Vibrio mimicus strains W-26768 (stool isolate) and N-1301 (environmental isolate) and Vibrio fluvialis strains AA-18239 (stool isolate) and M-940 (environmental isolate) were studied for virulence properties and lipopolysaccharide composition. All four strains were hydrophobic, produced cytotoxin, adhered to HeLa cells and showed mannose-sensitive agglutination of guinea pig erythrocyte. The strains were negative for enterotoxin production and were mostly susceptible to the common antibiotics. The environmental and clinical isolates of both species were antigenically unrelated to each other. Lipopolysaccharide antigen analysis showed that O-antigen polysaccharides of two strains of V. fluvialis and two strains of V. mimicus differed with respect to the sugar components. Only LPS from V. mimicus W-26768 showed the presence of an unusual sugar, 3, 6-dideoxy-3-acetamido-hexose. The sugar compositions of these V. fluvialis and V. mimicus strains differed from those of previously reported Japanese isolates. These differences probably reflect differences in the serogroup of strains.

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A conjugative aminoglycoside resistance plasmid pST2 has been isolated from Escherichia coli K-12 14R525, which was mated with a clinical isolate of Salmonella typhimurium S24. A novel resistance gene of aminoglycoside 6'-N-acetyltransferase [AAC(6')] was cloned from plasmid pST2 on a 1, 393 kilobase (kb) of SphI-SalI fragment into vector pACYC184 and pUC18. This novel AAC(6') gene in plasmid pST2 acetylated kanamycin, amikacin, dibekacin, tobramycin, gentamicin, netilmicin, and sisomicin. The complete nucleotide sequence of the novel AAC(6') gene and its neighboring sequences were also determined. Minicell experiments detected only one protein of 24.7 kilodaltons (kDa) translated from an open reading frame of the 618 base pairs (bp) gene.

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Through hemagglutination tests two isogenic strains of Salmonella enteritidis were shown to possess type 1 fimbriae (strain V) and type 1 and type 3 fimbriae (strain A). The two strains bound to human buccal and mouse small intestine epithelial cells. Strain A attached to the epithelial cells more readily and in larger numbers in comparison to strain V. Adherence of both strains were sensitive to the presence of D-mannose and pretreatment of the epithelial cells with tannic acid did not promote D-mannose resistant type binding of strain A S. enteritidis to human buccal and mouse small intestine epithelial cells. Furthermore, results from LD₅₀ study indicated that, when the tests were carried out through oral inoculation of the mice the highly fimbriated stain A appeared to be more virulent. However, when the tests were carried out through intraperitoneal inoculation strain V was more virulent. These results indicate that adherence is a major contributing factor to the virulence of S. enteritidis and both type 1 and type 3 fimbriae contribute to this phenomenon.

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Bacteriostatic and bactericidal activities of rifampicin, isoniazid, streptomycin, enviomycin and ethambutol against Mycobacterium tuberculosis, Mycobacterium avium-M. intracellulare complex and Mycobacterium kansasii were studied in different growth phases. Bacteriostatic activities of the drugs were similar in different growth phases, except isoniazid. M. tuberculosis was much less susceptible to isoniazid in the lag phase than in the log and the stationary phases. In contrast, bactericidal activity was influenced by the growth phase. M. tuberculosis was killed by isoniazid, streptomycin and rifampicin. The bactericidal activity of isoniazid was strongest. The bactericidal activity of isoniazid and streptomycin was most marked in the log phase. M. avium complex and M. kansasii resisted the bactericidal activity, but some strains of M. avium complex were killed by streptomycin and enviomycin, and the activities of these two drugs were most marked in the lag phase.

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Rat defensins were purified and tested for in vitro bactericidal assay against gram-positive and gram-negative bacteria. Staphylococcus aureus (209P, Cowan I, Smith diffuse and Smith compact) were resistant to defensins, whereas Staphylococcus epidermidis, Staphylococcus saprophyticus, Micrococcus lysodeikticus and Bacillus subtilis were less sensitive. Gram-negative bacteria, such as Pseudomonas aeruginosa (mucoid and K) and Klebsiella pneumoniae (Chedid, 277, and 8N3 which were heavily capsulated, moderately capsulated and noncapsulated, respectively) were all very sensitive to defensins and killed within 20min. Escherichia coli was moderately sensitive and the rough mutants of lipopolysaccharide (LPS) of Salmonella typhimurium LT2, such as Ra, Rc, Rd, and Re were equally sensitive to defensins, being killed within 40min. Lysozyme did not show any bactericidal activity except against M. lysodeikticus and B. subtilis, whereas it enhanced the bactericidal activity of defensins against P. aeruginosa, E. coli, and K. pneumoniae and suppressed the killing activity of defensins against S. typhimurium and S. aureus. With regard to the three synthetic rabbit defensins, NP1, NP4, and NP5, NP1 showed strong bactericidal activity against K. pneumoniae 277, comparable to that of rat defensins, Neither NP4 nor NP5 showed any bactericidal activity, while NP5 rather enhanced the bactericidal activity of NP1 against K. pneumoniae 277.

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Biliverdin (BV), a bile pigment, was examined for its antiviral activity against human herpesvirus-6 (HHV-6) in vitro. BV (10μg/ml) markedly inhibited HHV-6 replication in MT-4 cells when the cells were treated during a virus adsorption period. Its antiviral effect was weakened when cells were treated after adsorption. Treatment of cells with BV (40μg/ml) 3hr after virus infection had no inhibitory effect on virus replication. Virus replication was also significantly inhibited by treatment of MT-4 cells with BV (10μg/ml) before infection, while the virions were not inactivated by BV (20μg/ml). Bilirubin and urobilin, metabolic derivatives of BV, showed slight inhibitory effects on virus replication in the cells. On the other hand, BV had no potent inhibitory activity in the replication of herpes simplex virus-1 or human cytomegalovirus. These observations suggest that BV could interact with MT-4 cells to inhibit an early stage of HHV-6 infection in a virus-specific manner.

