Barrier-fixing function in germ-free animals]

The state of barrier-fixative function was studied in germfree and conventional guinea pigs and rats. Under conditions of conventional animals contamination with E. coli 055 (in doses of 500 million and 10 milliard microbial bodies for subcutaneous and oral inoculation, respectively) only an early transitory bacteremia developed at the early postinfection periods. As to bacteriemia in gnotobiotes, it increased progressively leading to the animal death in the course of 2 to 3 days. A decreased fixative and bactericidal capacity of the regional lymphatic apparatus and deep structures of the mononuclear-phagocytic system was revealed in germfree animals. An experimental confirmation of the participance of antibodies in the manifestation of the barrier-fixative function to E. coli was obtained. These studies demonstrated an important role of the microbial factor in the formation of the macroorganism barrier-fixative function.     

Nonspecific resistance in germ-free and E. coli monocontaminated miniature piglets]

Phagocytic activity of leukocytes, as well as the complement, properdin, and lysozyme levels in the blood serum of miniature piglets, germfree and monocontaminated with E. coli 055 and E. coli 083, were studied. E. coli 055 phagocytosis was decreased in the presence of autologous serum and complement and increased under the effect of specific opsonins (antibodies to E. coli 055). Complement, properdin, and lysozyme levels were decreased in the germfree, in comparison with conventional animals. In the E. coli contaminated piglets properdin and complement production was stimulated most, and lysozyme formation--less. No antibodies to E. coli 055 were revealed in monocontaminated piglets. The highest lysozyme levels were found in the ex-germfree animals, this indicating the participation of factors other than E. coli contamination in lysozyme stimulation. It is concluded that microbial contamination played an important role in the development of cellular and humoral factors of the organism resistance.     

Influence of indigenous microbiota on activities of alkaline phosphatase, phosphodiesterase I, and thymidine kinase in mouse enterocytes.

An indigenous microflora introduced into the gastrointestinal tracts of animals in a population of germfree mice affected in different ways three enzymes in small bowel enterocytes. Cells were obtained by techniques designed for sequentially removing enterocytes from the tip of the villus to the crypts of Lieberkühn. The specific activity of alkaline phosphatase, a component of the enterocyte microvillous membrane, did not differ in cells isolated from germfree mice and from those associated with a microflora, while that of phosphodiesterase I, also a part of the microvillous membrane, was approximately 1.5-fold greater in the suspensions from all levels of the villi in germfree mice than in those from the associated animals. By contrast, the specific activity of thymidine kinase, a cytosol enzyme, in suspensions in which the cells were isolated from the lower portion of the villi and crypts was about one-half as great in cells from germfree mice as in those from the same regions of animals with a microbiota. These results support the hypothesis that activities of certain enzymes involved in metabolism, uptake, and incorporation by enterocytes of components of dietary nuclei acids are influenced by a microflora.     

Influence of certain indigenous gastrointestinal microorganisms on duodenal alkaline phosphatase in mice.

Alkaline phosphatase activity was assayed in duodenal homogenates or extracts from adult specific pathogen-free (SPF) and germfree mice and gnotobiotic mice monoassociated with a Lactobacillus sp., a Bacteroides sp., or a coliform strain indigenous to SPF mice. Activity levels of the enzyme were much higher in the preparations from germfree mice than in those from the SPF controls. In the gnotobiotes monoassociated either with a freshly isolated Lactobacillus sp. or a Bacteroides sp., the levels of alkaline phosphatase activity were intermediate between the values for germfree and SPF mice. By contrast, in the gnotobiotes monoassociated with a coliform strain, alkaline phosphatase activity remained at high germfree levels. Butanol extracts of duodenal tissue from SPF mice, germfree mice, and exgermfree mice associated with an indigenous microflora from SPF mice (conventionalized) were subjected to acrylamide gel electrophoresis. A stain for alkaline phosphatase activity revealed three major bands in the gels prepared with extracts from SPF and conventionalized mice, but only two in the gels prepared with extracts from germfree mice. All three bands may have been present in the latter gels. One of the bands (the middle one) may have been obscured, however, by high activity in the slowest moving band. As determined by densitometric scanning, the slowest moving band had much higher activity in the preparations from germfree animals than in those from SPF or conventionalized mice. These findings suggest that the indigenous microbial flora affects not only quantitatively, but also qualitatively, the activity of alkaline phosphatases in the mouse intestinal mucosa.     

