Lactobacilli as effectors of host functions: no influence on the activities of enzymes in enterocytes of mice.

Preparations of lactobacilli are often used as dietary supplements to improve the growth and efficiency in utilizing food of animals of commercial value. We tested in an experimental model whether the effects of lactobacilli on growth of and food utilization by animals may be due to alteration of the activities of absorptive enzymes in the small bowel. Germfree mice housed in isolators under tightly controlled conditions were monoassociated with one of four strains of indigenous Lactobacillus spp. From 1 to 5 weeks later, the activity of alkaline phosphatase was assayed in homogenates of segments of the upper small intestines of the associated animals and of matched germfree controls. The specific activity of the enzyme was the same in the mice in the two groups. In other experiments, epithelial cells were isolated from the upper small intestines of mice associated with eight Lactobacillus strains (octa-associated) and from those of matched germfree mice and assayed for alkaline phosphatase, phosphodiesterase, and thymidine kinase activities. The epithelial cells were harvested sequentially from the tips of the villi toward the crypts of Lieberkühn of the intestines. In all preparations, mice of both types yielded an equivalent mass (wet weight) of cells. The protein content of the cells reflected the mass. The activities of the microvillous membrane enzymes alkaline phosphatase and phosphodiesterase and the cytosol enzyme thymidine kinase were the same whether or not the animals contained the bacteria. Therefore, any effects on animal growth and food utilization observed when lactobacilli are used as dietary supplements may not be due to a direct alteration by the bacteria of the absorptive enzymes of the host animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactobacilli as effectors of host functions: no influence on the activities of enzymes in enterocytes of mice

Preparations of lactobacilli are often used as dietary supplements to improve the growth and efficiency in utilizing food of animals of commercial value. We tested in an experimental model whether the effects of lactobacilli on growth of and food utilization by animals may be due to alteration of the activities of absorptive enzymes in the small bowel. Germfree mice housed in isolators under tightly controlled conditions were monoassociated with one of four strains of indigenous Lactobacillus spp. From 1 to 5 weeks later, the activity of alkaline phosphatase was assayed in homogenates of segments of the upper small intestines of the associated animals and of matched germfree controls. The specific activity of the enzyme was the same in the mice in the two groups. In other experiments, epithelial cells were isolated from the upper small intestines of mice associated with eight Lactobacillus strains (octa-associated) and from those of matched germfree mice and assayed for alkaline phosphatase, phosphodiesterase, and thymidine kinase activities. The epithelial cells were harvested sequentially from the tips of the villi toward the crypts of Lieberkühn of the intestines. In all preparations, mice of both types yielded an equivalent mass (wet weight) of cells. The protein content of the cells reflected the mass. The activities of the microvillous membrane enzymes alkaline phosphatase and phosphodiesterase and the cytosol enzyme thymidine kinase were the same whether or not the animals contained the bacteria. Therefore, any effects on animal growth and food utilization observed when lactobacilli are used as dietary supplements may not be due to a direct alteration by the bacteria of the absorptive enzymes of the host animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Genetic transformation in Lactobacillus sp. strain 100-33 of the capacity to colonize the nonsecreting gastric epithelium in mice

Lactobacillus isolates able to colonize the surfaces of the nonsecreting epithelia in the stomachs of monoassociated ex-germfree mice were derived from Lactobacillus acidophilus 100-33. Strain 100-33 was originally isolated from pig feces and is unable to colonize the murine gastric epithelium. In experiments involving attempts genetically to transform the capacity to colonize the epithelium, cells of strain 100-33 were treated with muralytic enzymes and mixed with polyethylene glycol and genomic or plasmid DNA extracted from Lactobacillus fermentum RI. Strain RI was originally isolated from a conventional mouse and has the capacity to colonize the nonsecreting gastric epithelium. The mixtures containing cells, polyethylene glycol, and DNA were plated on a regeneration medium. After overnight incubation, the cells were washed from the plates and introduced by gastric gavage into germfree mice. Only mice that received regenerated 100-33 cells previously mixed with genomic DNA from strain RI had layers of gram-positive bacteria on the keratinized epithelia of their stomachs. Six isolates cultured from the washed gastric tissues of these animals were characterized. When a culture of each or a pool of cultures of the six were orally administered to germfree mice, layers of gram-positive bacterial cells were visible on the keratinized gastric epithelia of the animals within 1 to 3 weeks. Cells of all six, but not of strain 100-33, reacted with antibody made in rabbits to L. fermentum RI cells, as determined by an enzyme-linked immunosorbent assay. Nevertheless, all six had fermentation profiles identical to that of strain 100-33.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic transformation in Lactobacillus sp. strain 100-33 of the capacity to colonize the nonsecreting gastric epithelium in mice.

