The minimal inhibitory concentrations (MICs) of an antibiotic are present for only a certain period of time, after which they become sub-inhibitory concentrations (sub-MICs). These sub-MICs are still active because they can interfere with the mechanism of bacterial adhesion, which is the first step in the sequence of events leading to infection. The purpose of the present study was to investigate the effects of sub-MICs of ceftibuten, a new third-generation cephalosporin, on the adhesion of Escherichia coli (E. coli) to human buccal cells. The degree of inhibition was maximal at 1/2 MIC and then gradually returned toward to the control values at 1/128 the MIC. The differences were statistically significant from 1/2 to 1/32 MIC. Since the MIC was 0.5μg/ml, concentrations from 0.25 to 0.015μg/ml significantly reduce bacterial adhesion. Ceftibuten also caused marked elongation of E. coli. These findings could help to explain the efficacy showed by ceftibuten in the treatment of respiratory and urinary tract infections when administered once daily.

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Using a mouse monoclonal antibody (MAb) 2F raised against Vibrio cholerae non-O1 heat-stable enterotoxin (NAG-ST) which also recognizes a shared epitope of Yersinia enterocolitica heat-stable enterotoxin (Y-ST), a competitive enzyme-linked immunosorbent assay (ELISA) was developed for independent detection of NAG-ST and Y-ST. There was good concordance between the Y-ST ELISA and the suckling mouse assay (SMA) for detection of Y-ST from test strains of Y. enterocolitica, and the Y-ST ELISA can effectively replace the SMA for routine detection of Y-ST. On the contrary, evaluation of the NAG-ST ELISA and the SMA using 139 strains of V. cholerae non-O1 showed discordant results and this was attributed to the presence of the suckling mice active factor(s) such as El Tor hemolysin and to the production of low amounts of NAG-ST. Concentration of culture supernatants of V. cholerae non-O1 followed by heating at 100C was essential to obtain reproducible results by both the NAG-ST ELISA and the SMA. The ELISA developed in this study can be used for the identification of biologically active strains. While recently genetic methods such as polymerase chain reaction became available and were very reliable and simple techniques, the ELISA in this study has an advantage in detecting biologically toxic gene products of the strains. The genetic methods cannot differentiate silent STa genes which we often encounter in the case of Y. enterocolitica.

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Biphasic germination induced by inosine in the presence of Ca²⁺ was examined using Bacillus cereus T spores treated with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) at pH 10. The first phase of the germination was stimulated by Ca²⁺ in the concentration-dependent manner, showing the optimal concentration at 0.5-1.0mM. The second phase appeared to be insensitive to the cation. The optimal temperatures for the first and the second phase were 25C and 40C, respectively; the optimal pHs for the two phases were 7-9 and around 7.5, respectively. Heat resistance and dipicolinic acid of the SDS-DTT-treated spores were lost mostly during the first phase. A Ca²⁺-specific chelator, glycoletherdiamine-N, N, N', N'-tetraacetic acid (GEDTA), inhibited the first phase evoked by Ca²⁺, while it had no inhibitory effect on the second phase. In contrast, the divalent cations examined, except Mg²⁺ and Sr²⁺, affected not only the first phase but also the second phase. The order of inhibitory effect on the first phase was Hg²⁺>Zn²⁺>Ba²⁺, Co²⁺, Cu²⁺>Mn²⁺; on the second phase, it was Hg²⁺>Cu²⁺>Zn²⁺>Co²⁺>Mn²⁺>Ba²⁺.

