Gelatin intake increases the atheroma formation in apoE knock out mice.

The effect of gelatin ingestion on cholesterol metabolism and on atheroma formation was evaluated in both wild type (n=14) and apoprotein E (apoE) knock out (apoE(-/-)) (n=20) C57BL/6 7-week-old mice. Animals were fed a cholesterol-free isoproteic semi-purified diet containing 20% of casein (control diet) or 10% of casein plus 10% of gelatin (gel diet) for 8 weeks. In wild type mice, dietary gelatin caused a reduction in the serum triacylglycerols levels associated with an increase in the fecal excretion. No difference in blood cholesterol was seen at the sixth week of experiment. At the eighth week of experiment, there was a modest but significant reduction of serum total and high density lipoprotein (HDL) cholesterol in apoE(-/-) mice fed on gel diet compared to the control. Total cholesterol/HDL cholesterol ratio was 2-fold higher in the gel group than that seen in the control group (14.39 and 7.84, respectively). Histological analyzes showed a 2.2-fold increase in the dimension of the atherosclerotic plaques in the proximal aorta in apoE(-/-) mice fed on a gel diet compared to those fed on a control diet. The gel diet also promoted a reduction in the fecal excretion of bile acids. Hepatic cholesterol was similar in both groups. In conclusion, although gelatin reduced total serum cholesterol, this reduction was associated to a decrease of HDL cholesterol and consequent increase of total cholesterol/HDL cholesterol ratio, resulting in an acceleration of atherogenesis.

Bacteriocin-like activity of Bacteroides fragilis group isolated from marmosets.

The ability of strains of the B. fragilis group, isolated from the oral cavity and intestine of marmosets, to produce bacteriorin-like substances in solid medium, in terms of auto-, iso- and heteroantagonism, was evaluated. Antagonistic activity was exhibited by 52% of the intestinal strains, 3 of which showed autoantagonistic activity. Three out of 9 oral strains isolated, tested against themselves, showed simultaneous isoantagonism to 4 indicator strains; but not autoantagonism. The same 9 oral strains, when tested against 16 reference strains, revealed interspecific activity only against 2 Gram-positive microorganisms. Higher activity, evaluated by the size of the inhibition halo, was observed in BHI-S agar, and greatest inhibition was obtained after 72 h of incubation.

Extraction, partial purification and characterization of a bacteriocin (fragicilin) produced by a strain of Bacteroides fragilis isolated from Callithrix penicillata.

A strain of Bacteroides fragilis, isolated from the marmoset Callithrix penicillata, produced protein(s) with bacteriocin activity (fragicilin). Two active fractions (36 and 150 kDa) were isolated by chromatography. The bacteriocin exhibited iso- and heteroantagonism. It remained stable between pH 3 and 10 and at 60 degrees C for 24 h. Pronase, trypsin, proteinase K and type VII protease inactivated the bacteriocin, giving evidence of its protein nature.

Microbiological and histological study of the gastrointestinal tract of germ-free mice infected with Helicobacter trogontum.

Helicobacter spp. have been the focus of considerable research because of the role of this genus in gastrointestinal diseases. We infected NIH germ-free mice with Helicobacter trogontum, a recently described intestinal bacterium of rats, in order to study the distribution of this bacterium in the gastrointestinal tract and the histopathological changes it can induce in this host. Sixteen mice were challenged with a single dose of H. trogontum (test group) and killed one and six weeks after inoculation (eight animals at each point). Eight animals were challenged with 0.85% saline alone (control group) and killed at the same time points (four at each point). Fragments from the gastric and intestinal mucosa were obtained for microbiological and histological examination. H. trogontum was isolated from the cecum and colon of all test mice and also from the gastric mucosa of several of them. All infected animals presented histological changes in at least one region of the bowel. Alterations in the gastric mucosa were also observed mainly in the six-week-infected group. The predominant histological change observed was a moderate diffuse inflammatory infiltrate of mononuclear cells in the lamina propria, often accompanied by a mild infiltration of polymorphonuclear cells. Two animals presented focal infiltration of inflammatory cells in the liver, although no bacteria were found in the liver of any animal. H. trogontum is an intestinal species that is able to elicit inflammatory responses in other regions of the gastrointestinal tract such as the gastric mucosa and the liver of gnotobiotic mice.

Antagonism against Vibrio cholerae by diffusible substances produced by bacterial components of the human faecal microbiota.

