Yoshiyuki Morishita

Effects of age and starvation on the gastrointestinal microflora and the heat resistance of fecal bacteria in rats.

The microflora of the gastrointestinal tract in rats 1 day to 100 weeks old, of the cecal contents and wall in starved rats, and of heat‐treated feces of normal rats was determined by cultural examination. Streptococci, staphylococci, lactobacilli, actinobacilli, and coliforms colonized the tract during the 1st week of life. Bacteroidaceae, veillonellae, catenabacteria (composed of eubacteria and anaerobic lactobacilli), clostridia, bifidobacteria, anaerobic gram‐positive cocci, fusiform bacteria, curved rods, and spirochetes appeared when the rats were 2 to 4 weeks old. Yeasts were slower in colonizing the tract than any other organism. Dramatic changes occurred in the microflora of rats 2 to 4 weeks of age. There was a time lag between the changes in enterococcal and coliform populations. The enterococcal population was depressed over a period from 2 to 6 weeks of age. Bifidobacteria showed a larger population at 4 to 9 weeks than at any other age. The microflora of the stomach was the same as that of the small intestine, with some exceptions. It differed markedly from that of the cecum. The ratio of total aerobic count to anaerobic count gradually increased in the stomach, but decreased in the cecum, with advance in age. The microorganisms distributed in the tract could be divided roughly into 3 types. The population of each organism, except spirochetes, in the cecal wall was approximately 1/1,000 of that in the cecal contents. One of the 2 types of spirochetes was found only in the cecal wall and in a high incidence, forming a large population. In rats starved for 48 hr, coliforms, Proteus spp., anaerobic gram‐positive cocci, Clostridia, and some bacteroidaceae showed an increase in population in the cecum, but lactobacilli, veillonellae, and spirochetes decreased. The major organisms cultured from the heat‐treated feces were fusiform and curved bacteria, some members of Bacillus, minor anaerobic cocci, and straight rods.

HLA-DRB1*0410-Restricted Recognition of XAGE-1b37-48 Peptide by CD4 T Cells

XAGE‐1b belongs to cancer/testis (CT) antigens, and has been shown to be expressed frequently in lung cancers and to elicit an antibody response in patients with XAGE‐1b‐expressing tumors. In this study, we investigated an XAGE‐1b peptide recognized by CD4 T cells. CD4 T cells were purified from PBMC of a healthy donor and stimulated with pooled 25‐mer peptides overlapped with 15 amino acids spanning the entire XAGE‐1b protein. The generation of XAGE‐1b‐specific CD4 T cells was shown by IFN7 secretion assay. A CD4 T cell clone OHD1 was obtained by limiting dilution. OHD1 recognized two overlapping peptides, XAGE1‐b33–49 and XAGE‐1b37–52, by ELISPOT assay. A peptide XAGE‐1b38–46 which was included in both XAGE‐1b33–49 and XAGE‐1b37–52 was predicted to be a DRB1*0410‐restricted 9‐mer peptide by a computer‐based program. We identified the 12‐mer peptide XAGE‐1b37–48 as a new XAGE‐1b epitope restricted to HLA‐DRB1*0410.

Changes in the Intestinal Microflora in Association with Colon Tumorigenesis in Rats Treated with 1,2‐Dimethylhydrazine Hydrochloride

