phenylobacterium-increased in relative abundance following alfalfa amendment. Functional gene profiling revealed elevated expression of genes related to microbial activity and biomass production. Genes with potential roles in the breakdown of complex carbon structures (e.g., xylanases/chitin deacetylases) and in the transformation of aromatic compounds (e.g., ring-cleaving dioxygenases) were revealed. We conclude that complex carbon amendments can enhance the microbial activity, promoting the biotransformation of diflufenican in urban gravel environments. These findings provide new insights into the interactions between microbial community dynamics, gene expression profiles, and pesticide biotransformation in non-agricultural matrices.IMPORTANCEPesticides used on urban areas, e.g., gravel paths, are likely to have different effects and fates than when these are used on agricultural soils. Hence, studies into the degradation of pesticides applied to urban matrices are needed. We have previously shown that metabolites of the persistent pesticide diflufenican are even more persistent in urban soils, and it has also previously been shown that these metabolites leach from gravel surfaces. The reasons behind this are that the urban gravel provides an environment very different from agricultural soils; i.e., it is both lower in carbon and microbial activity. In the present study, we, therefore, endeavored to stimulate the degradation of the pesticide diflufenican added to urban gravel microcosms amended with dry alfalfa to increase microbial activity, concurrently studying the changes in the active microbiome by Total RNA-metatranscriptomics.

Biodegradation, Environmental↗

A bacterial protease perturbs the paracellular barrier function of transporting epithelial monolayers in culture.

Tight junctions between cells and adhesion to the substratum maintain the barrier function of epithelia throughout the body. Damage to the epithelial barrier by microbial products allows penetration of bacteria and promotion of infection. We studied the effects of Pseudomonas elastase (PE) on the barrier function of epithelia by using Madin-Darby canine kidney (MDCK) epithelial cells; these cells form tight junctions (zonula occludens [ZO]) in vitro. PE decreased electrical resistance across the monolayers in a concentration- and time-dependent manner. Immunostaining of selected proteins of the ZO and zonula adherens was used to explore the effects of PE on junctional proteins. PE-treated monolayers of MDCK cells had markedly decreased immunostaining of ZO-1, a protein of the ZO, but light microscopy of PE-treated cells revealed no obvious morphologic changes. A chromium release assay indicated that, even with marked changes in transmonolayer electrical resistance, the permeability defect was not due to membrane disruption. Fluorescence staining of F-actin indicated diminution of cellular microfilaments in PE-treated cells, but E cadherin (uvomorulin), a protein of the zonula adherens, was unaffected by the enzyme. Elastases from porcine pancreas and human leukocytes with similar enzymatic activity (6 U/ml) did not decrease transmonolayer electrical resistance or degrade ZO-1. These results suggest that PE disturbs the barrier function of epithelial monolayers, in part, by changing the cell architecture and altering at least one protein of the ZO.

Animals↗

Histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow (Capricornis crispus, Artiodactyla, Mammalia) with special reference to glandular structures.

The histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow ( Capricornis crispus ) was studied by light microscopic histochemical methods, particularly lectin histochemistry. The eccrine glands present exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine, alpha-D-galactose and N-acetyl-neuraminic acid) especially in the cells of the secretory acini, the free surface of the collecting duct cells also showed distinct positive reactions with most of the histochemical methods. The thick epidermis of the nasolabial skin contained smaller amounts of glycoproteins. The results obtained are discussed with regard to possible functions of the glandular secretions. This substance mixture may particularly improve water retention on the skin surface, and protect against physical damage as well as microbial contamination. There seem to be no basic differences of muzzle functions between wild and domesticated bovine species.

Animals↗

An immunohistochemical study on organized lymphoid cell infiltrates in fetal and neonatal pancreases. A comparison with similar infiltrates found in the pancreas of a diabetic infant.