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Escherichia coli heat-stable enterotoxin Ip (STp) is synthesized as the 72-amino-acid residue precursor consisting of three regions: pre region (amino acid residues 1 to 19), pro region (amino acid residues 20 to 54), and mature ST (mST) region (amino acid residues 55 to 72). We examined the role of the pro sequence of STp in enterotoxigenicity of a strain by deleting the gene fragment encoding amino acids 22 to 57. This deletion caused a remarkable reduction of its enterotoxic activity of culture supernatant. In order to analyze the sequence responsible for the function of the pro region, two additional deletion mutants were made. The deletion of the sequence covering amino acids 29 to 38, which is conserved in all sequences of ST reported, brought about a significant reduction of enterotoxic activity but the deletion of the non-conserved sequence (amino acids 40 to 53) did not. This result shows that conserved sequence is mainly responsible for the function. Subsequently, to examine the mechanism of action of the pro region, plasmids carrying DNA sequences of hybrid proteins consisting of pre-pro-nuclease, pre-mST-nuclease, pre-pro-mST-nuclease and pre-pro-nuclease-mST were constructed. Amino acid sequence determination and SDS-polyacrylamide gel analysis revealed that these fusion proteins were cleaved between pre sequence and pro sequence during secretion and the cleaved fusion proteins were accumulated in periplasmic space. But the amount of hybrid protein accumulated in the periplasmic space varied among the strains. That is, the amount of the pre-pro-nuclease gene product that accumulated in the periplasmic space was the highest of all fusion gene products. These results indicate that the existence of the mST region strongly interferes with the translocation of the gene product into the periplasmic space and that the pro region functions to guide the mST region into the periplasmic space.

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The serodiagnostic tests, beta-glucan, mannan, candida antigen by Cand-Tec, and D-arabinitol were evaluated in 10 patients with candidemia, 14 patients with suspected fungemia, and 10 healthy persons. By blood culture or lysis centrifugation, C. albicans was isolated from 5 patients, C. parapsilosis from 4, and C. tropicalis from 1 patient; no organisms were isolated from the 14 patients with suspected fungemia or the 10 healthy subjects. Beta-glucan was measured by the difference between two chromogenic limulus tests (Endotoxin test-D® and Endospecy®), which was more than 60pg/ml in 7 of 9 (78%) candidemic patients and 1 of 12 (8%) patients with suspected fungemia. Mannan was positive in 6 of 10 (60%) candidemic patients and 1 of 13 (8%) patients with suspected fungemia. Both antigens were very sensitive and highly specific for candidemia. However, the Cand-Tec assay was less specific, because titers of more than 4 were observed in 5 of 14 (34%) patients with suspected fungemia. D-Arabinitol was the least sensitive, because a D-arabinitol/creatinine ratio greater than 2.0μmol/mg was observed in only 2 of 7 (29%) candidemic patients. The titers of serodiagnostic tests decreased after successful treatment with an anti-fungal agent. Our results show that the combined use of the assays in necessary for accurate serological diagnosis of candidemia.

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The prophylactic effects by mouse nasal inoculation of 31 kinds of organic dye-inactivated Sendai viruses were investigated by contact exposure experiment that used mouse nasally infected with 10^5.8 EID₅₀, immunofluorescent examination of the entire respiratory tract, and check of rise of serum HI titer postexposure. The relative merits of the dye-structures for inciting nasal immunogenicity were determined. Of 14 azo-dye-inactivated vaccines, only azo blue- and amido black 10B treated ones brought about nearly complete protection, while the other 5 dye-groups provided partial protection and the remaining 7 dye-groups the least protection. Of 6 thiazole dye-vaccines, primuline-, thioflavine S-, and thioflavine T-vaccines induced complete or almost complete protection, and the others moderate or the least protection. With 3 quinoline-vaccines, both pinacyanol- and quinaldine red-inactivated ones provided complete protection, but not with quinoline blue-vaccine. Of 4 reactive dye-vaccines, both cibacron brilliant yellow 3G-P-and reactive blue 4-treated ones brought about nearly complete protection, but the remaining 2 vaccines induced regional infection. Nevertheless, all 4 naphthol group (AS, AS-BS, AS-BI, and AS-MX)-treated vaccines yielded complete or almost complete protection. The thirteen most effective vaccine groups suppressed marked rise of high or low serum HI titers developed through nasal vaccination postexposure. In short, specified dyestuffs having a great affinity for cellulosic fibers, evidently incited mucosal immunogenicity, probably by major union of the dyes to viral core ribose.