Resistance of germfree rats to indomethacin-induced intestinal lesions.

Indomethacin given orally to conventional rats produced in three days a syndrome, often fatal, of intestinal lesions characterized by multiple ulcers and peritonitis. Male germfree rats were found to be resistant to this effect of indomethacin, while female germfree rats developed very mild lesions. Germfree rats became sensitive again to such lesions when monocontaminated with E. coli. In such animals, however, the lesions were less severe than in conventional animals, presumably because more than one microorganism is necessary for the full syndrome to develop. These results suggest that microorganisms are necessary for the development of indomethacin-induced intestinal lesions. Secondary bile acids, absent in germfree animals, may also be necessary. The prostaglandin deficiency caused by indomethacin appears to weaken the resistance of the intestinal mucosa to microorganisms and/or their toxins. The latter may then penetrate the mucosa, damage the cells and produce ulcers and perforations. Since several prostaglandins also protect against indomethacin-induced lesions, the hypothesis is advanced that certain prostaglandins may protect the mucosa ("cytoprotection") by preventing the spread of microorganisms and/or their toxin through the intestinal wall.     

Opsonizing activity of the serum of gnotobiotic guinea pigs contaminated with individual representatives of intestinal microflora]

The influence of contamination of germfree guinea pigs with individual representatives of the intestinal microflora (Bac. mesentericus, Bac. subtilis, S. albus, and S. faecalis) on the formation of the serum opsonic activity was studied. An increase of the opsonic activity to all the microorganisms on the 11th day after a corresponding monocontamination and a stimulating influence of the serum on the intracellular digestion of Bac. mesentericus and Bac. subtilis microbes was noted. As to the pathogenic microorganisms (E. coli 055), S. Faecalis only were capable of stimulating the serum opsonic activity. The results indicated the presence of an association between the microflora composition and the opsonic activity of the animal blood serum. The value of this index also depended on the properties of the phagocytosis object.     

Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier

Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that FcgammaRIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.     

实验性痴呆动物的肠道菌群和粘附性研究

用ＡＦ６４Ａ复制实验性痴呆动物模型,分析该动物的肠道菌群,并以双歧杆菌和大肠杆菌作为肠道菌的代表,初步探讨它们对实验性痴呆动物肠道粘膜上皮细胞表面的粘附特性。结果表明,实验性痴呆动物的肠道菌群是紊乱的,二种试验菌均能粘附到正常小鼠肠上皮细胞上,双歧杆菌的粘附率明显高于大肠杆菌,而双歧杆菌对实验性痴呆小鼠肠上皮细胞的粘附率明显低于对照组小鼠,大肠杆菌则相反。     

Trypsin and chymotrypsin activity of the intestinal content in germfree, monoassociated and conventional rabbits.

Trypsin (T) and chymotrypsin (CHT) activities in luminal contents of the ileum, caecum and sigmoideum were followed in conventional (6 animals), monoassociated (5) and germfree (5) rabbits by pH-stat automatic titration using p-toluenesulphonyl-L-arginine methylester and acetyl-L-tyrosine ethylester as substrates. In conventional rabbits with complete microbial flora an aborally increasing decline of both proteolytic activities of luminal contents was determined (ileum T 198.2 - CHT 100.0; signmoideum T 10m.2 - CHT 68.8 mrg/g of intestinal content). Monoassociated animals represent a group different from both germfree and conventional animals. Trypsin and chymotrypsin of intestinal contents were not significantly altered by the presence of megacaecum in germfree rabbits (ileum T 219.2 - CHT 160.2; sigmoideum T 208.8 - CHT 110.8 mug/g of intestinal content). Chymotrypsin in the intestinal contents appears more labile and more affected by microbial flora than trypsin.     