Lactobacillus isolates able to colonize the surfaces of the nonsecreting epithelia in the stomachs of monoassociated ex-germfree mice were derived from Lactobacillus acidophilus 100-33. Strain 100-33 was originally isolated from pig feces and is unable to colonize the murine gastric epithelium. In experiments involving attempts genetically to transform the capacity to colonize the epithelium, cells of strain 100-33 were treated with muralytic enzymes and mixed with polyethylene glycol and genomic or plasmid DNA extracted from Lactobacillus fermentum RI. Strain RI was originally isolated from a conventional mouse and has the capacity to colonize the nonsecreting gastric epithelium. The mixtures containing cells, polyethylene glycol, and DNA were plated on a regeneration medium. After overnight incubation, the cells were washed from the plates and introduced by gastric gavage into germfree mice. Only mice that received regenerated 100-33 cells previously mixed with genomic DNA from strain RI had layers of gram-positive bacteria on the keratinized epithelia of their stomachs. Six isolates cultured from the washed gastric tissues of these animals were characterized. When a culture of each or a pool of cultures of the six were orally administered to germfree mice, layers of gram-positive bacterial cells were visible on the keratinized gastric epithelia of the animals within 1 to 3 weeks. Cells of all six, but not of strain 100-33, reacted with antibody made in rabbits to L. fermentum RI cells, as determined by an enzyme-linked immunosorbent assay. Nevertheless, all six had fermentation profiles identical to that of strain 100-33.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colonization of gnotobiotic mice by Roseburia cecicola, a motile, obligately anaerobic bacterium from murine ceca.

In scrapings of mouse cecal mucosae, motile bacteria outnumbered nonmotile bacteria by a ratio of 2:1. Obligately anaerobic bacteria were obtained from such scrapings through the use of techniques designed for the selective isolation of motile bacteria. One of the isolates, Roseburia cecicola, was rapidly motile in broth by means of 20 to 35 flagella arranged in a fascicle on each cell. R. cecicola cells colonized germfree mice (3 x 10(9) to 1 x 10(10) CFU/g of cecum) within 11 days after the animals were inoculated intragastrically with 2 x 10(8) CFU per mouse. In such monoassociated gnotobiotes, the bacteria were found primarily in the cecum, dispersed in the lumen among particles of digesta, and in the mucus over the epithelial surface. Between 2 and 3 weeks after birth, offspring of monoassociated adult mice were colonized by the bacterium (2 x 10(9) to 1 x 10(10) CFU/g of cecum). These results indicate that R. cecicola is suitable for studies of the ecology of host-associated microorganisms, particularly for investigation of the role of motility and possibly also chemotaxis in bacterial colonization of the mammalian gastrointestinal tract.     

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.     

Host specificity of the colonization of murine gastric epithelium by lactobacilli

Abstract Strains of Lactobacillus isolated from animals of several species were examined for their capacity to colonize the lumens and form layers on the keratinized nonsecreting epithelium in the stomachs of monoassociated ex-germfree mice. All strains tested could be cultured at comparable population levels from the stomachs of the mono-associated mice. With one exception, however, only strains previously isolated from rodents were able to form thick continuous layers on the gastric epithelial surface. The exception was a strain isolated from calf feces. This strain formed a layer on the epithelial surface, comparable to layers seen in animals associated with strains from rodents.     

Interactions between gut-associated lymphoid tissue and colonization levels of indigenous, segmented, filamentous bacteria in the small intestine of mice.

