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The intestinal microflora regulates cytokine production positively in spleen-derived macrophages but negatively in bone marrow-derived macrophages.

Besides its role as a barrier against potential pathogens, intestinal flora is presumed to protect the host by priming the immunological defense mechanisms. In this respect, the influence of intestinal flora on macrophage precursors was examined, and its modulating effect was compared on LPS-induced cytokine production by macrophages derived from bone marrow and spleen precursors (BMDM and SDM respectively). The regulation of IL-1, IL-6, TNF-alpha and IL-12 production in macrophages from germ-free and from three groups of flora-associated mice, conventional, conventionalized and E. coli-mono-associated mice, was investigated. The whole flora inhibited IL-1, TNF-alpha and IL-12 secretion by BMDM, whereas it had a stimulatory effect on IL-12 secretion by SDM. Implantation of E. coli alone enhanced cytokine secretion by BMDM but had a more limited effect than whole flora on SDM, enhancing only TNF-alpha and IL-12 secretion. Study of expression of mRNA showed a correlation with protein secretion for IL-6 but not for TNF-alpha and IL-1. IL-12 enhancement in BMDM seemed to be dependent on regulation of p35 mRNA expression while it was correlated to increased p40 mRNA expression in SDM. The results demonstrated that intestinal flora modulated bone marrow and spleen macrophage cytokine production in a differential manner and suggested a role for bacteria other than E. coli among the whole flora. The contrasting effects exerted by the intestinal flora on bone marrow and spleen precursors are an interesting observation in view of the different functions of these organs in immunity. The finding that intestinal flora enhanced IL-12 production in spleen is also potentially important since this cytokine is implicated in the determination of the relative levels of Th1 and Th2 responses and plays a pivotal role in host defense against intracellular microorganisms.

Animals↗

[The diagnosis of campylobacteriosis].

Results of assays for Campylobacter in patients with intestinal dysfunction are presented for the period from 1984 to 1991. 4147 cases were analyzed in 144 of which (3%) Campylobacter was detected in feces. Children constituted 12% of this group. They were distributed by age in the following way: 1 year and younger--1%, from 1 to 2 years--4.2%, from 3 to 6 years--3.8%, from 7 to 16 years--2.3%. 15 Campylobacter-positive patients were established to discharge other enteropathogenic microorganisms. 4% of children suffered from severe form of campylobacteriosis associated with exsicosis, 80% had moderate form of the disease. In order to study reservoirs of the infection 102 samples of intestinal content in livestock were analyzed for Campylobacter 57.1% of which proved to be positive.

Acute Disease↗

Effects of iron and phytic acid on production of extracellular radicals by Enterococcus faecalis.

Enterococcus faecalis is a human intestinal commensal that produces extracellular superoxide, hydrogen peroxide, and hydroxyl radical while colonizing the intestinal tract. To determine whether dietary factors implicated in colorectal cancer affect oxidant production by E. faecalis, radicals were measured in rats colonized with this microorganism while on diets supplemented with iron or phytic acid. Hydroxyl radical activity was measured by assaying for aromatic hydroxylation products of D-phenylalanine using reverse-phase high-performance liquid chromatography and electrochemical detection. In vitro, as expected, iron enhanced, and phytic acid decreased, hydroxyl radical formation by E. faecalis. For rats colonized with E. faecalis given supplemental dietary iron (740 mg elemental iron as ferric phosphate per kg diet) or phytic acid (1.2% w/w), no differences were found in concentrations of urinary ortho- or meta- isomers of D-phenylalanine compared to rats on a basal diet. Aqueous radicals in colonic contents were further assessed ex vivo by electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide as a spin trap. Mixtures of thiyl (sulfur-centered) and oxygen-centered radicals were detected across all diets. In vitro, similar spectra were observed when E. faecalis was incubated with hydrogen sulfide, air-oxidized cysteine, or an alkylsulfide, as typical sulfur-containing compounds that might occur in colonic contents. In conclusion, intestinal colonization with E. faecalis in a rat model generates both thiyl and oxygen-centered radicals in colonic contents. Radical formation, however, was not significantly altered by short-term dietary supplementation with iron or phytic acid.