Endotoxin in germfree, gnotobiotic, or conventional-flora Sprague-Dawley rats.

The Limulus assay for bacterial endotoxin was performed on serum and (or) plasma from animals monoassociated with Clostridium species, Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Enterobacter agglomerans, Bacteroides fragilis, Klebsiella pneumoniae, or Candida albicans. Plasma from animals monoassociated with the gram-negative bacteria or C. albicans consistently showed a positive Limulus test while conventional-flora controls, germfree rats, and gnotobiotic animals monoassociated with gram-positive bacteria or E. agglomerans were negative. Germfree and conventional rats were injected (intraperitoneal (i.p.)) with Salmonella typhosa lipopolysaccharide (LPS). Although no endotoxin was detectable in either group prior to the injection, by 1 h post injection endotoxin was in the plasma of all groups. The germfree rats appeared to clear the LPS quicker than their conventional-flora counterparts. Generally, LPS-injected rats (conventional and germfree) showed clumping and decreased number of platelets, a decrease in their lymphocyte counts, and increased polymorphonuclear leukocyte (PMN) counts.     

Course of the post-irradiation syndrome after irradiation with lethal doses in newborn conventional,germ-free andEscherichia coli-monoassociated piglets

The influence of whole-body irradiation with lethal doses of ionizing radiation (⁶⁰Co) was studied in conventional, germ-free andEscherichia coli-monoassociated newborn piglets. The dose 1,200 R produced an acute intestinal death (i.e. within 3–4 days) in conventional animals, whereas survival was three times as long in their germ-free counterparts. Artificial colonization of the intestinal tract of germ-free piglets with non-pathogenic strain ofEscherichia coli, prior to irradiation with the same dose, produced the conventionalization of these animals and reduction in the survival time almost to the level of conventional animals. In conventional animals, profound focal regressive changes of the epithelium accompanying the denudation of intestinal villi were found already on the 2nd–3rd day after irradiation with 1,200 R. On the other hand, the intestinal epithelium of germ-free piglets, irradiated with 1,200 R, was found to be intact on the 7th–9th day of post-irradiation, and the first signs of damage started to occur around the 9–10th days. The morphological characteristics of the intestinal mucous membrane ofEscherichia coli-monoassociated piglets were comparable to those of conventional, irradiated piglets. The role of the presence of the microbial factor for the turnover and radiosensitivity-resistance of enterocytes, and for the survival-death rate of animals irradiated with doses producing the post-irradiation gastro-intestinal syndrome, is discussed.     

Obtaining and rearing germ-free miniature piglets for biomedical research

The method of obtaining and rearing germfree miniature piglets for medical-biological research is described. The gnotobiotes were reared in rigid organic glass isolation cells up to 3 months. Milk diets supplemented with minerals and vitamins were used for nutrition. The gnotobiotes gained weight well during the whole observation period.     

Effect of human immunoglobulins on mouse intestinal microflora in dysbacteriosis

Human immunoglobulins, injected into mice subcutaneously after their irradiation or at the beginning of antibiotic therapy, protected the mucous membrane of the proximal section of the small intestine from the penetration of enterobacteria. The formation of the protective barrier was observed when immunoglobulin preparations with the titer of antibodies to Escherichia coli O14 between 1:16 and 1:256 was used. The preliminary exhaustion of immunoglobulin preparations with E. coli strain O14 led to the complete loss of their protective properties.     

