Production and Purification of Acid Protease from the Thermophilic Fungus, Penicillium duponti K1014

The use of combusted natural gas as atmosphere for cultivation of the anaerobic bacterial flora from the cecum of mice is described. The combusted gas (inert gas) continuously flushes a glove box in which it is possible to cultivate a number of strict anaerobes from the cecum of mice.     

Gas supersaturation in the cecal wall of mice due to bacterial CO2 production.

PCO2 in the lumen and serosa of cecum and jejunum was measured in mice. The anesthetic used was a fentanyl-fluanisone-midazolam mixture. PCO2 was recorded in vivo and postmortem. PCO2 was 409 +/- 32 Torr (55 +/- 4 kPa) in the cecal lumen and 199 +/- 22 Torr (27 +/- 3 kPa) on the serosa in normal mice. Irrigation of the cecum resulted in serosal and luminal PCO2 levels of 65-75 Torr. Cecal PCO2 was significantly lower in germ-free mice (65 +/- 5 Torr). Cecal PCO2 increased significantly after introduction of normal bacterial flora into germ-free mice. Introduction of bacterial monocultures into germ-free mice had no effect. After the deaths of the mice, cecal PCO2 increased rapidly in normal mice. The intestinal bacteria produced the majority of the cecal PCO2, and the use of tonometry in intestinal segments with a high bacterial activity should be interpreted with caution. We propose that serosal PCO2 levels >150-190 Torr (20-25 kPa) in the cecum of mice with a normal circulation may represent a state of gas supersaturation in the cecal wall.     

Susceptibility of Germ-free Mice to Infectious Megaenteron

Germ-free (GF)-ICR mice were shown to be less susceptible to oral inoculation with a pathogenic strain of Escherichia coli (E. coli 0115a, c: K(B)) than GF-CF#1 mice. In GF-CF#1 mice a large number of organisms were recovered from the intestinal wall from the cecum to the rectum 3 to 7 days after inoculation. Unlike those in GF-CF#1 mice, lesions in GF-ICR mice were localized in a part of the cecum and organisms were recovered only from the cecal wall and rarely from organs other than those of the alimentary tract. In both strains of mice, however, organisms were recovered in large number from the intestinal contents. Histopathology and immunofluorescence revealed organisms closely attached to the surface of the cecum, colon and rectal epithelia in GF-CF#1 mice but only in a part of the cecal epithelium in GF-ICR mice. After being in contact with conventional CF#1 mice for 21 days and then inoculated orally with the pathogenic E. coli, ex-GF-CF#1 mice died within 14 days with severe intestinal lesions, but ex-GF-ICR mice survived without lesions.     

Importance of cecum inClostridium botulinum colonization of mice and relationship of organism to large bowel

Germ-free mice, tested more than two weeks after removing their cecum, were at least 10⁴ times more resistant than controls to intestinal colonization byClostridium botulinum type A or B. Histologic examinations were done with the cecum and colon taken fromC. botulinum-monoassociated mice. Specimens from mice with severe botulism had normal appearance. In specimens from chronically monoassociated mice, the pathogen was in the lumen, but not attached to the intestine. Scanning electron microscopy suggested a possible association of the pathogen with the mucous gel lining.     

Effect of Bacterial Contamination on Cecal Size and Cecal Contents of Gnotobiotic Rodents

In the present investigation the effect of various bacterial contaminations of gnotobiotic mice and rats on cecal size is presented. Of the species tested, Bacteroides oralis and Fusobacterium nucleatum did not establish in germ-free mice. Streptococcus mutans, Clostridium difficile, a Neisseria strain and two recent cecal isolates established, but failed to exert an effect upon the cecum of mice. A group K streptococcus and B. fragilis increased the cecal size apparently by increasing the levels of water-soluble protein, peptides, and carbohydrates in the cecal contents. Mixed ileal bacteria decreased the cecal size by preventing accumulation of soluble proteins and carbohydrates in the cecum. A Peptococcus strain caused a reduction by lowering the levels of insoluble material in the cecum. When this strain was combined with two Clostridium isolates and introduced into gnotobiotic rats, 50 to 65% cecal reduction was observed. This polycontamination did not decrease the per cent water of the cecal contents but caused lower levels of both soluble and insoluble material to accumulate in the cecum. No net nitrogen absorption from the distal small intestine occurred in either the germ-free or polycontaminated rats.     