Animals↗

Double-blind, placebo-controlled study comparing the effect of azithromycin with clarithromycin on oropharyngeal and bowel microflora in volunteers.

The purpose of this double-blind study was to assess the effect of azithromycin and clarithromycin on oral and fecal microflora. Bacterial species from fecal samples and throat washes from healthy volunteers were identified and quantified before, during and after receipt of either placebo ( n=6), azithromycin (500 mg once daily for 3 days; n=6) or clarithromycin (500 mg twice daily for 7 days; n=6). In both antibiotic groups, the changes in oropharyngeal aerobic microflora following antibiotic administration were minor. Antibiotics neither changed the bacterial load of Streptococcus spp. compared with placebo, nor did macrolide-resistant streptococci emerge. In the fecal aerobic microflora, the number of organisms of the family Enterobacteriaceae decreased slightly after antibiotic administration in both the clarithromycin and the azithromycin groups, but levels normalized by day 21 after therapy. No colonization with nonfermenters or Clostridium difficile was seen, and the total number of anaerobic bacteria was not affected in any study group. In conclusion, there were no significant differences between azithromycin and clarithromycin in their effect on human oropharyngeal and intestinal microflora, nor was the use of these antibiotics associated with colonization by resistant, gram-positive organisms or overgrowth of opportunistic microorganisms.

Adolescent↗

Lactobacillus casei DN-114 001 inhibits the increase in paracellular permeability of enteropathogenic Escherichia coli-infected T84 cells.

Probiotics are living microorganisms which, when ingested in adequate amounts, exert health benefits toward the host. For instance, probiotics might act through reinforcement of the intestinal epithelial barrier function. The goal of the present study was to determine whether Lactobacillus casei DN-114 001 could abrogate the increase in paracellular permeability induced by enteropathogenic Escherichia coli. We used the human colon T84 cell line infected with a wild-type enteropathogenic E. coli (strain E2348/69). Paracellular permeability was followed by monitoring transepithelial electrical resistance variations and by observing zonula occludens-1 distribution. Two infection procedures were used: co-incubation (the pathogenic and probiotic strains were simultaneously incubated with T84 cells) and post-infection (the probiotic was added in the presence of pathogenic bacteria 3 h after the beginning of the infection). We also investigated the effect of L. casei on enteropathogenic E. coli adhesion. L. casei DN-114 001 inhibited, in a dose-dependent-manner, the decrease in enteropathogenic E. coli-induced transepithelial electrical resistance and zonula occludens-1 redistribution using two different infection procedures. However, L. casei did not inhibit pathogenic strain adhesion. L. casei DN-114 001 inhibited the increase in EPEC-induced paracellular permeability. This property could partially explain the previously observed health benefits of this probiotic for human natural defenses, such as those associated with prevention of diarrhea.

Bacterial Adhesion↗

Cell culture modeling of specialized tissue: identification of genes expressed specifically by follicle-associated epithelium of Peyer's patch by expression profiling of Caco-2/Raji co-cultures.

Peyer's patch follicle-associated epithelium (FAE) regulates intestinal antigen access to the immune system in part through the action of microfold (M) cells which mediate transcytosis of antigens and microorganisms. Studies on M cells have been limited by the difficulties in isolating purified cells, so we applied TOGA mRNA expression profiling to identify genes associated with the in vitro induction of M cell-like features in Caco-2 cells and tested them against normal Peyer's patch tissue for their expression in FAE. Among the genes identified by this method, laminin beta3, a matrix metalloproteinase and a tetraspan family member, showed enriched expression in FAE of mouse Peyer's patches. Moreover, the C. perfringens enterotoxin receptor (CPE-R) appeared to be expressed more strongly by UEA-1(+) M cells relative to neighboring FAE. Expression of the tetraspan TM4SF3 gene and CPE-R was also confirmed in human Peyer's patch FAE. Our results suggest that while the Caco-2 differentiation model is associated with some functional features of M cells, the genes induced may instead reflect the acquisition of a more general FAE phenotype, sharing only select features with the M cell subset.

