A new method of preparing a chlamydial complement fixation (CF) antigen by treatment with β-propiolactone (BPL) is presented. Chlamydia psittaci strains Pigeon-1041 and Budgerigar-No. 1, and Chlamydia trachomatis strain L2/434/BU, propagated in L-929 cell monolayers, were inactivated with BPL. This BPL-treated antigen was useful for detecting CF antibodies in both human and pigeon sera, and it did not cause false-positive reactions, as are sometimes observed between some human sera and phenol-treated antigen derived from eggs. When this CF antigen was treated with potassium periodate and tested for reactivity with mouse immune ascitic fluid, it was found that the antigen contained type- or strain specificity as well as genus specificity. Immunization with the BPL-treated antigen elicited type- or strain-specific neutralizing antibody.

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Viable cells of Saccharomyces cerevisiae 4484-24D-1 mutant strain were treated with an Arthrobacter sp. β-1, 3-glucanase, Zymolyase-60, 000, in the presence of a serine protease inhibitor, phenylmethylsulfonyl fluoride. Fractionation of the solubilized materials with Cetavlon (cetyltrimethylammonium bromide) yielded a purified mannan-protein complex, which had a molecular weight of ca. 150, 000, approximately three times higher than that of the mannan isolated from the same cells by the hot-water extraction method at 135C. The amino acid composition of the mannan-protein complex was found to be very similar to that of the mannan-protein complexes of S. cerevisiae X2180-1A wild and S. cerevisiae X2180-1A-5 mutant strains, indicating the presence of large amounts of serine and threonine. It was unexpected that the antibody-precipitating activity of this complex against the homologous anti-whole cell serum was about twice as great as that of the mannan isolated by hot-water extraction. Treatment of this complex with 100mM NaOH, hot water at 135C, and pronase, respectively, gave degradation products having the same molecular weight and antibody-precipitating activity as those of the hot-water extracted mannan, allowing the assumption that the protein moiety participated in a large part of this activity.

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The growing process and the fine structure of the cross wall of Staphylococcus were investigated by electron microscopy. Examination of the tangentially sectioned cross wall revealed that it was initially synthesized as a thin cell wall layer by an invaginated cytoplasmic membrane. The wall thickness soon increased by additional synthesis of the wall from the cytoplasmic membrane located at the side region of the cross wall. Scanning electron microscopic observation of sodium dodecyl sulfate-treated and mechanically separated cross walls revealed that the outer surface of the cross wall exhibits regular circular structures and the inner surface showed has an irregular surface. This indicates that cell wall materials were arranged in a regular circular manner in the initially synthesized thin layer. It is conceivable that in Staphylococcus spp. two cell wall synthesizing systems are present: wall-elongation synthesis in which wall materials are arranged in a regular circular manner and wall-thickening synthesis in which wall materials are arranged in an irregular manner.

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When 117 strains of Yersinia were grown in serial broth culture at 22 or 30C, 44 (38%) formed a broad-spectrum, mannose-resistant hemagglutinin (MR/Y-HA) associated with thick (8nm) channelled (MR/Y) fimbriae; eight other strains (7%) formed a narrow-spectrum, mannose-resistant hemagglutinin (MR/K-like HA) associated with thin (4nm) non-channelled (type 3-like) fimbriae. The distribution of these two fimbrial hemagglutinins in different Yersinia species is discussed and their properties are compared with those of other reported mannose-resistant hemagglutinins. The thick fimbriae of four Yersinia species were antigenically similar as judged by immunoelectronmicroscopy. Likewise, the thin fimbriae of three Yersinia species were antigenically similar, though different from the thick fimbriae.

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The specificity of endotoxin (lipopolysaccharide, LPS) in the carbocyanine dye reaction was investigated, and then a stoichiometric study of the dye-LPS interaction was conducted with attention to the relationship of biological activities of LPS to the reactivity with the dye. Absorption maxima of some bacterial components in the dye reaction were as follows; LPS from both Escherichia coli and Pseudomonas aeruginosa and lipid A from E. coli LPS, 465nm; Shigella flexneri LPS, 460nm; Salmonella minnesota R595 glycolipid, 470nm; polysaccharide from E. coli LPS, 650nm; yeast RNA, 620nm: streptococcal M protein and pyrogenic exotoxin, 610nm; and free fatty acids, 445-450nm. The absorbance at 465nm was increased approximately threefold by sonicating LPS for 1-3min, which roughly paralleled the decrease in turbidity of the LPS aqueous solution. The Limulus amoebocyte lysate (LAL) gelation activity of LPS increased 10-fold when LPS was sonicated for 0.5-5min, but it decreased to the control level after further treatment. This decrease, however, was overcome by sonication in the presence of 5mmol of L-ascorbic acid used as an antioxidant. The LAL gelation activity of LPS was inactivated in parallel with an increase in the ratio (w/w) of dye to LPS from 1.73 to 6.90 in the dye-LPS mixture. Pyrogenicity of LPS was also clearly inactivated when the ratio was over 1.73. The ratios of the height of the β band at 465nm (dye-LPS complex) to that of the α band at 510nm (free dye) were increased by sonicating LPS, indicating that the binding character, or stacking tendency, was increased by sonicating LPS.