Luminal mucin in the large intestine of mice,rats and guinea pigs

Summary The luminal and epithelial mucin was studied histochemically in the large intestine of mice (Mus musculus), rats (Rattus rattus) and guinea pigs (Cavia porcellus) using freeze-substitution and vapor-fixation methods. Neutral mucin decreased and acid mucin increased in the epithelium from the cecum to the distal colon. Vacuolated cells contained more acid mucin than goblet cells. Luminal mucin always contained neutral mucin, which formed the main constituents in the cecum and in the proximal colon. Sialo-mucin increased from the cecum to the distal colon. Sulfo-mucin appeared only in the distal colon. Except in the cecum a luminal mucin layer (LML) was found at the epithelial surface. In the proximal colon LML was not entirely continuous and varied in composition and thickness (182.4 ± 170.1, 150.5 ± 110.4, 30.0 ± 28.9 (m), in mice, rats and guinea pigs, respectively), and contained many bacteria. In the distal colon LML was compact, homogeneous and thin (33.6 ± 18.8, 16.1 ± 7.3, 29.1 ± 20.0 (m), in mice, rats and guinea pigs, respectively) containing few bacteria. Possible functions of the luminal mucin and their regional differentiations were discussed.Supported by a grant from Deutsche Forschungsgemeinschaft (En 65/9). A preliminary part of this study was presented at 5th ISRP (September 1979), Clermond-Ferrand, France. Authors thank Miss G. Becker for her technical assistance     

The pattern of neural crest advance in the cecum and colon

Neural crest cells leave the hindbrain, enter the gut mesenchyme at the pharynx, and migrate as strands of cells to the terminal bowel to form the enteric nervous system. We generated embryos containing fluorescent enteric neural crest-derived cells (ENCCs) by mating Wnt1-Cre mice with Rosa-floxed-YFP mice and investigated ENCC behavior in the intact gut of mouse embryos using time-lapse fluorescent microscopy. With respect to the entire gut, we have found that ENCCs in the cecum and proximal colon behave uniquely. ENCCs migrating caudally through either the ileum, or caudal colon, are gradually advancing populations of strands displaying largely unpredictable local trajectories. However, in the cecum, advancing ENCCs pause for approximately 12 h, and then display an invariable pattern of migration to distinct regions of the cecum and proximal colon. In addition, while most ENCCs migrating through other regions of the gut remain interconnected as strands; ENCCs initially migrating through the cecum and proximal colon fragment from the main population and advance as isolated single cells. These cells aggregate into groups isolated from the main network, and eventually extend strands themselves to reestablish a network in the mid-colon. As the advancing network of ENCCs reaches the terminal bowel, strands of sacral crest cells extend, and intersect with vagal crest to bridge the small space between. We found a relationship between ENCC number, interaction, and migratory behavior by utilizing endogenously isolated strands and by making cuts along the ENCC wavefront. Depending on the number of cells, the ENCCs aggregated, proliferated, and extended strands to advance the wavefront. Our results show that interactions between ENCCs are important for regulating behaviors necessary for their advancement.     

Sex differences in susceptibility of ICR mice to oral infection with Corynebacterium kutscheri.

Sex difference in susceptibility to oral infection with Corynebacterium (C.) kutscheri was experimentally studied in ICR mice. Immature (4-week-old) and adult (14-week-old) mice were inoculated with two infecting doses of C. kutscheri, and necropsied for bacteriological and serological survey 4 weeks after the bacterial infection. No macroscopic lesions at necropsy were demonstrated, except for one adult male given 10(9) bacteria. In immature mice, C. Kutscheri isolated from the oral cavity and cecum with FNC agar, were recovered in only 40.0% of female mice but in 90.0% of male mice given 10(6) bacteria (p < 0.05), and in only 55.6% of female mice but in 80.0% male mice given 10(8) bacteria. In adult mice given 10(9) bacteria, the organism were recovered in only 45.5% of female mice but in 90.9% of male mice (p < 0.05), furthermore, the mean number of organisms in the cecum of male mice harboring the organism was significantly higher than that in females (p < 0.01). Castration caused an increase in host resistance in adult male mice. These results indicated that ICR male mice were more susceptible than females, in terms of bacterial colonization in the cecum and the oral cavity, to oral infection with C. kutscheri.     