Unlike most other indigenous bacteria, segmented filamentous bacteria (SFB) are potent activators of the mucosal immune system. SFB are strongly anchored to the epithelial cells of the small intestine where they have a preference for mucosal lymphoid epithelium. Since SFB are only present in high numbers shortly after weaning, it was investigated whether an SFB-induced immune reaction results in the removal of these bacteria from the small intestine. A correlation was found between age and colonization levels in the small intestines of SFB monoassociated Swiss mice. Five-week-old athymic BALB/c (nu/nu) mice showed lower colonization levels than their heterozygous littermates, but the opposite was found at the age of 12 weeks. However, SFB inoculation of germfree Swiss mice resulted in higher colonization levels in 5-week-old mice when compared with 4-month-old mice. We conclude that SFB colonization levels in the small intestine are likely influenced by the activity of the mucosal immune system. However, an additional age-dependent factor that modulates SFB colonization levels cannot be excluded.     

The size pH, and redox potential of the cecum in mice associated with various microbial floras.

Cecal size and in situ redox potential and pH of cecal contents were determined in conventionally reared mice and mice reared under a variety of gnotobiotic conditions: germfree, monoassociated with a cecal Clostridium sp., hexaflora-associated and thermoduric polyflora-associated. The mean Eh was approximately +200 mV in germfree and -200 mV in conventional mice. The Eh was close to zero in the monoassociated mice, thus occupying a position intermediate between the germfree and conventional mice. The potentials observed in the hexaflora and the thermoduric flora groups were indistinguishable from those of conventional animals. The degree of normalization was more advanced with respect to the redox potential than to the cecal size in the various gnotobiotic groups. In the thermoduric polyflora-associated group, normalization was observed in both cecal size and redox potential. This demonstrates that normalization can be accomplished with a relatively simplified microflora, at least with regard to the parameters studied.     

Constitutive delivery of bovine beta-lactoglobulin to the digestive tracts of gnotobiotic mice by engineered Lactobacillus casei

The gut microbiota is critical for maturation of the immune system. Recent evidence suggests that early establishment of lactobacilli in the intestinal microbiota, during neonatal colonization or by probiotic supplementation, could prevent the development of allergic disorders. Postnatal maturation of the gut immune system with allergen-producing lactobacilli colonizing the digestive tract could then affect the development of further allergic sensitization. In this paper, we describe construction of a recombinant Lactobacillus casei strain that can constitutively deliver bovine beta-lactoglobulin (BLG), a major cow's milk allergen, to the guts of gnotobiotic mice. The blg gene was inserted into the L. casei chromosome downstream of an endogenous promoter. BLG production was improved by fusing the propeptide LEISSTCDA (LEISS) to the BLG mature moiety. This led to a 10-fold increase in LEISS-BLG production compared to the production obtained without the propeptide and also led to enhanced secretion corresponding to 5% of the total production. After inoculation into germfree C3H/HeN mice, the genetic stability of the recombinant strain and in vivo BLG production were confirmed for at least 10 weeks. BLG stimulation of spleen cells from mice monoassociated with the BLG-producing lactobacilli induced secretion of the Th1 cytokine gamma interferon and, to a lesser extent, the Th2 cytokine interleukin-5. No BLG-specific immunoglobulin G1 (IgG1), IgG2a, or IgA was detected in sera or in fecal samples. These results suggest that gut colonization with allergen-producing lactobacilli could provide a useful model for studying the modulation of allergic disorders.     

Influence of Indigenous Microbiota on Amount of Protein and Activities of Alkaline Phosphatase and Disaccharidases in Extracts of Intestinal Mucosa in Mice

The protein content and the activities of alkaline phosphatase, maltase, and sucrase were measured at 0800, 1000, 1200, 1400, and 1600 in saline extracts of the proximal small bowels of germfree and of ex-germfree mice colonized with an indigenous microbiota. In extracts prepared from germfree mice, the total activities of all of the enzymes were relatively constant throughout the sampling period. Likewise, the total activity of alkaline phosphatase in extracts prepared from associated mice varied little as a function of time. By contrast, the total activities of maltase and sucrase in the extracts from these latter animals varied significantly from sample to sample. The total activity levels in extracts from germfree mice were approximately twofold greater than the levels in extracts from associated mice. The specific activities of alkaline phosphatase and sucrase did not vary from sample to sample in extracts prepared from either type of mouse. In contrast, the specific activity of maltase in extracts prepared from both germfree and associated mice differed significantly from sample to sample. The specific activities of all three enzymes were greater in extracts from germfree animals than in those from associated animals. The protein content of extracts prepared from germfree mice also was greater than that of extracts prepared from associated animals at every sampling time. The amount of protein extractable from the mucosa of the small bowels of the former animals varied significantly at different sampling times during the day, whereas the amount of protein extractable from the tracts of associated animals remained relatively constant throughout the day. The indigenous microbiota apparently stabilizes in some way the amount of protein extractable from the mucosa of the mouse small bowel.     