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The protective antigen present on the flagella of Clostridium chauvoei was studied by the mouse protection test. A partially purified flagella preparation (PPF) showed protective antigenicity after two intraperitoneal injections of 2μg as protein, while the protective antigenicity of nonflagellated mutants (NFM) was 100-fold less than that of the flagellated parent strain. Although the protective effect of antisera against the whole cells and PPF, in terms of ED₅₀ values, was mostly lost after absorption with the parent strain, that of antisera after absorption with NFMs showed no appreciable loss. These results suggest that the flagella of Cl. chauvaei play some role in inducing protective immunity in mice.

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A human T-lymphoblastoid cell line, TCL-Fuj, constitutively produced a large amount of human gamma interferon (IFN) in culture fluids and has sustained stable IFN production for more than two years. When cells were incubated in RPMI-1640 medium with 10% fetal calf serum for three days, IFN activity was detectable at a cell density of 6×10⁴ cells/ml, whereas 2, 000-16, 000 units of IFN per ml were produced at 5-10×10⁵ cells/ml. IFN production was also detected even in serum-free medium and as early as 2hr after cultivation in fresh medium. IFN was inhibited by treatment of cells with either actinomycin D or cycloheximide, indicating the requirement of IFN-mRNA and protein for de novo synthesis. The molecular weight of the IFN was 45, 000-60, 000 as determined by Sephacryl S200 gel filtration. Two activity peaks corresponding to molecular weights of 22, 000 and 39, 000 were obtained by SDS-polyacrylamide gel electrophoresis, Analysis by isoelectric focusing revealed charge heterogeneity with four species at pIs of 6.0, 7.1, 8.6, and 9.3. Conventional IFN-gamma inducers, concanavalin A and 12-O-tetradecanoyl-phorbol-13-acetate, further enhanced the production of IFN in this cell line.

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Newborn mice and rats were inoculated intracerebrally (ic) or intraperitoneally (ip) with Hantaan virus (76-118 strain) or HFRS-related virus (B-1 strain). The mortality and the influence on the increase of body weight in newborn mice were higher in the groups infected with the 76-118 strain than in the groups infected with the B-1 strain, while the B-1 strain was more virulent in rats than the 76-118 strain. Virus isolation from rats inoculated with either strain was attempted 7 and 11 weeks after inoculation. Virus could be isolated from various organs of rats infected with the B-1 strain, while it was recovered from only the brain and lungs of rats infected with the 76-118 strain. Viral antigen was readily detected in various organs of rats infected with the B-1 strain, but the amount and distribution of antigens were less in rats infected with the 76-118 strain. Our results suggest that the virulence of HFRS-related virus is variable, depending on the species of infected animals as well as on the virus strains. The virus also persists in the injected animals with high titers of antibodies for at least 11 weeks.

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To examine the temporal relationship between the DTH skin reaction to HBsAg and antibody response to HBsAg, serial skin tests for HBsAg were carried out on seven individuals immunized with HB vaccine.
All cases developed both a skin reaction and antibody response. In many cases, the DTH skin reaction appeared four weeks before the antibody response. These data suggest that DTH skin reaction may be a more sensitive method than humoral antibody assessment for monitoring the success of vaccination.

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We examined the antigenic relationship between Candida parapsilosis and C. albicans serotype B with respect to antigenic factors 13 and 13b, specific for the former species and common to both species, respectively. Acetolysis of C. albicans serotype B cell-wall mannan gave six oligosaccharides. Their chemical structure was determined by ¹H-nuclear magnetic resonance (NMR) spectroscopy, methylation analysis, and partial acid hydrolysis. The structure of the hexasaccharide derived from C. albicans serotype B mannan was α-D-Manp-(1-2)-α-D-Manp-(1-3)-α-D-Manp-(1-2)-α-D-Manp-(1-2)-α-D-Manp-(1-2)-D-Man (M6) which is identical to that from C. parapsilosis mannan. Inhibition of two precipitin reaction systems (anti-C. albicans serotype B serum and anti-C. parapsilosis serum to the respective homologous mannan), by oligosaccharides from homologous and heterologous mannans indicated that M6 from either C. albicans serotype B or C. parapsilosis was the most effective inhibitor. Moreover inhibition of the agglutination reaction between factor serum containing anti-factors 13 and 13b and C. albicans serotype B or C. parapsilosis cells by oligosaccharides from both mannans also indicated that the M6s were the most effective inhibitors. These results suggest that the M6s derived from the two species are identical in their chemical structure, although the structures of the whole mannans of the two species are not identical as demonstrated by gel diffusion precipitation patterns, and that M6s may be involved in the specificities of antigenic factors 13 and 13b. The amount of M6 is larger in C. parapsilosis cell-wall mannan, suggesting that high repeating frequency of M6 fragment may induce the antibody specific for C. parapsilosis.

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The mechanisms of “panning, ” a simple positive cell separation technique, were examined. Using monoclonal antibodies to “pan” for T cells (T101⁺, T4⁺, and T8⁺), we obtained enriched populations with 90% purity and viability from unfractionated human peripheral blood lymphocytes (PBL). We found that the “panning” method reflects an active process. It is sodium azide inhibitable and independent of the divalent cation concentration. Effective panning does not require capping, patch formation, or DNA synthesis. The cell yields are unaffected by mitomycin-C or microtubule and microfilament blocking reagents such as concanavalin A, colchicine, and cytochalasin B. This economical technique provides large numbers of functionally intact monoclonal antibody-specific cells within a relatively short time for further functional and biochemical characterization.

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