Effects of Intestinal Contents from Normal and Immunized Mice on Sporozoites of Eimeria falciformis1

The interaction of Eimeria falciformis sporozoites with the intestinal epithelium and with the intestinal contents from the cecum and colon of normal and specifically immunized mice was studied by light (LM) and scanning electron (SEM) microscopy. Fecal (FM) and enterocyte-associated (EAM) mucus were removed from the cecum and colon of normal mice and mice that had been immunized 1, 6, 12, or 20 days earlier with a series of oral inoculations of E. falciformis oocysts. Sporozoite-specific IgA, but neither IgM nor IgG, was detected by the immunofluorescent antibody test in FM and EAM from immunized mice. No sporozoite-specific immunoglobulin was detected in normal mice. When examined by LM, sporozoites exposed to all FM and EAM preparations exhibited greater motility and excystation from sporocysts. At 4 h after incubation in FM or EAM from normal or immune mice, about 10% of the sporozoites appeared damaged, being non-motile and non-refractile. Immune FM and EAM caused agglutination of sporozoites and sporocysts and oocyst walls of E. falciformis. but did not agglutinate those of E. ferrisi. Scanning electron microscopy of in vitro interactions between E. falciformis sporozoites and intestinal contents revealed that sporozoites exposed to immune EAM were coated with particulate material whereas those exposed to normal EAM were relatively clean. Sporozoites exposed to immune FM were usually embedded within the mucus whereas those exposed to normal FM were situated on top of the mucus. No significant differences occurred between the length/width (L/W) ratios of sporozoites incubated in normal FM and EAM or in PBS. Sporozoites exposed to immune FM had significantly greater L/W ratios than those exposed to normal FM whereas those exposed to immune EAM had significantly shorter L/W ratios than ones exposed to normal EAM. Few of the sporozoites observed on the luminal surface of the colon and cecum of normal mice were covered by mucus and none was altered in shape or showed pellicular damage. Only a few sporozoites were observed on the luminal surface of the colon and cecum of immunized mice. Most of these were covered by mucus and some exhibited pellicular alterations.     

Characteristics of Bacteroides isolates from the cecum of conventional mice.

Bacteroides isolates from the cecum of conventional mice were characterized and grouped according to their ability to ferment or hydrolyze carbohydrates and other compounds believed to be present in the intestinal ecosystem. The isolates were divided into 11 groups on the basis of the fermentation of glucose, cellobiose, gum arabic and xylan (hemicelluloses), N-acetylglucosamine, and dextrin; the hydrolysis of starch and casein (proteolysis); and the production of indole. Stock cultures of B. fragilis, B. distasonis, B. ovatus, B. vulgatus, and B. ruminicola were characterized in the same way. The strains isolated most frequently from the mouse cecum resembled B. fragilis (except that arabinose was fermented) and B. thetaiotaomicron.     

Oral lactoferrin treatment resolves amoebic intracecal infection in C3H/HeJ mice

Entamoeba histolytica is a protozoan parasite that causes amoebiasis, an illness that affects many people around the world. We have previously reported that lactoferrin is able to kill E. histolytica in in vitro cultures. The aim of the present study was to evaluate the therapeutic effect of orally administered bovine lactoferrin in the control of intestinal amoebiasis of susceptible C3H/HeJ mice. The results showed that 20 mg lactoferrin/kg orally administered each day for 1 week was able to eliminate the infection in 63% of the mice, since neither trophozoites nor evidence of epithelial damage and (or) swelling were found in tissue sections of the cecum. The rest of the treated animals (37%) showed a decrease in trophozoite numbers and mucus secreted to the lumen, as compared with untreated and infected mice (p < 0.05). By immunohistochemistry, the profile of secreted cytokines in the cecum revealed that infected but untreated animals showed a mixed Th1/regulatory cytokines profile, whereas the cecum of mice treated (cured) showed a Th2 cytokine profile (IL-4) and expression of the multifunctional IL-6. In addition, cytokines and increasing cecal production of total IgA antibodies were found associated with little inflammation and disease control observed in the cecum of lactoferrin-treated animals. These results suggest that oral administration of lactoferrin can control intestinal amoebic infection probably by killing amoebas or favoring their removal and reestablish the antiinflammatory intestinal environment.     

