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.     

Modulation of the rats' immune status by monoassociation with anaerobic bacteria

The capacity of a pure culture of anaerobic intestinal bacteria to influence the host's cellular and humoral immune systems was investigated with germfree, monoassociated, and conventionally reared rats. Monoassociation of germfree rats with Bacteroides fragilis stimulated the production of serum gamma globulin, agglutinating antibodies, and an apparent IgG (immunoelectrophoresis) band. A comparison of the in vitro blastogenic potential of lymphocytes (spleen cells and mesenteric lymph node cells) from germfree, monoassociated, and conventionally reared rats indicated the following: (1) the microbial flora had no obvious effect on the capacity of nonstimulated lymphocytes to incorporate [3H]thymidine; (2) spleen cells from conventionally reared rats responded to phytohemagglutinin, concanavalin A, or pokeweed mitogen better than splenocytes from germfree rats; (3) colonization of germfree rats with Fusobacterium necrophorum increased the responsiveness of splenocytes to photohemagglutinin and concanavalin A; and (4) monoassociation of germfree rats with B. fragilis, but not with F. necrophorum or propionibacterium acnes, increased splenocyte blastogenesis to homologous (i.e., colonizing) bacterial antigens. This study indicated that some intestinal bacteria can modulate the immune status of the host; the extent and nature of this modulation depended on the particular species of colonizing bacteria.     

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.     

Rapid Detection of Gram-Negative Bacteriuria by Use of the Limulus Endotoxin Assay

The Limulus in vitro endotoxin assay was evaluated as a possible method for the prompt detection of significant gram-negative bacteriuria in children. This assay is capable of detecting endotoxin associated with intact cell walls of viable gram-negative bacteria as well as free endotoxin. Quantitative results are obtained following a 1-h incubation of Limulus lysate and 10-fold dilutions of otherwise untreated urine. A standard curve of Limulus activity and viable cell counts of Escherichia coli and Klebsiella pneumoniae in urine demonstrated that a positive Limulus reaction at a dilution of 1:100 or 1:1,000 indicated a colony count of at least 100,000 bacteria/ml. A positive Limulus reaction only from undiluted urine or at a dilution of 1:10 indicated less than 100,000 cells/ml. These experimental observations were confirmed by comparing the Limulus test with quantitative plate counts on 209 urine specimens from a mixed pediatric population. These results indicate that the Limulus assay is a simple, accurate method for rapid presumptive detection of gram-negative bacteriuria in patients where an immediate diagnosis is needed. This test would also seem promising for screening large patient populations for bacteriuria or for monitoring the effectiveness of treatment of urinary tract infections.     

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.     

Studies on the sensitivity and specificity of the Limulus amebocyte lysate test and rabbit pyrogen assays.

The sensitivity and specificity of the Limulus amebocyte lysate test and rabbit pyrogen assay were studied by means of artificially contaminated parenterals. Various gram-negative and gram-positive bacterial strains were used as was one strain of the yeast Candida albicans. The numbers of organisms needed to elicit positive responses in distilled water and normal saline were recorded and compared. The sensitivity and specificity of the Limulus amebocyte lysate assay for the detection of bacterial endotoxin from gram-negative bacteria were demonstrated. Variable results were recorded with gram-positive bacteria and Candida albicans.     

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.     

Silicon Deprivation Does Not Significantly Modify the Acute White Blood Cell Response but Does Modify Tissue Mineral Distribution Response to an Endotoxin Challenge

