The method of detecting petroleum ether-soluble sulfolipids by thin-layer chromatography after incubation with [³⁵S]sulfate is useful for differentiation between mycobacterial species. Rapidly growing mycobacteria, including two subspecies of Mycobacterium chelonei, were differentiated by this method. Most species of slowly growing mycobacteria were characterized by the pattern of distribution of radioactive sulfolipids in the thin-layer chromatograms. Mycobacterium nonchromogenicum was clearly differentiated from Mycobacterium terrae and Mycobacterium triviale by the presence of a sulfolipid. The latter two did not contain any significant amount of sulfolipids. Mycobacterium avium contained a large amount of sulfolipids, whereas Mycobacterium scrofulaceum did not contain any detectable sulfolipid. Rapidly growing, scotochromogenic mycobacteria, except for Mycobacterium flavescens, did not contain any remarkable amounts of sulfolipids.

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The fecal flora of 35 breast-fed and 35 bottle-fed babies was determined. Bifidobacteria were the predominant fecal bacteria in both groups. Conversely, the counts of most of the other bacteria, such as bacteroides, eubacteria, peptococci, veillonella, clostridia, enterobacteria, streptococci, and bacilli in the bottle-fed group were significantly higher than those in the breast-fed group. The frequencies of occurrence of lecithinase positive clostridia, clostridia-others, pseudomonas and bacilli in the bottle-fed group were significantly higher than those in the breast-fed group. Twenty-one genera and 103 species or biovars of microorganisms were isolated from the feces of the breast-fed group and 20 genera and 97 species or biovars from the bottle-fed group. The organism that showed the highest number and the highest frequency of occurrence in both groups was Bifidobacterium breve. Bifidobacterium infantis, which was formerly the most prevalent Bifidobacterium species in baby feces, was never isolated in this study. Further, the counts and incidences of Clostridium paraputrificum, C. perfringens, and Bacillus subtilis, the counts of C. clostridiiforme, Bacteroides vulgatus, Veillonella parvula, Lactobacillus acidophilus, Escherichia coli, Streptococcus bovis, S. faecalis, and S. faecium and the incidences of C. difficile, C. tertium, and Pseudomonas aeruginosa in the bottle-fed infants were significantly higher than those in the breast-fed infants.

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A tumoricidal effect was observed when protein A-bearing Staphylococcus aureus organisms were adsorbed on Ehrlich ascites tumor cells previously sensitized with antiserum from a rabbit immunized with Ehrlich ascites tumor cells. Electron micrographs showed that staphylococci were firmly attached to the tumor cells, which might explain how effectively the attached cocci killed the tumor cells. The tumoricidal effect was confirmed not only by an in vitro experiment but also by an in vivo one. The possible applications of the tumoricidal adsorption as an indicator for staphylococcal virulence or for selective anti-tumor action was discussed.

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A simple polysaccharide, the neutral mannan from Saccharomyces cerevisiae wild type strain (WNM) was found to kill ddY strain mice by intravenous administration, showing a LD₅₀ value of 12.2mg/kg. On the other hand, the acidic mannan fraction from the same yeast containing phosphate (WAM025), and chemically phosphorylated WNM (WNM-P) were practically non-toxic. Concerning the relationship between chemical structure and lethal effect of these mannans, it was demonstrated that a mannan possessing a highly branched structure exhibited stronger lethality than those with less branched structures. Against C3H/HeJ strain mice with no responsiveness to lipopolysaccharide, the LD₅₀ value of WNM was as high as 75mg/kg. Pretreatment with 500mg/kg of D-mannose, N-acetyl-D-glucosamine, D-galactose, and L-fucose prevented mice from the lethal effects of WNM. However, WNM (LD₁₀₀) did not show any lethal effect in mice for 2 to 12hr after treatment with dexamethasone, an anti-inflammatory steroid.

