The aqueous layer was isolated from Leptospira interrogans serovar canicola strain Moulton by the hot phenol-water method. After ultracentrifugation, the precipitate was designated as lipopolysaccharide-like substance (LLS) fraction and the chemical composition was compared with that of bacterial LPS. The LLS fraction consists of 35.2% carbohydrate, 3.8% amino sugar, 36.4% lipid, 15.2% protein, and 0.3% phosphorus. Neutral sugars were detected as rhamnose, arabinose, xylose, 4-O-methylmannose, mannose, galactose, and a small amount of erythrose, fucose and glucose by gas-liquid chromatography (GLC), but 2-keto-3-deoxyoctonic acid was not detected in the LLS by thiobarbituric acid test and high voltage paper electrophoresis. Fatty acids detected by GLC were decanoic acid (C10:0), dodecanoic acid (C12:0), dodecenoic acid (C12:1), tridecenoic acid (C13:1), tetradecanoic acid (C14:0), hexadecanoic acid (C16:0), hexadecenoic acid (C16:1), and octadecenoic acid (C18:1). With SDS-polyacrylamide gel electrophoresis, bacterial LPS showed many orderly bands, while the banding pattern of the leptospiral LLS was very simple. These findings demonstrate that the physicochemical properties and chemical composition of LLS fraction from Leptospira are different from those of LPS extracted from gram-negative bacteria such as Enterobacteriaceae, and suggesting that Leptospira has no typical LPS.

View full abstract

The biological activities of lipopolysaccharide-like substance (LLS) extracted from Leptospira interrogans serovar canicola strain Moulton by the hot phenol-water method were studied in mice. The addition of 12.5μg/ml or more of LLS fraction increased the incorporation of [³H]thymidine into in vitro cultured spleen cells of C57BL/6 mice, while the activity of the LLS fraction was about 20 times weaker than that of Salmonella typhimurium lipopolysaccharide (LPS). Pretreatment of murine spleen cells with rabbit anti-mouse thymocyte antiserum did not diminish the mitogenic activity of leptospiral LLS, and the LLS could not increase the incorporation of [³H]thymidine into thymocytes, suggesting that LLS acts on a B-lymphocyte population of lymphocytes. When sheeperythrocytes and LLS fraction were injected intraperitoneally into BALB/c mice, LLS exhibited an enhancing effect on antibody response in vivo. However, lethal toxicity of the LLS fraction was about 500 times lower than that of LPS in C57BL/6 mice loaded with galactosamine. No antitumor activity of leptospiral LLS (250-1, 000μg/mouse) against the ascites form of Ehrlich carcinoma in ddY mice was observed. The biological activities of the LLS fraction from the organism were weaker than those of gram-negative bacterial LPS, suggesting that Leptospira possesses no typical LPS.

View full abstract

The plasmid pNR333 is a kanamycin-resistant, deletion derivative of pNR 113 with an extremely high copy number in Escherichia coli and in Proteus mirabilis. In order to determine the usefulness of pNR333 as a replication gene of vector, the genes encoding chloramphenicol acetyl-transferase (CAT) and β-galactosidase (β-gal) were cloned individually into both pNR333 and other low-copy-number plasmids. The expression of the cloned genes was compared by measuring the specific activity of each enzyme and the amounts of the proteins produced. A hybrid plasmid pNR333-cat expressed 53 times as much activity of CAT as the low-copy plasmid S-a which had a copy number of four. The lacZ gene cloned in pNR333 produced 17 times as much β-gal as in the low-copy-number plasmid pNR1150. These results suggest that pNR333 is a useful vector plasmid for producing a large amount of polypeptides in E. coli hosts.