Cholera vibrios sometimes survive, probably in low-level silent populations, in the small intestine of chronic carriers or pass through the gastrointestinal tract of a few individuals without causing diarrhoea or colonisation. To understand these situations, the present study used plate cultures (ex-vivo test) to investigate the frequency of appearance of an inhibitory halo against Vibrio cholerae produced by faecal specimens from 92 healthy volunteers (40 females, 52 males) aged 4-61 years. The frequency of inhibitory halo was 20.6% in the whole group. An apparently higher percentage (27.3%) was observed in the age range 20-40 years when compared with the range 4-19 years (10.7%), but not the range 41-61 years (20.0%). Frequency was significantly higher in males (30.8%) than females (7.5%). The dominant microbiota of a volunteer whose faeces produced an inhibitory halo was isolated by plate culture of decimal dilutions in an anaerobic chamber. Potential isolates of 26 apparently different morphologies were associated with germ-free NIH mice. One week later, the inhibitory test showed an antagonistic halo around the faeces from the associated animals, but not from the axenic mice. Of the 26 bacteria isolated, two (Lactobacillus sp. and Peptostreptococcus sp.) produced a compound antagonistic against V. cholerae in an in-vitro assay. When bi-associated with germ-free mice those strains eliminated the vibrio from the intestinal ecosystem in c. 5 days.

A method of decontaminating Strongyloides venezuelensis larvae for the study of strongyloidiasis in germ-free and conventional mice.

To study the possible influence of intestinal micro-organisms on the course of strongyloidiasis in mice, a method was developed to obtain axenic infective larvae of Strongyloides venezuelensis. Cultured larvae from conventional mice were treated with sodium hypochlorite 0.25% for 10 min, washed in distilled water and then exposed to various combinations of antibiotics for 30 or 60 min. Success was achieved with a combination of penicillin 180 mg/L and ceftazidime 1 mg/ml. Decontamination of the larvae was determined by aerobic and anaerobic culture and by inoculation into gnotobiotic mice. Viability was established by subcutaneous inoculation of larvae into germ-free and conventional mice. Preliminary results showed that gnotobiotic mice were more susceptible than conventional mice to infection with axenic S. venezuelensis larvae as judged by faecal egg excretion, recovery of worms in the small intestine and histopathological examination of the duodenal mucosa. These results suggest that the normal intestinal flora protects the host against experimental infection with S. venezuelensis.

Experimental root canal infections in conventional and germ-free mice.

A small animal model was evaluated to study the interrelationships between microorganisms after their implantation in root canals (inferior central incisors) using germ-free (GF) and conventional (CV) mice. The selected microorganisms were: Porphyromonas endodontalis (ATCC 35406), Eubacterium lentum (ATCC 25559), Peptostreptococcus anaerobius (ATCC 27337), Fusobacterium nucleatum (ATCC 10953), Escherichia coli (ATCC 25922), and Enterococcus faecalis (ATCC 4083). Only P. anaerobius, E. coli, and E. faecalis, respectively, were able to colonize when inoculated alone into the root canal of both CV and GF mice. E. lentum, when inoculated alone colonized only in CV animals. P. endodontalis and F. nucleatum were unable to colonize in CV and GF animals after single inoculation. It is concluded that the experimental animal model presented herein is valuable for ecological studies of root canal infections and that only some strict anaerobic bacteria are able to colonize mice root canals when inoculated by themselves alone in pure culture.

Evaluation of microbial infiltration in restored cavities--an alternative method.

This work evaluated the efficacy of an improved method used to determine the frequency of bacterial infiltration and bacterial population levels and morphotypes in cavities restored with adhesive composites in conventional mice. By using the alternative methodology suggested in this work, bacteria from microleakage were recovered and identified in cavities subjected to restoration procedures that used acid etching of the dentin and dentin adhesives used with light-curing resin. The methodology presented herein seems to be more effective than the one normally used to investigate the presence of bacteria, which uses acid demineralization of dental structures for the histological processing of tissues. The results suggest that the methodology presented in this work made it possible to recover and identify Gram-negative and Gram-positive bacteria from microleakage. Frequencies of microleakage and bacterial population levels in restored cavities using two different adhesive systems were not statistically different (p < 0.05).

Heat or gamma irradiation sterilization affects the hypocholesterolemic effect of guar gum in axenic and heteroxenic rats.