Indigenous bacteria influence the number of Salmonella typhimurium in the ileum of gnotobiotic mice.

Gnotobiotic BALB/c mice associated with indigenous Lactobacillus, Bacteroides, and two fusiform-shaped Clostridium strains had fewer S. typhimurium present in the ileum 3 days after intragastric challenge with the pathogen than did similarly challenged germfree mice. Acetic and butyric acids were detected in the caecal contents of the gnotobiotic mice, but in smaller concentration than was present in conventionalized mice. No difference in the motility of the small intestine was detected between germfree, gnotobiotic, and conventionalized mice.     

Translocation ofLactobacillus murinus from the gastrointestinal tract

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other extraintestinal sites. The translocation rate of a newly described species of indigenous bacteria,Lactobacillus murinus, was compared with the translocation rates of indigenousLactobacillus acidophilus and nonindigenousSalmonella enteritidis. Groups of germfree or antibiotic-decontaminated, specific pathogen-free mice were monoassociated with each of these bacterial strains and tested at various intervals for translocation to the mesenteric lymph nodes. The translocation rates of the various bacteria expressed in decreasing order as the numbers of translocating bacteria per gram mesenteric lymph node wereS. enteritidis, L. murinus, andL. acidophilus. The degree of histologic damage to the gastrointestinal mucosa after monoassociation with these strains followed the same pattern. Thus,L. murinus translocates from the GI tract at a surprisingly high rate for an indigenous bacterial strain, and its translocation appears to be associated with mucosal alterations.     

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.     

Cell Proliferation and Ageing In Mouse Colon

The descending colon of 4 month and 2 year old mice was exposed to 1250 rad X-rays. This killed most of the epithelial cells. the surviving cells formed new crypts and surface epithelium in animals of both ages. Not all the crypts were replaced. the irradiated area contained no more than 80% of the control number of crypts per section for at least 6 weeks after irradiation. In the young mice new crypts were much larger and the labelling index (LI) was much higher than in unirradiated animals during the first week after irradiation. In the old mice the overshoot in LI and crypt size began later and continued longer than in young animals. This may be because the control of cell proliferation was much less precise in old than in young mice. The irradiation was repeated, in an attempt to age prematurely the epithelial cells by increasing the number of divisions they underwent. the overshoot in LI and cells per crypt was smaller after a second dose than after the first in both young and old mice. There was almost no overshoot after a third dose was given to young mice. Increasing the number of divisions undergone by the surviving epithelial cells did not change the timing of repopulation in young mice compared to that found in old mice. Little evidence was found for the presence of a limited proliferative lifespan in colon epithelial cells.     

Morphometric and Kinetic Studies On the Changes Induced In the Intestinal Mucosa of Rats By Intraperitoneal Administration of Quinacrine

The intraperitoneal administration of large doses of quinacrine in rats results in a state of enteromegaly affecting mainly the distal small bowel, caecum and proximal colon. This enteromegaly is associated with mucosal crypt hyperplasia, and hypertrophy and hyperplasia of the Muscularis propria. In order to investigate the changes in the intestinal mucosal crypts, morphometry and a metaphase-arrest experiment with vincristine were undertaken on a group of rats given 12 mg of quinacrine hydrochloride by intraperitoneal injection daily for 5 days 2 weeks previously, and comparisons drawn with a group of control animals. In the quinacrine-treated animals there was marked enteromegaly affecting the distal small bowel, caecum and proximal colon, and in these segments there was clear crypt hyperplasia. Proximal and distal to the dilated bowel hyperplasia was not seen. No consistent pattern of change in crypt-cell birth rate was evident. the mechanisms by which quinacrine may effect kinetic and morphometric changes in the intestinal crypts are considered.     