Animals↗

[Anthrax due to deliberate infection].

Anthrax is a zoonosis which is particularly prevalent in cattle, goats and sheep and is caused by Bacillus anthracis, a Gram-positive spore forming aerobic microorganism. The endospores can survive outside of the body for many decades. The natural form of anthrax has a cutaneous, pulmonary and intestinal form. The pulmonary form can be rapidly fatal but is difficult to recognise due to an initially non-specific, flu-like clinical picture. As a result of spores being inhaled, a mediastinal lymphadenitis arises from which a systemic disease develops with a violent toxaemia, damage to the vascular endothelium, oedema, internal haemorrhages and circulatory collapse. Anthrax is diagnosed by demonstrating the presence of the bacteria in the cutaneous abnormality, in blood or another sterile body component such as cerebrospinal fluid, by means of a direct preparation, immunofluorescence or surface antigens, molecular diagnostics with PCR, or by means of culturing. B. anthracis is sensitive to quinolones, clindamycin and tetracyclines, and often to penicillin. Although naturally acquired cutaneous anthrax can be effectively treated with a short antibiotic cure, it is nevertheless advised in the USA to complete the full 60-day cure and to regard the cutaneous manifestation as a telltale sign of possible respiratory exposure. Anthrax is not transmitted from one person to another.

Adult↗

[Essential oils for the nutrition of poultry, swine and ruminants].

Essential oils are very complex mixtures of volatile, lipophilic compounds originating from plants. Due to their lipophility they posses a good intestinale and percutane absorption. Under external application essential oils demonstrate antiphlogistic or rubefacient to pro-inflammatority activities. Orally intake they stimulate the secretion of digestive enzymes and increase gastric and intestinal motility. Moreover they show spasmolytic, expectorative and diuretic activities. Besides antimicrobial properties on bacteria and fungi have been observed in vitro and in vivo. Due to their various effects essential oils increasingly gain attention in animal nutrition and are discussed to be alternatives to antibiotic growth promoters. In some studies essential oils have been reported to reduce intestinal pathogens in broilers and piglets. When administered to ruminants essential oils decrease ruminal ammonia production by suppression the growth of deaminative microorganisms. However, the observed effects on growth performance are inconsistent among studies. In conclusion, there are still some unanswered questions concerning the mode of action, metabolic pathway and optimal dosage of essential oils in different animal species. Further scientific research is therefore needed to use essential oils effectivly in livestock feeding.

Animal Feed↗

[A study of human fecal microbiocenosis by experimental in vitro modeling of constipation].

A study of human fecal microbiocenosis using experimental in vitro modeling of constipation was carried out. The study revealed certain dynamics of the incidence, proportion and ratio of microorganisms isolated from the feces at various stages of cultivation. The character of the changes of these parameters depended on the type of the microorganism. According to the results of the experiment, transitory flora (staphylococci; candidas) begins to disappear within the first day of cultivation. If intestinal evacuation is retarded for more than 2 or 3 days, distortion of the microbiological characteristics of the residential microflora, bifidobacteria and escherichiae, is possible. These processes may lead to a false positive result of dysbiosis analysis. The authors conclude that dysbiotic changes in constipation are caused by fecal retention. That is why correct diagnostics of dysbiosis is possible only after regular everyday stool is reestablished.

Adult↗

Driving force for peptide transport in mammalian intestine and kidney.