1. Twenty-two axenic (germfree) or thirty heteroxenic (axenic colonized with human flora) 2.5-3.5 months old female Fisher rats were fed for four weeks either a hypercholesterolemic (HYPER) diet or a HYPER diet containing 5% guar gum (GG) sterilized by heat or by gamma irradiation. 2. Axenic rats fed the irradiated GG diet had higher cholesterolemia than their counterparts fed an autoclaved diet (4.50 vs 2.29 mmol/l), whereas the method of sterilization had no effect on plasma cholesterol in axenic HYPER or heteroxenic animals (7.35 vs 6.51 mg/dl). 3. The levels of hepatic esterified cholesterol were higher in heteroxenic animals fed the irradiated GG diet than in their counterparts fed the autoclaved GG diet (5.65 vs 3.57 mmol/g tissue). 4. The composition of volatile fatty acids in the cecal content of heteroxenic rats was dependent on the method of sterilization regardless of the presence of fiber: the levels of butyrate were 2.88 and 0.85 mumol/g for rats fed the autoclaved and irradiated diets, respectively. 5. Gamma irradiation abolished the cholesterol-lowering effect of guar gum, whereas sterilization by heat preserved this effect. 6. The hypocholesterolemic effect of guar was reduced by gamma irradiation sterilization and was probably mediated by qualitative changes in the intestinal microflora which interfered with bile acid absorption.

Parasitic infections in germfree animals.

The association of vertebrate hosts with the indigenous microbiota and its effect on the response to infections has long been a subject of scientific curiosity. From the first theory supported by Louis Pasteur that life would be impossible in the absence of associated microorganisms to the development of germfree mammals for research, a lot was learned about how the normal microbiota influences the environment in which pathogens may find themselves. In the present review, we attempt to summarize the more recent results from our group and others on the influence of the normal microbiota on the outcome of parasitic infections. Our results and those of others point to a complex relationship between the mammalian system and its indigenous microbiota, leading to greater resistance to some infections and enhanced susceptibility to others.

Intragastric infection of germfree and conventional mice with Salmonella typhimurium.

The present study investigates the lethality of intragastric Salmonella typhimurium infection of germfree (GF) and conventional (CV, not germfree) mice. The introduction of only 10 viable S. typhimurium into the gastrointestinal tract of GF mice resulted in death of all animals within 8 days of inoculation. In contrast, the 50% lethal dose for CV mice was 4.7 x 10(3) viable organisms. The results demonstrate that resident microbes protect mice from the pathogenic effect of S. typhimurium infection.

Intragastric infection of conventional and germfree mice with Giardia lamblia.

The effects of experimental infection with Giardia lamblia were studied in 30-day old conventional and germfree CFW mice (7 animals in each group) of both sexes. Cysts were observed in the feces of both groups 6 to 7 days after intragastric infection of each animal with about 2.5 x 10(5) G. lamblia trophozoites. Fecal cyst level was statistically higher in germfree mice (about 10(5) cysts/g feces) when compared with the conventional group (about 10(4) cysts/g feces). The peak of infection in the conventional group apparently occurred on the 10th day after infection as indicated by an increase of fecal weight and by histopathological examination. Intense infiltration of the lamina propria and high reactional hyperplasia of the lymphoid component were observed in the conventional group. There was no infiltration or hyperplasia in germfree infected mice and fecal weight was relatively constant throughout the experiment. These results suggest that, as is the case for other intestinal pathogenic protozoa, the intestinal microflora is indispensable for the expression of the pathogenicity but not for the multiplication of G. lamblia.

The acute phase of experimental infection with Trypanosoma cruzi is more severe in mice monoassociated with strict anaerobic bacteria.

1. The influence of some components of the normal human intestinal flora on the acute phase of experimental infection with strain CL of Trypanosoma cruzi was studied in 30-day-old germ-free or gnotobiotic CFW (LOB) mice monoassociated with Bacteroides fragilis, Peptostreptococcus sp or Clostridium sp by intragastric inoculation of 10(6) bacteria 10 days before the intraperitoneal infection with 5 x 10(3) trypomastigotes/g body weight. 2. Significantly earlier parasitemia peak and mortality were observed in Bacteroides fragilis- and Clostridium-associated mice (16.75 +/- 0.96 and 15.00 +/- 1.15 days, respectively) when compared with germfree animals (18.83 +/- 1.17 days). More precocious mortality (10.40 +/- 2.06 days) and, curiously, much lower blood parasitemia were observed in Peptostreptococcus-associated mice than in other gnotobiotic mice. 3. The extent of cardiac tissue parasitism decreased in the following order: germfree, B. fragilis-associated, Clostridium-associated, and Peptostreptococcus-associated animals. The levels of inflammatory reaction decreased in the following order: germfree, Peptostreptococcus-associated, Clostridium-associated, and B. fragilis-associated mice. 4. These results show that the acute phase of experimental infection with T. cruzi was more severe in mice associated with strict anaerobic bacteria when compared with germfree animals. This suggests that a normal intestinal flora may be another factor, in addition to nutritional and genetic factors, responsible for the different susceptibility of organisms of the same species infected with T. cruzi.