Lymphoid cell infiltrates were analyzed using immunohistochemical techniques on 5 normal fetal and 6 normal neonatal pancreases. Data were compared to data obtained analyzing the lymphoid cell infiltrates in the pancreas of an 8 months old diabetic infant. In the normal fetal and neonatal pancreases islets were intact and not infiltrated. In the diabetic infant beta-cells had vanished in almost all islets, the remaining islets showed a minor infiltration with primarily T-cells, a few B-cells, and some classical macrophages. It appeared that a widespread infiltration of the exocrine pancreas with single dendritic-like cells, and T-cells, and little clusters of these cells were normal features of fetal and neonatal pancreases. In the diabetic case these infiltrative patterns were more pronounced. Larger accumulations of such lymphoid cells could also be detected in the normal fetal and neonatal pancreases and these consisted mainly of T-cell zones, sometimes containing HEV's, with intermingled interdigitating dendritic cells and a few macrophages. This architecture is reminiscent of peripheral lymphoid tissue, such as bronchus-or gut-associated lymphoid tissue. The function of this fetal/neonatal intrapancreatic lymphoid tissue (which disappears in later life) is unknown. Various possibilities are suggested such as a yet unknown ubiquitous fetal/neonatal microbial infection, tolerance induction towards islet cell antigens, an endocrine regulatory function of infiltrated lymphoid cells, and a normal ontogenetic process.

Age Factors↗

[Physiology of microflora in the digestive tract].

The microflora of the digestive tract is a complex microbial ecosystem, well balanced, which in an aboral direction undergoes specific changes as to the ratio of aerobic and anaerobic microorganisms the functions of which supplement each other--the aerobes ensure for the whole ecosystem the scavenger effect. The microbial profile of the digestive tract is typical by the absence of anaerobic microorganisms in the stomach and conversely their absolute predominance in the distal colon. The basic physiological functions of the microflora of the digestive tract can be characterized as follows: 1. microbial barrier against pathogens and potential pathogens, 2. formation of products of the microflora and their influence on the blood supply of the intestinal mucosa and peristaltics, 3. stimulation of the immune system in the gut, 4. reduction of bacterial translocation, 5. production of vitamins. To this problem in the world literature, contrary to ours, deserved attention has been paid for many years. Knowledge of the problem opens the door to expedient manipulation with the microbial flora of the digestive tract by the use of diet or prebiotics, probiotics as well as antibiotic treatment. The author presents a summary of basic findings which developed on the subject of physiological microflora of the digestive tract successively up to their present shape.

Bacteria↗

Germ-free and conventional animal models for intestinal carbohydrate disposition.

Under conventional (CONV) conditions, the microbial flora is capable of breaking down most dietary derived carbohydrates as well as complex carbohydrates (glycolipids, glycoproteins, etc.) from the host. The major end products of these processes are short-chain fatty acids (SCFAs). In non-ruminants, most of the fermentation takes place in the large bowel. In ruminants, however, most acids are formed more proximal in the gastrointestinal (GI) tract. These microbial derived products may influence upon many host related functions, locally in the GI tract as well as elsewhere in the body. In germ-free (GF) animals, neglectable amounts of SCFAs are found in faeces. Parallel studies in GF and CONV animals given a standardized diet and ex-GF animals, receiving the same diet and mono/poly-associated with known microbial species, represent excellent models for answering such questions as (i) what can the microbes do? and (ii) what have the microbes done? These models allow detailed studies of the complex interplay between the host, his diet and his GI flora.

Animals↗

Microbial community structure and biomass in developing drinking water biofilms.

Traditional techniques to study microbes, such as culturable counts, microbial biomass, or microbial activity, do not give information on the microbial ecology of drinking water systems. The aim of this study was to analyze whether the microbial community structure and biomass differed in biofilms collected from two Finnish drinking water distribution systems (A and B) receiving conventionally treated (coagulation, filtration, disinfection) surface water. Phospholipid fatty acid methyl esters (PLFAs) and lipopolysaccharide 3-hydroxy fatty acid methyl esters (LPS 3-OH-FAs) were analyzed from biofilms as a function of water residence time and development time. The microbial communities were rather stabile through the distribution systems, as water residence time had minor effects on PLFA profiles. In distribution system A, the microbial community structure in biofilms, which had developed in 6 weeks, was more complex than those grown for 23 or 40 weeks. The microbial communities between the studied distribution systems differed, possibly reflecting the differences in raw water, water purification processes, and distribution systems. The viable microbial biomass, estimated on the basis of PLFAs, increased with increasing water residence time in both distribution systems. The quantitative amount of LPS 3-OH-FAs increased with increasing development time of biofilms of distribution system B. In distribution system A, LPS 3-OH-FAs were below the detection limit.

Bacteria↗