View full abstract

Legionella pneumophila in sterile distilled water was not detected after ultraviolet irradiation by FLONLIZER®, a new-type sterilizer, at a flow rate of 82.5 to 364.8 liters/hr. When irradiated by FLONLIZER® at a flow rate of under 324.0 liters/hr, no viable cells of legionellac, other heterotrophic bacteria and bacterivorous protozoa were detected in the cooling tower water, which was found to contain L. pneumophila. No viable cells of L. pneumophila and L. bozemanii suspended in sterile distilled water were detected after the irradiation with UV-doses of over 6.16×10³μW•sec/cm². At the irradiation of low UV-doses under 1.06×10⁴μW sec/cm², the viable count of legionellae recuperated by photoreactivation from UV-damage increased with the exposure time under a white fluorescent lamp. However, in the samples irradiated with UV-doses of over 3.52×10⁴μW•sec/cm², equal to the FLONLIZER®, legionellae did not recuperate even after 18hr illumination with a white fluorescent lamp. FLONLIZER® is thus expected to act as a sterilizer which can control the legionellae inhabiting cooling tower systems placed in outdoor space.

View full abstract

A new method to evaluate the adhesive ability of Vibrio cholerae O1 was proposed. Broth cultured V. cholerae O1 and a piece of formalin-fixed rabbit intestinal wall were incubated together in KRT buffer and the number of adhered organisms was counted under a scanning electron microscope. This method was much less laborious than other methods that have been used so far, and most significantly, constant results were obtained in repeated experiments. The adhesive properties of toxigenic V. cholerae O1 evaluated by this method correlated well with its observed experimental pathogenicity.

View full abstract

Effects of systemic and intestinal local immune responses in mice fed ad libitum and forcedly with live or heat-killed Vibrio cholerae on the elimination of vibrios from the intestine were investigated. In mice fed with live vibrios, ad libitum feeding could induce potential delayed-type hypersensitivity and rapid production of vibriocidal antibody in the serum whereas forcedly feeding suppressed the delayed-type hypersensitivity and retarded the antibody production. In contrast, when killed microorganisms were used as antigens, significant delayed-type hypersensitivity and rapid response of vibriocidal antibody were induced in forcedly fed mice although ad libitum feeding suppressed the induction of the delayed-type hypersensitivity and retarded the production of vibriocidal antibody. The elimination of vibrios from the intestine of mice was promoted in both mice groups fed ad libitum and forcedly with live vibrios but not with killed microorganisms. Total IgA in the intestinal contents of mice fed with live vibrios both ad libitum and forcedly were higher than those of mice fed with killed antigens. In addition, when the extracts of intestinal contents were absorbed by live antigens, IgA contents in mice fed with live vibrios were reduced more markedly than those in mice immunized orally by the feeding with killed antigens. These findings suggested that the elimination of vibrios from the organ was closely related to local IgA antibody response to heat-labile substance of live Vibrio cholerae.

View full abstract

Bovine enteroviruses isolated from cattle and other ruminants in various areas of the world were classified into three distinct serotypes by cross-neutralization tests using criteria established for the differentiation of human enteroviruses. According to Western blot analysis, however, immunodominant structural polypeptides VP1 of the viruses tested have common epitopes, recognized by antisera to each of the three serotypes. These findings indicate that non-neutralizing epitopes on VP1 are generally conserved. It is, therefore, conjectured that bovine enteroviruses were derived from a common ancestor.

View full abstract

We describe the quantitative measurement of antigen-specific clusters formed by antigen-pulsed macrophages and immunized T cells in mice. We have found the peripheral blood T cells show very little non-specific adhesion to macrophages in mice. By using this population of lymphocytes in the peripheral blood as the source of immunized T cells, we could quantitate antigen-specific cluster formation. On OVA-pulsed monolayers of peritoneal exudate macrophages from normal BALB/c mice, syngeneic peripheral blood T cells from donors immunized with the same antigen develop 20-40 clusters per 1, 000 macrophages, whereas the same T cells on non-pulsed monolayers develop only 0-5 cluster-like accumulations of cells. On antigen-pulsed monolayers of macrophages from allogeneic (C57BL/6 or A/J) mice, clusters are developed only in the negative range (0-5/1, 000 macrophages). Considering the observation by Braendstrup et al, these data seem to suggest that histocompatibility between macrophages and T cells is required to develop antigen-specific T cell clusters on antigen-pulsed macrophage monolayers, and that the genetic restriction of immune responsiveness may be directly expressed in this initial form of cellular interaction between antigen-bearing macrophages and specific T cells.

