An anaerobic continuous flow (CF) culture method was used in order to study the effect of Peptostreptococcus magnus and Streptococcus parvulus, anaerobic gram-positive cocci which are members of intestinal bacterial flora, on growth and cytotoxin-activity of Clostridium difficile. The growth- and the cytotoxin activity-patterns of C. difficile in an established CF culture of P. magnus were similar to those of C. difficile alone. On the other hand, in the mixed culture system of C. difficile and S. parvulus, the cytotoxin levels were significantly lower as compared with C. difficile alone in spite of the fact that no differences existed between growth of C. difficile in mixed and single culture systems. The culture filtrate of P. magnus did not influence the growth and cytotoxin production of C. difficile, nor did that of S. parvulus have any effect on growth of C. difficile in static culture. The cytotoxin activity of C. difficile was, however, suppressed by the culture filtrate of S. parvulus. Furthermore, when P. magnus or S. parvulus was statically cultured in a medium containing cytotoxic culture filtrate of C. difficile, the toxin in the medium was not inactivated.

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Numerous lymphoblastoid cell lines (LCLs) which secreted antibodies against Pseudomonas aeruginosa (all Fisher's immunotypes and Homma's immunotype 1) were established by Epstein-Barr virus (EBV)-transformation of lymphocytes. Five LCLs were established as long-term culture lines and their properties were determined. These LCLs produced monoclonal antibodies to Fisher's immunotype 1 and 4 and Homma's immunotype 1, and their immunoglobulin classes were IgM, IgG, and IgA. We found that three monoclonal antibodies (G3-1, H7-2, and E10-1) among them successfully protected mice from the corresponding immunotype of P. aeruginosa infection. Their protective dose (PD₅₀) values were 0.5, 2.6, and 3.1μg immunoglobulin/mouse. These human monoclonal antibodies against P. aeruginosa prepared by EBV-transformation method will be a valuable aid for the treatment for severe P. aeruginosa infections.

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A method for specific removal of [³²P]orthophosphate (Pi) as phosphomolybdate-triethylamine complex was slightly modified by repeating the Pi precipitation procedures in the presence of unlabeled Pi, which resulted in a complete removal of ³²Pi (>99.98%). Using this modified method, we determined ³²P incorporation into acid-soluble compounds in order to evaluate the ATP-forming ability of Bacillus megaterium spores at the very early stage of germination. Addition of fructose as a substrate started the ³²P incorporation later than a few min after triggering germination. This delay of a few min was well coincident with the onset of 3-phosphoglycerate (3PGA) breakdown, indicating that fructose metabolism and the accompanying aerobic ATP formation were initiated only after fructose phosphorylation by the ATP derived from anaerobic breakdown of endogenous 3PGA. In contrast, addition of glucose started incorporation of ³²P into acid-soluble compounds immediately after germination. In the latter case, NADH generated by glucose oxidation to gluconate (catalyzed by glucose dehydrogenase) might serve as an initial ATP source without depending on 3PGA breakdown and glucose metabolism via the Embden-Meyerhof pathway.

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The effect of antibiotics on allergic reactions was studied in vitro using the release of histamine from human peripheral blood leukocytes (basophils) after incubation with anti-IgE. For the several antibiotics we tested, including beta-lactams and aminoglycosides, none had the capacity to enhance antigen-induced histamine release, but some of them (minocycline, polymyxin B, and fosfomycin) suppressed the release of histamine in a dose-dependent manner. Since fosfomycin has proved to be capable of suppressing IgE-mediated histamine release non-cytotoxi-cally, the effect of fosfomycin on histamine release induced by other secretagogues was further studied. The suppression of histamine release was also demonstrated when the leukocytes, preincubated with fosfomycin, were challenged with either Ca ionophore A 23187 or a synthetic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). We concluded that some antibiotics, particularly fosfomycin, have the capacity to suppress histamine release mediated by various secretagogues, suggesting they may possess an anti-allergic property as well as a bactericidal activity.

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With a view to clarifying the actual state of inapparent infection of tsutsugamushi disease, inhabitants of endemic and nonendemic areas were screened for anti-Rickettsia tsutsugamushi antibody (anti-Rt antibody) by the indirect immunofluorescence test. The anti-Rt antibody-positive rate in the inhabitants of the endemic area (about 50%) was statistically significantly higher than that in the nonendemic area (14.7%). The antibody titer in the inhabitants of the endemic area was 10-160, and the number of inhabitants showing a high antibody titer was 2-4 times larger than that of the nonendemic area. A total of 257 volunteers in the endemic area were analyzed for the changes in anti-Rt antibody titer over 1.5-2 years on an individual basis. An increase in the antibody titer was found in 20 inhabitants. There was no difference in the anti-Rt antibody-positive rate between male and female in either the endemic or the nonendemic area. The positive rate was also compared as to the distribution by 10 years of age. In the endemic area, there were no significant differences in the positive rate between any pair of 10-year age groups from 30s to 60s, whereas in the nonendemic area, the positive rate in the teen-age group was significantly lower than those in the age groups of 20 years or older. In Yamada district, the numbers of serum samples obtained from each age group were about the same, and the distribution of the positive rates showed a normal distribution. The nurse students having their homes in Toyama Prefecture were plotted on the map as for their anti-Rt antibody and geographical distribution. The results showed that many of them having homes in the endemic area were positive for the antibody, while some antibody-positives were scattered all over Toyama Prefecture.