An experiment with rats was conducted to determine whether silicon deprivation affects the acute-phase immune response to an endotoxin challenge. Weanling female rats were assigned to two weight-matched groups of 24; one group was fed a basal diet containing about 1.9 µg Si/kg; the other group was fed the basal diet supplemented with 35 µg Si/kg as arginine silicate inositol complex. After being fed their respective diets for 8 weeks, 12 rats in each group were injected subcutaneously with 1 mg lipopolysaccharide (LPS)/kg body weight; the other 12 rats in each group were injected with deionized water. Two hours after injection, the rats were anesthetized with ether for collection of blood (for plasma), liver and femurs, and then euthanized by decapitation. LPS injection decreased total white blood cell, lymphocyte, monocyte, eosinophil, and basophil counts by 80–90%, but did not affect neutrophil counts. LPS injection also increased plasma tumor necrosis factor-α and osteopontin and decreased plasma hyaluronic acid. Silicon deprivation did not significantly affect any of these responses to LPS. Silicon in liver and silicon, iron, and zinc in femur were increased by LPS injection only in silicon-deprived rats. Silicon deprivation also increased monocyte counts and osteopontin and decreased femur zinc in rats not injected with LPS. The findings indicate that silicon deprivation does not affect the acute-immune phase decrease in inflammatory cell numbers and increase in inflammatory cytokines in response to an endotoxin challenge. Silicon deprivation, however, apparently causes slight chronic inflammation and might influence inflammatory cell proliferation in the chronic-phase inflammatory response.     

Studies on the sensitivity and specificity of the Limulus amebocyte lysate test and rabbit pyrogen assays

The sensitivity and specificity of the Limulus amebocyte lysate test and rabbit pyrogen assay were studied by means of artificially contaminated parenterals. Various gram-negative and gram-positive bacterial strains were used as was one strain of the yeast Candida albicans. The numbers of organisms needed to elicit positive responses in distilled water and normal saline were recorded and compared. The sensitivity and specificity of the Limulus amebocyte lysate assay for the detection of bacterial endotoxin from gram-negative bacteria were demonstrated. Variable results were recorded with gram-positive bacteria and Candida albicans.     

Response of the blood clotting system of the American horseshoe crab, Limulus polyphemus, to a novel form of lipopolysaccharide from a green alga

Lipopolysaccharide (LPS, endotoxin) is a component of Gram-negative bacteria and is the principal indicator to the innate immune systems of higher animals of a Gram-negative bacterial invasion. LPS activates the blood clotting system of the American horseshoe crab, Limulus polyphemus. By stimulating blood cell degranulation, LPS triggers the release of the proteins of the clotting system from the cells, and by activating a protease cascade that converts coagulogen, a soluble zymogen, to coagulin, the structural protein of the clot, LPS triggers the production of the fibrillar coagulin blood clot. Although originally thought to be restricted to the Gram-negative bacteria and the cyanobacteria, LPS, or a very similar molecule, has recently been described from a eukaryotic green alga, Chlorella. Here we show that, like LPS from Gram-negative bacteria, the algal molecule stimulates exocytosis of the Limulus blood cell and the clotting of coagulin. The coagulin clot efficiently entraps the cells of Chlorella in a network of fibrils. Invasion and erosion of the carapace by green algae is an important cause of mortality of Limulus, and it is suggested that the cellular response to aLPS may contribute to defense against this pathogen.     

Effects of vagotomy on serum endotoxin, cytokines, and corticosterone after intraperitoneal lipopolysaccharide

The vagus nerve appears to play a role in communicating cytokine signals to the central nervous system, but the exact extent of its involvement in cytokine-to-brain communication remains controversial. Recently, subdiaphragmatic vagotomy was shown to increase bacterial translocation across the gut barrier and thus may cause endotoxin tolerance. The current experiment tested whether or not vagotomized animals have similar systemic responses to endotoxin challenge as do sham-operated animals. Subdiaphragmatically vagotomized and sham-operated animals were injected intraperitoneally with one of three doses (10, 50, 100 microg/kg) of lipopolysaccharide (LPS) or vehicle, and blood samples were taken at 15, 30, 60, 90, and 120 min after the injection. The intraperitoneal injection of LPS increased circulating LPS levels at all time points examined. In addition, all three doses of LPS significantly increased serum interleukin (IL)-1beta, IL-6, and corticosterone in both control and vagotomized rats. In conclusion, vagotomy itself has no marked effect on circulating endotoxin levels or the production of IL-1beta, IL-6, or corticosterone in blood after an intraperitoneal injection of LPS.     