WSX-1: a key role in induction of chronic intestinal nematode infection

Chronic infection by the gastrointestinal nematode Trichuris muris in susceptible AKR mice, which mount a Th1 response, is associated with IL-27p28 expression in the cecum. In contrast to wild-type mice, mice that lack the WSX-1/IL-27R gene fail to harbor a chronic infection, having significantly lower Th1 responses. The lower level of Ag-specific IFN-gamma-positive cells in WSX-1 knockout (KO) mice was found to be CD4(+) T cell specific, and the KO mice also had increased levels of IL-4-positive CD4(+) T cells. Polyclonal activation of mesenteric lymph node cells from naive WSX-1 KO or wild-type mice demonstrated that there was no inherent defect in the production of IFN-gamma by CD4(+) T cells, suggesting the decrease in these cells seen in infected WSX-1 KO mice is an in vivo Ag-driven effect. IL-12 treatment of WSX-1 KO mice failed to rescue the type 1 response, resulting in unaltered type-2-driven resistance. Infection of WSX-1 KO mice was also associated with a reduction of IL-27/WSX-1 downstream signaling gene expression within the cecum. These studies demonstrate an important role for WSX-1 signaling in the promotion of type 1 responses and chronic gastrointestinal nematode infection.     

Anesthetic Gases and Water Structure : The effect of xenon on tritiated water flux across the gut

Pauling and Miller have independently proposed that the presence of an anesthetic gas in tissue induces a cage-like arrangement of hydrogen-bonded water molecules. The theories recognize that most gas-hydrate crystals would not form at the temperature and pressure that exist during anesthesia and propose that other components of tissue such as protein should have a stabilizing effect. Measurements of the behavior of water, rather than the anesthetic agent, would provide alternative information about the likelihood of hydrate crystal formation and this information could be such as to be applicable to body temperature and to pressures used for anesthesia. If the number of hydrogen-bonded water molecules in tissue is increased, then the movement of an average water molecule should be hindered. Movement of water through the tissue may be measured by tagging it with tritium and the anesthetic gas should then slow the movement of tritiated water through the tissue. The flux of tritiated water through rat cecum is indeed slowed when the cecum is exposed to the anesthetic gas, xenon, which can participate biochemically only by virtue of its van der Waals interaction. The decrement in water flux is in reasonable agreement with what could be expected theoretically from calculations based on the activation energy for the self-diffusion of water and the degree of hypothermia necessary to produce narcosis.     

Experimental infection of specific-pathogen-free mice with enterohemorrhagic Escherichia coli O157:H7.

By subcutaneous inoculation of 10(8) CFU of enterohemorrhagic E. coli O157:H7, specific-pathogen-free mice revealed most of the symptoms and histological changes observed in patients. The histological changes in intestine were mainly seen in the distal parts of small intestine and the cecum. Vacuolation of villi in the cecum was also observed. The histological changes in the kidneys of the infected mice were featured as the swollen epithelial cells of glomeruli and the marked thickening of glomerular capillaries with barely visible lumens. Unexpected findings in the bronchiole were characterized by sloughing of the epithelial cells of bronchiolar wall, leading to partial or complete obstruction of the lumens. Histological changes in the spleen, liver and lymphnodes were also observed. The bacteria were recovered from the feces, contents of small intestine, and samples taken from kidney, liver, heart, spleen, different parts of small intestine, cecum, and colon. By using peroxidase-antiperoxidase (PAP) assay with polyclonal antibodies against "O" antigen of E. coli O157:H7, it was observed that the samples taken from the brain, kidney, ileum, cecum, spleen, and liver gave positive reactions. Feces and contents of small intestine obtained from all of the infected animals were positive by occult blood test. These results show that the experimental infection of E. coli O157:H7 in this model is systemic in nature.     

Gaseous disinfection of Cryptosporidium parvum oocysts.