Transport of small intact peptides across the brush-border membrane of the epithelial cells of small intestine and renal proximal tubule is a well-established phenomenon. This peptide transport system recognizes dipeptides and tripeptides as substrates and it is distinct from the transport systems available for absorption of free amino acids. While there is no doubt that active transport of amino acids is energized by a Na+ gradient, the nature of the driving force that energizes peptide transport has been a subject of controversy for many years. Numerous studies with intact tissue preparations have shown that the dependence of peptide transport on a Na+ gradient is at best partial. Recent investigations with isolated brush-border membrane vesicles from small intestine and kidney have clearly established that a H+ gradient rather than a Na+ gradient drives the active transport of intact peptides in these tissues. Since the transport mechanism involves peptide-H+ cotransport, thus rendering the process electrogenic, a membrane potential also plays a role in the energization of this transport system. Nutrient transport driven by an electrochemical H+ gradient is of common occurrence in microorganisms, but peptide transport represents the first example of this kind in mammalian systems. There is strong evidence that a H+ gradient actually exists in vivo across the brush-border membrane of intestinal and renal epithelial cells. The activity of the Na+-H+ exchanger which is located predominantly in the brush-border membrane and which, under physiological conditions, couples the influx of Na+ into the cell with the efflux of H+ from the cell, is primarily responsible for the existence of this H+ gradient. The driving force for the exchanger, a transmembrane Na+ gradient, is in turn generated and maintained at the expense of metabolic energy by the (Na+-K+) ATPase, which is present in the basal-lateral membrane of these cells. Accordingly, peptide transport in small intestine and kidney can be classified as a tertiary active transport system.

Animals↗

[Prevention of dysbacteriosis in the early neonatal period using a pure culture of acidophilic bacteria].

In order to prevent dysbacterioses occurring in the early neonatal period, 60 neonates classified with the risk group were administered, when they were still at the puerperal room, pure cultures of specially selected strains of Lactobacillus acidophilus possessing marked anti-Klebsiella activity and antagonistic activity against Staphylococcus aureus. The cultures were instilled into the mouth and nasal passages. Forty neonates administered bifidum-bacterin and 54 neonates not given biopreparations were used as control. Application of the pure culture of Lactobacillus acidophilus noticeably raised the efficacy of the preventive measures. Over 60 percent of the neonates were discharged from the maternity home with a normally formed intestinal microflora, 60 percent had normal microflora in the fauces, 80 percent in the nose, and 70 percent of the neonates demonstrated normal skin microflora. The carriage of opportunistic microorganisms in the fauces and nose of the neonates had decreased substantially by the time of the discharge from the maternity home, while the incidence of pyo-inflammatory diseases had also reduced among them.

Administration, Intranasal↗

Results of experimental intestinal strangulation obstruction in germfree rats.

Experimental low ileal strangulation obstruction has been produced in germfree and conventional rats. The mean survival time was 240 hours in the germfree rats and 44 hours in the conventional controls. 4 of the 10 germfree rats survived 15 or more days, whereas the 10 conventional animals were all dead within 2(1/2) days. The strangulation obstruction fluid from the germfree animals was sterile and non-toxic when injected into mice even after a fourfold concentration. The same fluid from the conventional animals contained a great number of microorganisms and caused death within 24 hours when injected intraperitoneally into mice.

Animals↗

[Bacterial translocation and its surgical implications].

The passage of viable bacteria through epithelial mucosa into lamina propria and then to mesentheric lymphnodes, and possibly other tissues was defined bacterial translocation (BT) by Berg and Garlington. The transepithelial passage of bacteria out of the intestinal lumen seems to occur in a variety of surgical conditions, and it is suspected to be involved in systemic inflammatory response syndrome, sepsis and multiorgan failure. There is increasing evidence that many nosocomial infections are caused by enteric translocating microorganisms, although the exact incidence of BT in humans is difficult to establish. Consensus is emerging that the barrier function of the gut is relevant in established critical illness and in patients at risk of developing sepsis and clinical studies have provided strong evidence that systemic infections often originate from intestinal flora in high risk patients. So the suspect of BT is made when there is infection after trauma, burns, major surgery, chemotherapy and immuno suppression. Bacterial translocation is also associated with organ transplantation, especially with small bowel transplantation. The Authors have summarized published experimental and clinical studies that have tried to understand the occurrence, mechanisms and effects of this complex process. At the present time there is a near full understanding of the relevance of BT like an interplay of diverse factors in a physiologically, immunologically and microbiologically complex intestinal tract. However additional experimental and clinical studies are needed to clarify the relationship between these phenomena and the development of sepsis or multiple organ dysfunction syndrome.