Bifidobacteria and human health: regulatory effect of indigenous bifidobacteria on Escherichia coli intestinal colonization

Bifidobacteria are assumed to exert colonization resistance to enteric pathogens. We associated C3H germfree mice with either Bifidobacterium longum or Escherichia coli or both strains and studied how they settled in the gut and the lymphoid organs as well as their effect on mucus composition. Within 24 hE. coli colonized the gut of germfree or B. longum ex-germfree mice. In contrast,B. longum was established in the intestine of E. coli ex-germfree mice only 1 month after inoculation whereas it colonized the germfree gut within 24 h. Although B. longum did not exert colonization resistance to E. coli, the establishing of bifidobacteria in the gut partly prevented changes in the E. coli cell wall. After colonization of the germfree or B. longum mono-associated mice, E. coli lipopolysaccharide exhibited a higher concentration of Kdo and the O-antigen side chain disappeared. A reduction in Kdo content was observed within 1 month in E. coli-B. longum diassociated mice whereas it remained at a high level in E. coli mono-associated mice. Association in a second step with B. longum led to Kdo reduction. Changes in E. coli LPS might be related to mucus modification. Inoculation of either bacterium led to a slow increase in mucus protein content which was however twice as high after E. coli implantation. Inoculation of B. longum in a second step led to a reduction in protein content before B. longum colonized the intestine at a high level suggesting that the protein concentration in the mucus was controlled by the host itself. A new glycoprotein of 200-230 kDa detected during the period preceeding colonization seemed to be broken down by B. longum. The resulting end product might participate in the restoration of E. coli LPS. Finally,B. longum inoculation led to the disappearance of E. coli from kidneys, liver, spleen and lung. The organs were cleared of E. coli before B. longum highly colonized the intestine suggesting that high intestinal colonization by B. longum was not required. Regulation of E. coli invasion seemed to depend on the ability of B. longum to stimulate the immune system.     

Local Shwartzman phenomenon in germ-free guinea pig]

Eleven germfree and two monoassociated with the Citrobacter guinea pigs, and 25 conventional animals were injected with the heat-inactivated Citrobacter or E. coli for the induction of the local Schwartzman phenomenon. All the gnotobiotic pigs gave a positive reaction. Infiltration at the site of intracutaneous injection was found in all the conventionals, but in none of the gnotobiotics. The data are discussed from the aspect of primary and secondary sequelae of the absence of host microflora.     

The histochemistry of mucosaccharides in some organs of germfree rats.

In order to study the histochemical nature of mucosaccharides in germfree animals, the organs in natural contact with bacteria (stomach, small and large intestine) and those naturally remote from bacteria (tracheal and ear cartilage and aorta) were studied by means of light microscopic methods for mucosaccharides in germfree and conventional rats. In the stomach (surface and foveolar cells) of germfree rats the histochemical reactions for acid and neutral mucosaccharides were apparently less intense than in that of conventional rats, whereas in the small and large intestine (goblet cells) of germfree rats the reactions were significantly more intense than in those of conventional rats. In the cartilage (intercellular matrix, lacunar border and chondrocyte cytoplasm) and aorta (interelastic spaces) of germfree animals the reactions were less intense than in those of conventional animals. In addition, some differences in the histochemical nature of mucosaccharides between the organs of germfree and conventional rats were noted, as revealed by the effects of chemical modifications and digestions with enzymes upon the histochemical reactions studied.     

Intestinal flora of animal models of human diseases as an environmental factor

Genetically-engineered animals are known to be useful in clarifying the functions of many genes and as animal models for human diseases. However, it has been widely reported that pathophysiology is not expressed in these animals when they become germfree or SPF animals, i.e., the pathophysiology is not the result of genes alone and a combination of gene function and intestinal flora as an environmental factor are necessary. It is important to determine the roles of each of these two factors by pathophysiological analysis. Gnotobiotic mice were produced by establishment of specified bacterial species in germfree animals to form the intestinal flora of SPF animals and they were placed in barrier facilities. Measures have been taken against infections by bacteria such as Pseudomonas aeruginosa and Enterobacter cloacae. In addition, gnotobiotic mice with a highly normal physiology are required. Analysis of the effects of each bacterial species and combinations of bacteria on in vivo functions, i.e., the cross-talk between the host and intestinal flora, is essential in the creation of better laboratory animals. Monitoring of the intestinal flora, a key factor in the colonies produced, is a topic for future research.     