Purified oocysts of Cryptosporidium parvum suspended in approximately 400 microliters of phosphate-buffered saline or deionized water in microcentrifuge tubes were exposed at 21 to 23 degrees C for 24 h to a saturated atmosphere of ammonia, carbon monoxide, ethylene oxide, formaldehyde, or methyl bromide gas. Controls were exposed to air. Oocysts in each tube were then rinsed and resuspended in fresh, deionized water, and 1 million oocysts exposed to each gas were orally administered to each of three to six neonatal BALB/c mice in replicate groups. Histologic sections of ileum, cecum, and colon tissues taken from each mouse 72 h after oral administration of oocysts were examined microscopically to determine if infection had been established. All 15 mice given oocysts exposed to carbon monoxide had numerous developmental stages of cryptosporidium in all three intestinal segments. Of 10 mice given oocysts exposed to formaldehyde, 6 had a few developmental stages of cryptosporidium in the ileum. No mice given oocysts exposed to ammonia, ethylene oxide, or methyl bromide were found to be infected. These findings indicate the efficacy of these low-molecular-weight gases (ammonia, ethylene oxide, and methyl bromide) as potential disinfectants for C. parvum oocysts where soil, rooms, buildings, tools, or instruments might be contaminated.     

Long-term colonization levels of Helicobacter hepaticus in the cecum of hepatitis-prone A/JCr mice are significantly lower than those in hepatitis-resistant C57BL/6 mice

Helicobacter hepaticus infection causes hepatitis in A/JCr mice but mild or no disease in C57BL/6 mice. Colonization of H. hepaticus in the cecum of experimentally infected A/JCr and C57BL/6 mice was quantified by use of real-time polymerase chain reaction (PCR) analysis with primers for the H. hepaticus cdtB gene and mouse 18srRNA. Eight-week-old mice were experimentally (n = 48) or sham (n = 24) infected with H. hepaticus, then were necropsied 6 months after infection. Liver specimens from experimentally infected mice had negative results of PCR analysis for H. hepaticus; thus, real-time quantification was not attempted. Quantitative PCR analysis of H. hepaticus in cecal specimens indicated that C57BL/6 mice were colonized to a greater extent than were A/JCr mice (P < 0.006). Appreciable typhlitis was not observed, but was consistent with that of previous reports; A/JCr mice developed more severe parenchymal necrosis, portal inflammation, and phlebitis in the liver (P < 0.0001), with mild disease observed in infected C57BL/6 mice. Thus, hepatitis in A/JCr mice caused by H. hepaticus infection is associated with significantly lower colonization levels of H. hepaticus in the cecum, compared with those of hepatitis-resistant C57BL/6 mice. Host responses of A/JCr mice that limit cecal colonization with H. hepaticus may have important roles in the pathogenesis of hepatic lesions.     

An automatic carbon-14 gas analyzer for organic compounds and biological samples

The present paper concerns automatic measurement of the radioactivity of substances labeled with carbon-14 isotope. The organic compounds or biological samples are combusted in an oxygen chamber. The oxygen excess is converted by hydrogen into water vapor in a reactor filled with copper and copper oxide. The water is retained by condensation and the carbon dioxide is swept by means of a counting gas, preferably propane or butane or a mixture of such gases, into an anticoincidence counter. The carbon-14 gas analyzer allows the measurement of carbon-14 isotope with high accuracy and sensitivity in a fully automated way, entirely free of manual intervention.     

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.     

A murine model of smoke inhalation

The United States has one of the world's largest per capita fire death rates. House fires alone kill >9,000 Americans annually, and smoke inhalation is the leading cause of mortality from structural fires. Animal models are needed to develop therapies to combat this problem. We have developed a murine model of smoke inhalation through the design, construction, and use of a controlled-environment smoke chamber. There is a direct relationship between the quantity of wood combusted and mortality in mice. As with human victims, the primary cause of death from smoke inhalation is an elevated blood carboxyhemoglobin level. Lethal (78%) and sublethal (50%) carboxyhemoglobin levels were obtained in mice subjected to varying amounts of smoke. Mice exposed to wood smoke demonstrated more dramatic pathology than mice exposed to cotton or polyurethane smoke. A CD-1 model of wood smoke exposure was developed, demonstrating type II cell hypertrophy, cytoplasmic blebbing, cytoplasmic vacuolization, sloughing, hemorrhage, edema, macrophage infiltration, and lymphocyte infiltration. The bronchoalveolar lavage fluid of smoke-exposed mice demonstrated a significant increase in total cell counts compared with those in control mice. These findings are comparable to the lung tissue response observed in human victims of smoke inhalation.