View full abstract

6B2-B8 T cell hybridoma cells were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated RTH-7, reacted with 89.5% of rat thymocytes, 30.2% of rat spleen cells, and 42.3% of rat lymph node cells. The RTH-7 reacted with a subset of rat T cells but not with B cells. Double staining analysis demonstrated that RTH-7 stained a rat T cell subset distinct from R1-10B5-positive cells that were known to be equivalent to mouse Lyt-2. It was revealed that RTH-7 and W3/25 recognize different antigenic epitopes on the same molecule. The RTH-7 as well as W3/25 substantially inhibited the production of interleukin 2 by cells in mixed lymphocyte reaction and the lymphocyte proliferation induced by mixed lymphocyte reaction. The RTH-7 inhibited the lymphocyte proliferation induced by Con A whereas W3/25 failed to do so. The RTH-7 defined antigen has a molecular weight of 53, 000 under reducing condition and 47, 000 ander nonreducing condition. The RTH-7 defined antigen showed a wide range of heterogeneity in pI (6.2-8.8). The associated molecule of approximate molecular weight of 27, 000 was occasionally detected with the RTH-7 defined antigen in 6B2-B8 T cell hybridoma cells as well as peripheral T cells but not in thymocytes. Thus, RTH-7 detects a cell surface antigen of a functional T cell subset of rat origin.

View full abstract

The three murine monoclonal antibodies (MAb), D1G2, D9D10, and D13C8, are specific for human interferon-γ (IFN-γ), but not human IFN-α and IFN-β. They react weakly with heat-treated IFN-γ. The three antibodies recognize different epitopes of the IFN-γ molecule, as evaluated by antibody-binding inhibition experiments. We have used these three monoclonal antibodies to construct a sandwich enzyme-linked immunosorbent assay (ELISA). The best result was obtained when we used D1G2 or D9D10 MAb as a solid-phase immunosorbent and D1G2 or D9D10 MAb as a tracer. When we measured IFN-γ in sera by a combination of D1G2 (a solid-phase) and D1G2 (a tracer), a result similar to the one by a combination of D9D10 (a solid-phase) and D1G2 (a tracer), was obtained. This may suggest that human IFN-γ exists in oligomeric form. Recombinant human IFN-γ expressed in E. coli is detectable at a concentration of 1ng/ml in this sandwich ELISA. This assay can be employed for the analysis of the structural characteristics of the human IFN-γ molecule as well as measurement of IFN-γ in human sera and tissue culture fluids.

View full abstract

The anti-TNP IgM plaque-forming cells (PFC) were generated in the spleen and bone marrow of non-immunodeficient normal mice after intraperitoneal administration of TNP-LPS. Irradiation of normal mice while shielding bone marrow completely abrogated the generation of bone marrow PFC, indicating that they are derived from extramedullary sites. The bone marrow PFC response to TNP-LPS was low in X-linked immunodeficient CBA/N strain mice, while the spleen response was comparable to that seen in the normal mice. To further study the basis of the deficient bone marrow PFC response in CBA/N mice, spleen cells were adoptively transferred to irradiated syngeneic mice stimulated with TNP-LPS. While spleen cells from normal mice generated high numbers of PFC in recipient bone marrow and spleen, those from CBA/N strain mice could not generate bone marrow PFC. This result was obtained regardless of whether normal or CBA/N recipients were used. These results indicate that TNP-LPS administration normally results in the migration of B lymphocytes from the periphery into the bone marrow and that B cells from immunodeficient CBA/N strain mice bear an inherent defect in this migratory function. This migratory defect was shown to be X-linked, as are the other previously reported B cell defects in this inbred mouse strain. The possible relationship between this migratory defect and the maturational defects of B cell lineage as reported previously in CBA/N strain mice is discussed.

View full abstract

Nineteen strains of Newcastle disease virus (NDV) isolated from wild ducks in Japan were placed into 4 distinct antigenic groups on the basis of their reactivities to 8 monoclonal antibodies against the HN molecule of NDV in hemagglutination inhibition tests. The NDV strains of duck origin were antigenically distinct from NDV-B1 and NDV-Miyadera originated from chickens, and varied in their virulence to chicken embryos. No apparent correlation was found between the antigenicity of the HN molecule and virulence.

View full abstract