Involvement of calmodulin in the regulation of adenylate cyclase activity in guinea-pig enterocytes

The involvement of calmodulin as an activator of adenylate cyclase activity was examined in isolated guinea-pig enterocytes and in a membrane preparation. In enterocytes, which responded to prostaglandin E1, vasoactive intestinal peptide and cholera toxin with a significant increase in the rate of cAMP formation trifluoperazine, a calmodulin antagonist, completely inhibited cAMP formation. In a membrane preparation adenylate cyclase activity was stimulated 10-20-fold by the GTP analog, guanosine 5'-[beta-imido]5'-triphosphate (Gpp[NH]p). Prostaglandin E1 and vasoactive intestinal peptide enhanced cAMP formation in this system by 2-3- and 1.2-1.6-fold. respectively. Addition of 200 nM calmodulin to membranes, in which endogenous calmodulin was decreased from 1.4 microgram/mg protein to 0.5 microgram/mg protein by washing with buffer containing EGTA and EDTA, resulted in a 3-4-fold increase of adenylate cyclase activity. The absolute increment in adenylate cyclase activity caused by calmodulin (10-15 pmol cAMP/min per mg protein) was approximately the same in the absence or presence of Gpp[NH]p. The apparent Ka for Gpp[NH]p (6 . 10-7 M) was not significantly changed by the addition of calmodulin. Although endogenous calcium (approx. 10 microM) in the enzyme assay was adequate to affect stimulation by calmodulin, a maximal effect was observed at a calcium concentration of 100 microM. These findings indicate that a calmodulin-sensitive form of adenylate cyclase is present in guinea-pig enterocytes, and that stimulation of cAMP formation in the intestinal mucosa may involve a calmodulin-mediated mechanism.     

Interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn kids

The elaboration of heat stable enterotoxin (STa) is an important step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC), which causes severe diarrhea in newborn animals. In this study, the distribution of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from the anterior jejunum, posterior jejunum, ileum and colon of newborn kids was investigated. The density of STa-receptors on enterocytes and BBMVs was higher in the posterior jejunum than that in other segments of the kids' intestines. Additionally, the affinity of the posterior jejunum STa-receptors was higher than the affinity of receptors present on the epithelium of other intestinal segments. Our findings suggest that the posterior jejunum is a major target for STa within the intestinal tract of newborn kids.     

The histochemistry of mucosaccharides in some organs of germfree rats

Summary In order to study the histochemical nature of mucosaccharides in germfree animals, the organs in natural contact with bacteria (stomach, small and large intestine) and those naturally remote from bacteria (tracheal and ear cartilage and aorta) were studied by means of light microscopic methods for mucosaccharides in germfree and conventional rats. In the stomach (surface and foveolar cells) of germfree rats the histochemical reactions for acid and neutral mucosaccharides were apparently less intense than in that of conventional rats, whereas in the small and large intestine (goblet cells) of germfree rats the reactions were significantly more intense than in those of conventional rats. In the cartilage (intercellular matrix, lacunar border and chondrocyte cytoplasm) and aorta (interelastic spaces) of germfree animals the reactions were less intense than in those of conventional animals. In addition, some differences in the histochemical nature of mucosaccharides between the organs of germfree and conventional rats were noted, as revealed by the effects of chemical modifications and digestions with enzymes upon the histochemical reactions studied.This investigation was supported in part by a Grant-in-Aid from the Japanese Education Ministry (1975). A major part of this investigation has been presented at the 10th International Congress of Anatomists held in Tokyo (1975)