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)     

Serum amyloid A, cytokines, and corticosterone responses in germfree and conventional mice after lipopolysaccharide injection.

To determine why germfree mice are less susceptible to lipopolysaccharide (LPS) than conventional mice, we studied serum levels of serum amyloid A (SAA), tumor necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and corticosterone in mice after treatment with LPS. A single injection of LPS caused an elevation of SAA, an acute-phase protein in the mouse, in both conventional and germfree IQI mice, and the response was significantly less in germfree mice. LPS-induced elevations of serum TNF, IL-1, and IL-6 levels were also significantly less in germfree mice, while serum corticosterone levels were greater in germfree mice than in conventional mice. These results suggest that the lower susceptibility to LPS and a smaller response of SAA elevation by LPS in germfree mice may result from less elevation in serum of these cytokines in these mice, which are known to mediate the acute phase response of SAA. High levels of serum corticosterone in germfree mice may be partly responsible for the lower responsiveness of these inflammatory cytokines to LPS in these mice.     

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.     

Catabolism and elimination of cholesterol in germfree rats

Three-month old germfree and conventional male rats were maintained on a complete steam-sterilized, semisynthetic diet. After intravenous injection of cholesterol-26-(14)C the animals were housed in a plastic metabolism chamber for 72 hr. Expired CO(2) was collected throughout the period. The conventional rats released 50% more (14)C as (14)CO(2) than the germfree animals. The total amount of the label recovered as (14)CO(2) during the 72 hr period amounted to 30% and 19% respectively, of the original dose. In both conventional and germfree rats the release of (14)CO(2) accounted for approximately 75% of the (14)C recovered in forms other than the original cholesterol-26-(14)C; 15-20% was found incorporated in water-soluble and fat-soluble fractions other than 3Beta-OH sterol of liver and carcass while the remainder was excreted with feces and urine. After the 72 hr period the specific activities of the cholesterol in plasma and liver were lower in conventional than in germfree animals. The data express the accelerating effect of the intestinal microflora on systemic cholesterol catabolism. They demonstrate that the release of (14)CO(2) from cholesterol-26-(14)C in the intact rat is a suitable and convenient indicator of the oxidative catabolism of cholesterol.     

Endotoxemia in newborn rats attenuates acute pancreatitis at adult age.

Bacterial endotoxin (lipopolysaccharide, LPS), at high concentration is responsible for sepsis, and neonatal mortality, however low concentration of LPS protected the pancreas against acute damage. The aim of this study was to investigate the effect of exposition of suckling rats to LPS on the course of acute pancreatitis at adult age. Suckling rat (30-40g) received intraperitoneal (i.p.) injection of saline (control) or LPS from Escherichia coli or Salmonella typhi (5, 10 or 15 mg/kg-day) during 5 consecutive days. Two months later these rats have been subjected to i.p. cearulein infusion (25 microg/kg) to produce caerulein-induced pancreatitis (CIP). The following parameters were tested: pancreatic weight and morphology, plasma amylase and lipase activities, interleukin 1beta (IL-1 beta), interleukin 6 (IL-6), and interleukin 10 (IL-10) plasma concentrations. Pancreatic concentration of superoxide dismutase (SOD) and lipid peroxidation products; malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) have been also measured. Caerulein infusion produced CIP in all animals tested, that was confirmed by histological examination. In the rats, which have been subjected in the neonatal period of life to LPS at doses 10 or 15 mg/kg-day x 5 days, all manifestations of CIP have been reduced. In these animals acute inflammatory infiltration of pancreatic tissue and pancreatic cell vacuolization have been significantly diminished. Also pancreatic weight, plasma lipase and alpha-amylase activities, as well as plasma concentrations of IL-1beta and IL-6 have been markedly decreased, whereas plasma anti-inflammatory IL-10 concentration was significantly increased in these animals as compared to the control rats, subjected in the infancy to saline injection instead of LPS. Caerulein-induced fall in pancreatic SOD concentration was reversed and accompanied by significant reduction of MDA + 4 HNE in the pancreatic tissue. The effects of LPS derived from E. coli or S. typhi were similar. Pretreatment of suckling rats with LPS at dose of 10 mg/kg-day x 5 days resulted in the most prominent attenuation of acute pancreatitis at adult age, whereas LPS at dose of 5 mg/kg-day x 5 days given to the neonatal rats failed to affect significantly acute pancreatitis induced in these animals 2 months later. We conclude that: 1/ Prolonged exposition of suckling rats to bacterial endotoxin attenuated acute pancreatitis induced in these animals at adult age. 2/ This effect could be related to the increased concentration of antioxidative enzyme SO in the pancreatic tissue and to the modulation of cytokines production in these animals.     