Bacterial Infections↗

The gastrointestinal epithelium and its autochthonous bacterial flora.

Colonization of the gastrointestinal tract by bacteria of the normal flora was followed by bacteriological and special histological techniques in mice from several colonies. These histological techniques were designed to preserve the intimate associations that become established between particular strains of microorganisms and the epithelium of the mucosa of certain areas of the gut. The findings were as follows: 1. The various strains of bacteria of the normal flora became established in the different areas of the guts of infant mice according to a definite time sequence. 2. The first types of bacteria that could be cultured from the gut were lactobacilli and Group N streptococci. Within the first day after birth, these bacteria colonized the entire digestive tract and formed layers on the stratified squamous epithelium of the nonsecreting portion of the stomach and of the distal esophagus. 3. The bacterial types that appeared next were coliforms and enterococci. From about the 9th to the 18th day after birth, these bacteria could be cultured in extremely high numbers from the cecum and the colon. Histological sections of those organs taken during the first 2 or 3 days of that interval revealed microcolonies of Gram-positive cocci in pairs and tiny Gram-negative rods embedded in the mucous layer of the epithelium. The microcolonies were well separated from the mixture of digesta and bacteria that occupied the center of the lumen; they may have consisted of the coliforms and enterococci mentioned above; but this possibility remains to be proved. 4. Histological sections also revealed that, at about the 12th day after birth, long, thin Gram-variable rods with tapering ends were present, side by side, with the small Gram-negative rods and Gram-positive cocci in the mucous layer. By the 15th day after birth, the fusiform bacteria formed thick layers in the mucus, and seemed to be the only bacteria remaining in that location. It has not yet been possible to enumerate these tapered rods by culture methods, but as judged by visual appearances in the histological sections, they seemed to outnumber all other bacteria in the cecum and the colon by a factor of as much as 1000. It must be stressed that these bacterial layers are readily disrupted and even washed away by conventional histological techniques; their discovery was largely due to the use of the special histological techniques described in the text. The bacteriological and histological findings described here constitute further evidence for the hypothesis that symbiotic associations exist between microorganisms and animals, and that a very large percentage of the bacteria in the gastrointestinal tract constitutes a true autochthonous flora. The constant occurrence of several distinct associations of bacteria with the special histological structures of the animal host renders obsolete the notion that the intestine constitutes a chemostat in which the bacterial populations are randomly mixed. For a full understanding of the ecology of the normal microflora, it is necessary to think of body surfaces as distinct microenvironments in which virtually pure cultures of a few species of microorganisms interact with their host and the adjacent microbial populations. Experiments based on this hypothesis are admittedly difficult to design, but on the other hand studies based on the assumption that microorganisms exist as mixtures in the gastrointestinal tract will be only of limited value and may often be misleading.

Animals↗

Nitric oxide mediates intestinal pathology but not immune expulsion during Trichinella spiralis infection in mice.

The relationship between intestinal pathology and immune expulsion of gastrointestinal (GI) nematodes remains controversial. Although immune expulsion of GI helminth parasites is usually associated with Th2 responses, the effector mechanisms directly responsible for parasite loss have not been identified. We have previously shown that while the intestinal pathology accompanying the expulsion of the GI parasite Trichinella spiralis may be dependent on IL-4 and mediated by TNF, parasite loss is independent of TNF. In contrast, intestinal pathology in other disease models has been attributed to Th1 cytokines, although it closely resembles that seen in helminth infections. Whereas production of inducible NO synthase (iNOS) in the gut is important for both homeostasis of the epithelial layer and in protection against pathogenic microorganisms, overproduction of NO has been implicated in the pathogenesis of a number of inflammatory conditions. We therefore investigated the role of NO in T. spiralis infection using iNOS-deficient mice. iNOS-/- and iNOS-/+ mice were infected with T. spiralis, and parasite expulsion and intestinal pathology were followed. Parasite expulsion proceeded similarly in both groups of animals, but significant intestinal pathology was only observed in the heterozygous mice. Thus it appears that, although the protective effects of Th2 responses in GI helminth infection do not require NO, this mediator contributes substantially to the associated enteropathy. NO may therefore be an important mediator of enteropathy in both Th1- and Th2-inducing conditions.