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Twenty-one monoclonal antibodies were produced against promastigote antigens of Leishmania donovani. Five monoclonal antibodies (Hyb.17, 6, 5, 4 and 2) identifying molecules associated with various L. donovani antigenic determinants ranging from 42-116kDa were selected as ‘capture antibodies’ and compared with specific anti-leishmania antisera for detection of circulating leishmania antigens in kala azar patients' sera in a competitive-enzyme-linked immunosorbent assay system (ELISA). The anti-leishmania antisera could detect circulating antigen in 30% of kala azar cases while out of the five monoclonals, Hyb. 17 could effectively detect circulating leishmania antigen in 85.4%. The efficacy of Hyb.6 was however low (31.7%). The antigens recognized by these monoclonal antibodies in the western blot assay could possibly represent the ones circulating in sera of patients suffering from kala azar. A cocktail of these monoclonal antibodies may be more useful than the conventional polyclonal antisera in detection of circulating antigen for clinical diagnosis of kala azar.

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The 4D1D4 hybridoma cells were derived from the fusion of spleen cells from BALB/c nude mice with NS-1 mouse myeloma cells. The surface phenotypes of 4D1D4 hybridoma cells were Thy-1.2⁺, L3T4 (CD4)^-, Lyt-2 (CD8)^-, Asialo GM1⁺, and p-55 interleukin-2 (IL-2) receptor (CD25)^-. This phenotypic pattern was consistent with the surface phenotype of NK cells. The 4D1D4 cells showed the definite killer activity against a syngenic tumor cell line, RL_??_-1, but not against an allogenic YAC-1 line. The killer activity of the 4D1D4 cells was not affected by the addition of exogenous IL-2. It was, therefore, suggested that 4D1D4 cells might be representative of resting NK cells with expression of no functional IL-2 receptors. The hybridoma technology might be useful for establishment of the cloned NK cells.

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To investigate the initial stages of recognition of the self idiotype (Id) by T cells, we examined the early increase in cytoplasmic free calcium ([Ca²⁺]i) occurring in murine CD4⁺ T cells specific for a model Id, Id315, following their interaction with the Id. The changes in [Ca²⁺]i were monitored with stopped-flow fluorometry by loading T cells with fura 2, a Ca²⁺-binding fluorescent dye. An increase of [Ca²⁺]i in the Id-specific T cell line was dependent on the presence of both antigen-presenting cells (APC) and Id315. When T cells were mixed with APC pulsed with M315 for 90min at 37C, a significant increase in T cell [Ca²⁺]i was observed within one second. A pronounced elevation in [Ca²⁺]i was also observed in T cells after their interaction with APC which had been pulsed for 90min with V_L-315 Id-containing proteins (such as V_L-315, L³¹⁵, Fv-315 or Fab'-315 fragments). In contrast, pulsing APC for 5min with the V_L fragment produced little or no change in the [Ca²⁺]i. These results suggest that V_L must be further processed by APC before it can be recognized by T cells. Indeed, a synthetic V_L region peptide (positions 91-108, designated as P18) produced an elevation in T cell [Ca²⁺]i when mixed with APC without pulsing.

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A new simple purification method (I) for Vibrio cholerae non-O1 hemagglutinin/protease (NAG-HA/P) was developed. The method (I) requires only an immunoaffinity column chromatography using a monoclonal antibody against NAG-HA/P. The method (I) is much simpler than previously reported purification method (II) (Honda, T. et al, Infection and Immunity 57: 2799-2803, 1989) which required four or more complicated chromatographic procedures. Method (I) also gave an improved recovery rate (about 27%) compared with (II). The molecular weight of NAG-HA/P purified by method (I) was mainly 34 kilodaltons (kDa) with a little of 32kDa, whereas that of NAG-HA/P purified by (II) was usually 32kDa. Immunological analysis by the Ouchterlony double gel diffusion test and Western blotting test using polyclonal antibody against 32kDa protein revealed that the 34 and 32kDa proteins are immunologically indistinguishable and thus it is supposed that 34K protein is an isoform or a preform of the 32K protein.

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Spontaneous production of interferon-gamma (IFN-γ) was shown in several T-lymphoblastoid cell lines persistently infected with human T-lymphotropic virus (HTLV-1). However, the produced IFN-γ was not always associated with the induction of the antivirus state. The induction of oligo-2', 5'-adenylate synthetase (2-5AS) by IFN was studied in five human T-cell lines persistently infected with HTLV-I (MT-1, MT-2, SMT-1, HUT 102 and OKM-2). Four cell lines are able to produce IFN-γ spontaneously, while the OKM-2 cell line is not. Poor induction of 2-5AS was recognized in three (MT-1, MT-2 and SNIT-1) of the four cell lines producing IFN-γ, though the poor induction was improved after long-term cultivation of cells with IFN-α. On the contrary, in the OKM-2 cell line, significant activity of the enzyme was induced by IFN-α. Induction of 2-5AS was not correlated with cell growth inhibition, but with the antivirus state. Furthermore, an inverse relationship between IFN-γ production and 2-5AS induction was demonstrated in these cell lines with the exception of HUT 102 cells.

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