Distribution and metabolism of ingested NO3- and NO2- in germfree and conventional-flora rats.

Germfree and conventional-flora Sprague-Dawley rats were fed sodium nitrate or sodium nitrite in their drinking water (1,000 microgram/ml), and various organs, tissues, and sections of the intestinal tract were assayed for nitrate (NO3-) and nitrite (NO2-) by a spectrophotometric method. When fed NO3-, germfree rats had chemically detectable levels of NO3- (only) in the stomach, small intestine, cecum, and colon. Conventional-flora rats fed NO3- had both NO3- and NO2- in the stomach, but only NO3- in the small intestine and colon. When fed NO2-, germfree rats had both NO3- and NO2- in the entire gastrointestinal tract. Conventional-flora rats fed NO2- had both ions in the stomach and small intestine, but only NO3- in the large intestine. Conventional-flora rats fed NO3- or NO2- had lower amounts of these ions in the gastrointestinal tract than comparably fed germfree rats. Control (non-NO3- or NO2--fed) germfree and conventional-flora rats had trace amounts of NO3- (only) in their stomachs and bladders. These results, in conjunction with various in vitro studies with intestinal contents, suggest that NO3- or NO2- reduction is a function of the normal bacterial flora, whereas NO2- oxidation is attributable to the mammalian host. In addition, the distribution of these ions after their ingestion appears more widespread in the body than previously thought.     

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.     

Relationship between bacterial counts and endotoxin concentrations in the air of wastewater treatment plants.

The relationship between bacterial counts and endotoxin concentrations in air samples was studied. Selective EMB medium favored the growth of a larger portion of airborne gram-negative bacteria than LES Endo or MacConkey medium and was a good predictor of the endotoxin levels determined with a chromogenic Limulus assay of the air of wastewater treatment plants. The bacterial counts determined with nonselective media correlated poorly with airborne endotoxin levels; however, R2A medium yielded higher viable bacterial counts than TYG medium. Direct counting by epifluorescence microscopy yielded the highest bacterial counts, but no correlation was obtained between total bacterial counts and endotoxin concentrations.     

Relationship between bacterial counts and endotoxin concentrations in the air of wastewater treatment plants.

The relationship between bacterial counts and endotoxin concentrations in air samples was studied. Selective EMB medium favored the growth of a larger portion of airborne gram-negative bacteria than LES Endo or MacConkey medium and was a good predictor of the endotoxin levels determined with a chromogenic Limulus assay of the air of wastewater treatment plants. The bacterial counts determined with nonselective media correlated poorly with airborne endotoxin levels; however, R2A medium yielded higher viable bacterial counts than TYG medium. Direct counting by epifluorescence microscopy yielded the highest bacterial counts, but no correlation was obtained between total bacterial counts and endotoxin concentrations.