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A serological survey of simian virus 40 (SV 40) was conducted by an immune adherence hemagglutination (IAHA) test in breeding monkeys. Of a total of 356 monkeys tested, 168 (47.2%) were seropositive. All 168 seropositive monkeys were detected from 224 monkeys which were bred or kept in Japan for a long time. In contrast, none of the 132 monkeys which were newly imported from Southeast Asia was seropositive. If a comparison was made in the same breeding place, the positive rate of 80.4% (111/138) of Japanese monkeys was significantly (P<0.01) higher than the 59.5% (25/42) among rhesus monkeys. The positive rate and the IAHA titers were higher in older age group (>5 years) but similar in male and female. These results indicated that SV 40 was highly prevalent among breeding monkeys in Japan.

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Defective proviruses of bovine leukemia virus (BLV) in the genomes of infected cells were investigated by using Southern blotting hybridization analysis with various portions of a cloned BLV DNA as probes. When nine independent tumors of enzootic bovine leukosis with a single proviral copy per cell were examined, a single defective provirus of BLV was found in one tumor and also in a bovine B cell line derived from this tumor. Hybridization analysis of this defective provirus revealed that it underwent deletion between the pol and env genes and contained no major deletion in the other regions.

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A simple quantitative method for determining bacterial adherence to epithelial cells was devised. The method involved incubation of fluorescein-labeled bacteria with oral epithelial cells. Non-adherent bacteria were subsequently removed by Percoll density centrifugation. The degree of bacterial adherence was then determined fluorospectrophotometrically by measuring the fluorescein extracted from the adherent bacteria. The degree of adherence corresponded well with the microscopic counts of adherent bacteria. The method proved to be suitable for assaying adherence of both gram-positive and gram-negative bacteria to human as well as animal epithelial cells.

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The histological localization and biochemical properties of the autoantigens relevant to experimental autoimmune ophthalmitis and thyroiditis were studied using sera from mice hyperimmunized with the corresponding tissue extract of syngeneic mice and Klebsiella O3 lipopolysaccharide (KO3 LPS) as a potent adjuvant. Specific antigens were detected in the lens of the eyeball by immunofluorescence test with sera from mice in which ophthalmitis had been induced and the antigens were lenticular proteins with molecular weights (MW) of 15, 000 (15K) to 25K, and 45K. The lenticular proteins with MW of 15K to 25K correspond to the subunits of crystalline. These findings clearly demonstrated that our experimental model for autoimmune ophthalmitis was classified as the lens-induced uveitis. The colloids of the thyroid follicles and the follicular cells were markedly stained by sera from mice in which thyroiditis had been induced. One of the autoantigens detected in the thyroid gland was biochemically consistent with a thyroglobulin subunit. It was also shown that these autoantigens detected in the present study were organ-specific but not species-specific. The nature of autoantigens in the eye and the thyroid gland is discussed.

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Dialyzable factor (DF) prepared from a ribosomal fraction of Salmonella typhimurium was tested for its ability to induce interleukin 1 (IL 1) and 2 (IL 2) production, in relation to acquired resistance, after an intraperitoneal injection of DF. IL 1 production in vitro by peritoneal macrophages of DF-treated mice reached the maximum 4 days after injection, at the time when the nonspecific local resistance via macrophages directly activated with DF became apparent (Kita et al, Microbiol. Immunol. 28: 807, 1984). Concanavalin A-induced IL 2 production by splenocytes of DF-treated mice reached the maximal level between days 6 and 8, and it could be enhanced even on day 14. Antigen-induced blastogenic responses of splenocytes from DF-treated mice reached the maximal level 14 days after treatment. Although DF did not show the mitogenic activity to normal splenocytes, T cells of DF-treated mice could respond to S. typhimurium. On the contrary, T cells of normal mice could respond to heat-killed cells of S. typhimurium when they were cultured with macrophages which had been directly stimulated in vitro with DF. Furthermore, T cells from DF-treated mice could respond to antigens of different species of bacteria, and especially to Listeria monocytogenes. These results suggest that T cells of DF-treated mice, being at the intermediate stage of activation via monokines including IL 1 which is produced by macrophages stimulated with DF, are able to proliferate immediately after the administration of challenging organisms as a second signal, and also that the specificity of the response may be defined by the challenging organisms.

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