Ultrastructure of the enlarged cecum in germfree rats

Summary The cecum of germfree rats, as studied by light microscopy and scanning and transmission electron microscopy, differs in many respects from the cecum of conventional rats. Epithelial cells in germfree rats are taller and have larger nuclei and longer microvilli than similar cells in conventional rats. The cecal mucosa of germfree rats shows a larger variability in the arrangement of the crypts of Lieberkühn than does the mucosa of conventional rats. Some crypts are funnel-shaped and connected close to the mucosal surface with adjacent similar crypts to form long valleys. Less wide crypts open on elevated regions of the mucosal surface. The lamina propria of germfree animals is devoid of plasma cells but rich in mast cells. Germfree animals show hypertrophy of the tunica muscularis externa.In conventional rats the cecal lumen contains a large variety of morphologically different bacteria. However, the lumen of the crypts of Lieberkühn contains only one type of elongated bacteria, which are present in large amounts. This finding suggests that symbiotic relations may be of particular importance in the crypts of Lieberkühn in the cecum.Supported by research grants from the Swedish Medical Research Council (206), Knut och Alice Wallenbergs Stiftelse and Stiftelsen Therese och Johan Anderssons Minne.     

Gastrointestinal cells of IL-7 receptor null mice exhibit increased sensitivity to irradiation

IL-7 is a critical cytokine in the development of T and B cells but little is known about its activity on nonhematopoietic cells. An unexpected finding was noted in allogeneic bone marrow transplant studies using IL-7 receptor null (IL-7R alpha(-/-)) mice as recipients. These mice exhibited a significantly greater weight loss after total body irradiation compared with wild type, IL-7R alpha(+/+), mice. Pathological assessment indicated greater intestinal crypt damage in IL-7R alpha(-/-) recipients, suggesting these mice may be predisposed to gut destruction. Therefore, we determined the effect of the conditioning itself on the intestinal tract of these mice. IL-7R alpha(-/-) mice and IL-7R alpha(+/+) mice were irradiated and examined for lesions and apoptosis within the small intestine. In moribund animals, IL-7R alpha(-/-) mice had extensive damage in the small intestine, including marked ablation of the crypts and extreme shortening of villi following 1500 cGy total body irradiation. In contrast, by 8 days after irradiation, the small intestines of IL-7R alpha(+/+) mice had regenerated as distinguished by normal villus length and hyperplastic crypts. Following 750 cGy irradiation, IL-7R alpha(-/-) mice had a higher proportion of apoptotic cells in the crypts and an accompanying increase in the pro-apoptotic protein Bak was expressed in intestinal epithelial cells. These results demonstrate the increased radiosensitivity of intestinal stem cells within the crypts in IL-7R alpha(-/-) mice and a role for IL-7 in the protection of radiation-induced apoptosis in these same cells. This study describes a novel role of IL-7 in nonhematopoietic tissues.     

Influence of a cecal volume-reducing intestinal microflora on the excretion and entero-hepatic circulation of steroids and bile acids

From mouse fecal material we have isolated four strictly anaerobic bacteria which, when associated with germfree mice or rats, reduced the cecal volume by 80 and 60%, respectively. This cecal volume-reducing flora did not metabolize estrone-3-sulfate, taurolithocholate-3-sulfate or taurolithocholate but gnotobiotic rats associated with this particular flora (CRF-rats) excreted these compounds faster in feces plus urine than did germfree rats. The time needed for 50% excretion (t1/2) of orally administered estrone-3-sulfate was 32 h in germfree rats versus 13 h in CRF rats; for intraperitoneally injected taurolithocholate-3-sulfate the t1/2 was 63 h in germfree versus 17 h in CRF rats and for taurolithocholate the t1/2 was 199 h in germfree and 96 h in CRF rats. Association of germfree rats with the cecal volume-reducing flora did not change the cecal absorption rate of estrone-3-sulfate, but shortened the 50% small intestinal transit time of [14C]PEG from 10 to 3 h; a value also found in conventional rats. These results stress the important influence of the intestinal microflora on the absorption and excretion of steroids via its effect on the physiology of the whole intestinal tract and point to the deficiencies inherent to the use of germfree animals in excretion studies.