The present study was undertaken to determine whether the intestinal microflora change during the tumorigenic process in the colon of rats treated with 1,2‐dimethylhydrazone (DMH), and to compare the intestinal microflora of rats with colon tumors induced by DMH with that of rats with gastric tumors induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). When compared with those in the control animals, the numbers of streptococci and bacteroidaceae were moderately increased in the intestinal tract of DMH‐treated rats before the development of visible intestinal tumors. The DMH‐treated rats bearing small intestinal and colonic tumors were found to have markedly increased numbers of enterobacteriaceae, Clostridium perfringens, streptococci, bacteroidaceae, and bifidobacteria. In DMH‐induction the overgrowth of enterobacteriaceae and/or C. perfringens was found to correlate with the size and number of tumors in both the small intestine and colon. The increased number of streptococci in the DMH‐treated rats was principally due to an increase in the number of the streptococci which did not reduce triphenyltetrazolium chloride (TTC). On the other hand, in the rats with gastric tumors induced by MNNG the numbers of enterobacteriaceae and TTC‐reducing streptococci were remarkably increased in the intestinal tract of only the debilitated animals, and Pseudomonas aeruginosa was detected in all of them. The number of anaerobic gram‐positive cocci was significantly but not remarkably increased in the gastric tumor‐bearing rats compared with the controls. These results indicate that the intestinal microflora of rats may change depending on the gastrointestinal site where tumors develop and the degree of malignancy in tumorigenesis.

Mutagenicity of pyrolysates of salt-tolerant bacteria from food and cigarettes

Smoke condensates obtained by pyrolysis of the cells of microorganisms isolated from food and cigarettes and of laboratory cultures were shown to be mutagenic to Salmonella typhimurium TA98 and TA100 in incorporation with liver microsomal fraction S-9 mix. The smoke condensates from salt-tolerant bacteria, which belonged to Micrococcus, Bacillus and Corynebacterium spp., showed higher mutagenic activity than those from other less salt-tolerant organisms, fish flesh or pork. Smoke deriving from microorganisms, especially salt-tolerant ones, in food or cigarettes as well as from food components might take part in human carcinogenesis because of the correlation between mutagenicity and carcinogenicity.

Liability of the Athymic Nude Mouse to Intestinal Colonization by Pseudomonas aeruginosa

Brown and Balish (1) reported that there were no marked differences in the gastrointestinal flora between BALBjc athymic nude (nujnu) mice and their heterozygous (nuj +) littermates. Investigating the intestinal flora of BALBjc nujnu mice and their nuj+ littermates, however, we found that although there were no significant differences in the indigenous flora of the intestine between them, nujnu mice showed a remarkably high incidence of spontaneous colonization by Pseudomonas aeruginosa in their small intestines (91 % of 11 animals tested) and feces (100% of 13 animals) as compared with nuj+ mice (0% of 14 and 47% of 15, respectively) . The high incidence of P. aeruginosa colonization in the nujnu mice we observed might be due to a lack of a thymus-dependent immune system (6). Therefore, to confirm this finding nujnu mice were compared with their nuj+ littermates with respect to intestinal colonization by P. aeruginosa administered orally. BALBjc nujnu mice and their nuj+ littermates (6 to 10 weeks old) were used. They were produced in our laboratory by mating nude (nujnu) males and heterozygous (nuj +) females. The nujnu and nuj + mice were separately housed in metal cages with filter caps (Sanki Kagaku Kogei Co.) and kept on a rack with a laminar air filter unit (Sanki Kagaku Kogei Co.). They were given a 3-Mradirradiated diet (MB-3, Funabashi Nojyo) and given fresh tap water each day with the exception of the time of the administration of a P. aeruginosa culture. Cages and bedding were sterilized at 160 C for an hour. For administration of P. aeruginosa, animals were given drinking water containing, per ml, approximately 1 X 106 viable cells of P. aeruginosa grown overnight in brain heart infusion broth (Difco Laboratories). The drinking water bottles were autoclaved after two days of feeding. Feces and intestines with their contents, as bacteriological samples, were aseptically collected, weighed, homogenized, and diluted serially 10-fold in phosphate-buffered solution (pH 7.2). NAC agar (Eiken Chemical Co.) was used for enumeration of P. aeruginosa. P. aeruginosa strains were serologically identified with antisera obtained from Denka Seiken Co. Bacteria other than P. aeruginosa were not counted, because there would be no significant difference in their counts between the nujnu and nuj + mice as we found on preliminary examination.