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The liver has been demonstrated to be a major site for extrathymic differentiation of T cells. In this study, an identification of CD5⁺ B cells, which are responsible for the onset of autoimmune disease by virtue of autoantibody production, was performed in autoimmune (NZB× NZW) F₁ mice. An age-associated increase of CD5⁺ B cells was demonstrated in the liver of these mice. Although CD5⁺ B cells (i.e., CD5⁺IgM⁺ and CD5⁺B220⁺) constituted a minor population of hepatic mononuclear cells (MNC) (<5%) when mice were young (8 weeks), a large population of CD5⁺ B cells (10 to 30% of whole MNC) was identified in the liver of mice aged 25 to 30 weeks after the onset of disease. Such age-dependent increase of CD5⁺ B cells was not observed in any other strains including NZB, NZW, C3H/He and BALB/c mice. The phenotype of hepatic CD5⁺ B cells was the same as that of CD5⁺ B cells in the peritoneal cavity and spleen, showing dull-CD5, bright-IgM and dull-B220. High levels of CD5⁺ B cells were observed in the peritoneal cavity and liver, but not in the spleen nor in any other lymphoid organs in mice aged 30 weeks. Radioimmunoassay of autoantibodies in the 5-day culture supernatants demonstrated that hepatic MNC were unable to produce any amounts of IgM- and IgG-autoantibodies against double-stranded DNA and single-stranded DNA, despite the increased proportion of CD5⁺ B cells. On the other hand, peritoneal exudate cells produced only IgM-, but not IgG-, autoantibodies, whereas splenic cells were able to produce both IgM- and IgG-autoantibodies. These results suggest that the liver might support the generation of the most primitive CD5⁺ B cells in these mice and that such generation increases as a function of age, probably resulting in the onset of autoimmune disease.

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Borrelia duttonii strain 406K, a causative agent of relapsing fever, could not be cultivated in vitro in currently available media for borreliae. We have developed an in vitro cultivation system by using SflEp cell cultures. The average increases of the number of borreliae, when inoculated with 1.0×10⁵ organisms per ml from infected mice, were 23-fold and 150-fold in the primary culture and the 3rd subculture, respectively. Even a single borrelia could propagate in this cultivation system. This system will be useful for immunological and physiological studies on uncultivable Borrelia strains.

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Field isolates of measles virus (MV) during an 8-year period in four areas of Japan, i.e., Osaka, Nagoya, Tokyo and Akita, were classified into three types in regard to the electrophoretic mobility of the hemagglutinin (HA) proteins: S type with small (78K) HA, M type with intermediate (80K) HA and L type with large (82K) HA. The type of field isolates was closely related with the geographical location and the year of virus isolation. The S type strain was isolated only in an outbreak from 1983 to 1984, whereas the M and L type strains were isolated between 1983 and 1990. The HA genes of the M and L type strains of MV were found to have a nucleotide substitution which introduces a new potential glycosylation site. In addition, the matrix proteins of all field strains isolated after 1977 showed slower electrophoretic mobility of 42K than 39K of the Edmonston and Toyoshima strains. These results indicate that MV strains of different HA types existed concomitantly and that major populations of MV currently circulating in Japan are changing from those prevalent in 1983-1984.

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Recently it has been reported that Japanese encephalitis virus (JEV)-specific RNAs can be synthesized in vitro in the subcellular fraction including outer-nuclear membrane (Takegami and Hotta, 1989). The results of Western blot analysis and indirect immunofluorescence test using two kinds of monospecific antisera against JEV nonstructural proteins NS3 and NS5 showed that NS3 and NS5 were membrane-associated proteins and formed the complex at the perinuclear site in the infected cells. Both antisera against NS3 and NS5 inhibited in vitro RNA synthesis. These results suggest that NS5 and NS3 play important role(s) in flavivirus RNA replication.

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