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A hybrid cell line, 3G6, producing monoclonal antibody (mAb) against the polyglycerophosphate (PGP) backbone of lipoteichoic acids has been derived by the polyethylene glycol-induced fusion of mouse myeloma cells and spleen cells from mice immunized with partially purified glucosyltransferase from culture supernatant of Streptococcus mutans strain 6715. Immunodiffusion tests and ELISA revealed that the antibody reacted with purified PGP from group A Streptococcus pyogenes strain Sv as well as crude phenol-water and saline extracts of various gram-positive bacteria except for a few species such as biotype B S. sanguis, Micrococcus sp., and Actinomyces viscosus. Whole cells of serotype b S. mutans and Staphylococcus epidermidis were agglutinated upon addition of 3G6mAb, while those of most other species were not significantly affected by this procedure. A hapten inhibition study showed that glycerophosphate was only a potent inhibitor of passive hemagglutination reactions between LTA coated sheep erythrocytes and 3G6mAb.

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Twelve monoclonal antibodies against Negishi virus were obtained and characterized by hemagglutination inhibition (HI) and neutralization (NT) tests using five flaviviruses isolated in the pan-Pacific region. The reaction pattern of the antibodies showed that Negishi virus was most closely related to Langat virus, followed by 3-Arch, JE and Apoi viruses in that order. Hemagglutinating (HA) antigen of the virus had distinct HI relating sites which were Negishi virus specific, tick borne encephalitis (TBE) virus complex specific and flavivirus cross-reactive. Monoclonal anti-Negishi antibodies cross-reactive to Japanese encephalitis (JE) virus in the HI test had neutralizing activity to JE virus but no activity to homologous Negishi virus.

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IFNγ production in cultures of spleen cells obtained from mice sensitized with TH69, a live Streptococcus faecalis preparation, was examined to determine how macrophages participate. It was demonstrated that sensitized spleen macrophages participated in enhanced IFNγ production by T cells at an early stage (0-6hr) of incubation, and that this production is mainly dependent on Ia-bearing macrophages. In the reconstitution experiments where different combinations of spleen macrophages and T cells obtained from mice sensitized with TH69, OK-432, and BCG were used, T cells required that the identity between the sensitizing organisms in vivo and the stimulating organisms in vitro be the same for enhanced IFNγ production while macrophages did not. Macrophage-mediated production of IFNγ appears to be genetically restricted because IFNγ was only produced in cultures where the H-2 region of macrophages and T cells matched.
Further examination revealed that for macrophages to participate in enhanced IFNγ production, first contact between cycloheximide-treated macrophages and T cells was required. Second, enhanced IFNγ production occurred when culture supernatants of macrophages obtained from sensitized spleen cells were added to T cells. However, the addition of culture supernatant obtained from sensitized peritoneal macrophages resulted in inhibition of IFNγ production.
These results clearly showed the crucial role of macrophages in enhanced IFNγ production by spleen T cells in vitro.

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A new mouse monoclonal antibody (HIEI, IgG1 type) that reacts with a cell surface glycoprotein of human lymphocytes was isolated. Membrane immunofluorescence assay showed that HIEI, like the anti-Tac monoclonal antibody, reacted preferentially with activated normal human T-cells and adult T-cell leukemia (ATL) virus (ATLV)-carrying human T- and B-cell lines. However, an interesting difference between HIEI and anti-Tac antibody was that HIEI did not react with ATLV-transformed simian cell lines or those cultured with interleukin-2 (IL-2), whereas the anti-Tac antibody did. The immunoprecipitation assay showed that both HIEI and anti-Tac antibody precipitated a glycoprotein with a molecular weight of 60, 000 daltons (gp60) from activated normal T-cells and ATLV-positive T- and B-cells, and also gp53 from MT-2 and MT-2-related T-cell lines transformed with ATLV in vitro by the MT-2 cocultivation method. HIEI inhibited the IL-2-dependent proliferation of normal T-cells, but its inhibitory effect was much weaker than that of the anti-Tac antibody. The anti-Tac antibody interfered with the binding of HIEI to target cells, but HIEI did not block binding of the anti-Tac antibody to the cells. These observations indicate that HIEI antibody recognizes a new antigenic determinant of the human Tac antigen.

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