Immune responses of germfree mice to experimental infection with Trypanosoma cruzi.

1. The immune responses to Trypanosoma cruzi infection of germfree mice were compared to the responses of infected conventional mice. Two groups (40 animals in each group) of 2-month old female CFW germfree and conventional mice were used. The IgM and IgG which bound to the surface of T. cruzi epimastigotes determined by ELISA were significantly lower in germfree than in conventional mice (1/3 and 1/5 for IgM and IgG, respectively). 2. After infection there was a three-fold increase in the circulating levels of these immunoglobulins in germfree but not in conventional mice. Twenty-one days after T. cruzi inoculation, both IgG and IgM levels were similar in germfree and conventional animals. 3. Footpad swelling after T. cruzi-antigen inoculation was initially four-fold more intense in germfree than in conventional mice. 4. These results suggest that the reduced humoral immune response of germfree mice during the initiation of experimental Chagas' disease may be responsible for the more severe parasitism when compared to conventional mice.

Effect of an essential fatty acid deficient diet on experimental infection with Trypanosoma cruzi in germfree and conventional mice.

1. To investigate the possible role of essential fatty acid deficiency in host cell/parasite interaction, weanling germfree (GF) and conventional (CV) CFW mice maintained on an essential fatty acid-deficient (-) or a control (+) diet for 110 days were infected with Trypanosoma cruzi. 2. Blood parasitemia indicated that the disease was milder in the animals maintained on the essential fatty acid-deficient diet than in the GF and CV controls (maximum parasitemia: GF+ 33,300, GF-26,200, CV+ 17,100 and CV- 6,400 trypomastigotes/ml blood). 3. Survival 30 days after infection was 12% for GF+, 28% for GF-, 37% for CV+ and 65% for CV- mice. 4. Linoleic and arachidonic acid levels were significantly lower in animals kept on the essential fatty acid-deficient diet (GF-: 28.0 +/- 9.3, 23.4 +/- 8.6; CV-: 37.6 +/- 5.8, 19.9 +/- 3.6) than in controls (GF+: 164.4 +/- 48.8, 162.6 +/- 45.8; CV+: 147.1 +/- 26.5, 107.5 +/- 23.6) confirming the deficiency. 5. Before the infection, succinic dehydrogenase levels were higher in liver of all CV mice (4.52 micrograms phosphate/mg fresh tissue) than in GF mice (0.84 micrograms phosphate/mg fresh tissue), whereas the opposite was true for 5'-nucleotidase levels in brain and liver, respectively (GF: 2.84 and 3.18 micrograms phosphate/mg fresh tissue; CV: 1.25 and 1.54 micrograms phosphate/mg fresh tissue). 6. The disease was milder in deficient than in control animals in both the GF and CV groups on the basis of parasitemia and survival, indicating that fatty acid-deficient mice are partially protected against Chagas' disease. The mechanism underlying this phenomenon requires further investigation.

Monoassociation with Lactobacillus acidophilus UFV-H2b20 stimulates the immune defense mechanisms of germfree mice.

Probiotics are formulations containing live microorganisms or microbial stimulants that have some beneficial influence on the maintenance of a balanced intestinal microbiota and on the resistance to infections. The search for probiotics to be used in prevention or treatment of enteric infections, as an alternative to antibiotic therapy, has gained significant impulse in the last few years. Several studies have demonstrated the beneficial effects of lactic acid bacteria in controlling infection by intestinal pathogens and in boosting the host's nonspecific immune response. Here, we studied the use of Lactobacillus acidophilus UFV-H2b20, a lactic acid bacterium isolated from a human newborn from Viçosa, Minas Gerais, Brazil, as a probiotic. A suspension containing 10(8) cells of Lactobacillus acidophilus UFV-H2b20 was inoculated into groups of at least five conventional and germfree Swiss mice to determine its capacity to stimulate the host mononuclear phagocytic activity. We demonstrate that this strain can survive the stressing conditions of the intestinal tract in vivo. Moreover, the monoassociation of germfree mice with this strain for seven days improved the host's macrophage phagocytic capacity, as demonstrated by the clearance of a Gram-negative bacterium inoculated intravenously. Monoassociated mice showed an undetectable number of circulating E. coli, while 0.1% of the original inoculum was still present in germfree animals. Mice treated with viable or heat-killed Lactobacillus acidophilus UFV-H2b20 presented similarly improved clearance capacity when compared with germfree controls. In addition, monoassociated mice had twice the amount of Kupffer cells, which are responsible for the clearance of circulating bacteria, compared to germfree controls. These results suggest that the L. acidophilus strain used here stimulates a nonspecific immune response and is a strong candidate to be used as a probiotic.