Animals↗

A rare cause of anemia due to intestinal tuberculosis in a renal transplant recipient.

A renal transplant recipient with stable allograft function presented with massive hemorrhagic diarrhea and severe anemia. No microbial infection could be found in stool cultures. Early colonoscopy showed severe colitis with ulceration. Histological samples confirmed granulomatous inflammation with acid-resistant Ziehl-Neelson-positive microorganisms of mycobacterial type. Polymerase chain reaction (PCR) analysis of native mucosal biopsies specified the infectious organism as Mycobacterium tuberculosis complex. The patient responded well to antimycobacterial therapy and was still asymptomatic after 6 months with a stable graft function. Our case shows that tuberculosis can be a severe clinical problem in transplant recipients. Most of the patients with intestinal tuberculosis, reported to literature, were diagnosed post mortem or after explorative laparotomy and bowel resection. Thus, intestinal tuberculosis should be considered when a transplant recipient shows abdominal symptoms with no clear evidence of another infection. Proper diagnosis and treatment resulted in a beneficial outcome in our patient.

Anemia↗

Review article: antibiotics and probiotics in inflammatory bowel disease.

Treatment with antibiotics in inflammatory bowel disease has a long tradition and is widely used. The indications for antibiotic therapy are wide ranging, from specific situations such as abscesses or fistulae, to patients with severe disease (as an unspecific 'protective' measure), and to address the hypothesis that the enteric flora as a whole, or specific microorganisms such as mycobacteria, are involved in the pathogenesis of inflammatory bowel disease. The best-studied single antibiotic compound is metronidazole. However, overall, the scientific basis for the use of antibiotics is limited, which may reflect a lack of interest from sponsors within the pharmaceutical industry. Despite this weak evidence base, antibiotics are a globally established therapeutic tool in inflammatory bowel disease. Growing evidence from human and animal studies points towards a pivotal pathogenetic role of intestinal bacteria in inflammatory bowel disease. In view of these experimental findings, clinical trials have been undertaken to elucidate the therapeutic effects of probiotics in inflammatory bowel disease. Probiotics are viable nonpathogenic microorganisms which confer health benefits to the host by improving the microbial balance of the indigenous microflora. So far, of the many candidates, one specific strain (Escherichia coli Nissle 1917) and a mixture of eight different bacteria have demonstrated convincing therapeutic efficacy in controlled studies. Maintenance therapy in ulcerative colitis and prevention therapy, as well as the treatment of pouchitis, have emerged as areas in which probiotic therapy offers a valid therapeutic alternative to current treatments. Further investigations may detect additional clinically effective probiotics and other clinical indications.

Anti-Bacterial Agents↗

Role of motility, chemotaxis, and adhesion in microbial ecology.

This review emphasized the implications of recent data pertaining to the role that motility, chemotaxis, and adhesion play in microbial ecology. Some of these processes appear to promote colonization by allowing certain organisms to selectively "seek out" nutrients or sites of colonization. For example, chemotaxis to NO3- and NO2- may provide pseudomonads with such a strong competitive mechanism that it allows this group of bacteria to outcompete other members of the soil microbiota for these chemicals. Likewise, chemotaxis also allows other bacteria to enter and colonize the mucus gel lining the intestinal epithelium and thereby resist physical removal from the gut. On the other hand, the understanding of such mechanisms offers important new possibilities for the deliberate control of microorganisms for the benefit of man. To that end, much remains to be done before a thorough understanding of the ecology of the microflora of any ecosystem can be accomplished. It is hoped that this review will stimulate further work in this area, as well as to lead to collaboration between engineers and microbiologists, which should lead to fruitful and exciting research in the future.

Animals↗