Influences of alpha-tocopherol on cholesterol metabolism and fatty streak development in apolipoprotein E-deficient mice fed an atherogenic diet.

Although the role of oxidized lipoproteins is well known in atherogenesis, the role of vitamin E supplementation is still controversial. There is also little information about cholesterol metabolism (hepatic concentration and fecal excretion) in the new models of atherosclerosis. In the present study, we evaluated the effect of moderate vitamin E supplementation on cholesterol metabolism and atherogenesis in apolipoprotein E (apo E)-deficient mice. Apo E-deficient mice were fed an atherogenic diet containing 40 or 400 mg/kg of alpha-tocopherol acetate for 6 weeks. Total cholesterol in serum and liver and 3-OH-alpha-sterols in feces, and fecal excretion of bile acids were determined and histological analyses of aortic lesion were performed. A vitamin E-rich diet did not affect body weight, food intake or serum cholesterol. Serum and hepatic concentrations of cholesterol as well as sterol concentration in feces were similar in both groups. However, when compared to controls, the alpha-tocopherol-treated mice showed a reduction of about 60% in the atherosclerotic lesions when both the sum of lesion areas and the average of the largest lesion area were considered. These results demonstrate that supplementation of moderate doses of alpha-tocopherol was able to slow atherogenesis in apo E-deficient mice and to reduce atherogenic lipoproteins without modifying the hepatic pool or fecal excretion of cholesterol and bile acids.

Eggplant (Solanum melongena) infusion has a modest and transitory effect on hypercholesterolemic subjects.

Eggplant (Solanum melongena) is consumed extensively in Brazil. It has been believed that infusion of a powdered preparation of the fruit may reduce serum cholesterol. However, there are few documented reports on its effects on cholesterol metabolism and its possible hypocholesterolemic effect has not been proved by well-controlled studies. The aim of the present study was to observe the effects of S. melongena on the serum cholesterol and triglycerides of 38 hypercholesterolemic human volunteers ingesting S. melongena infusion for five weeks. Thirty-eight hypercholesterolemic subjects receiving either S. melongena infusion (N = 19) or placebo (N = 19) participated in two clinical experiments in which the effect of S. melongena infusion was studied with (N = 16) or without (N = 38) dietary orientation. Total cholesterol and its fractions, triglycerides, and apolipoproteins A and B were measured in blood at the beginning of the experiment and three and five weeks thereafter. No differences were observed compared to control. Intraindividual analysis showed that S. melongena infusion significantly reduced the blood levels of total and LDL cholesterol and of apolipoprotein B. After dietary orientation, no intra- or intergroup differences were seen for any of the parameters analyzed. The results suggest that S. melongena infusion had a modest and transitory effect, which was not different from that obtained with standard orientation for dyslipidemia patients (diet and physical activities).

Heat shock response reduces mortality after severe experimental burns.

The heat shock response has imparted protective effects in animal models of septic shock and endotoxemia. This study has tested the hypothesis that it could be protective in experimental burns. One hundred and fifteen adult male Fischer rats were randomly divided into four groups. Rats in the first group (n = 12) were anesthetized and shaved. In the second group (n = 15) rats were anesthetized and heated in a 45 degrees C water bath. In the third group (n = 44), rats were anesthetized, shaved and submitted to 26-30% body surface third-degree burns using a brass bar. In the fourth group (n = 44), rats were anesthetized, heated and, 1 day after, they were burnt. Mortality rates were measured at 3, 7, 15 and 25 days. Liver and lung samples were collected from all groups for heat-shock protein 70 detection. Heat-shock protein 70 was positive in heated animals. No animals died in the first or second group. Heated and burnt animals showed significantly decreased mortality at days 3 (p < 0.05, Fischer's exact test) and at days 7, 15 and 25 (p < 0.01) after burns, when compared to unheated burnt animals. In conclusion, eliciting the heat-shock response significantly reduced mortality rates in this model of experimental burns.

Effect of dietary chitin on cholesterol absorption and metabolism in rats.

The effect of chitin at the level of 5% in the diet on cholesterol absorption and metabolism was studied in Wistar rats fed on diet containing beef tallow (7%) and cholesterol (1%). When compared with pair-fed controls, rats fed on diet containing chitin had: (1) similar weight gain and feed efficiency, (2) lower apparent protein digestibility, (3) equivalent liver steatosis, (4) reduced levels of liver triglycerides and cholesterol, (5) similar levels of serum and fecal cholesterol, (6) higher excretion of triglycerides in feces.