The number of the nontypeable Haemophilus infuenzae (HiN) organisms that adhered to the primary mouse fetal lung cells was significantly more than type b Haemophilus influenzae (Hib) organisms. The average number of HiN organisms adherent to host cells was 2, 291/100 host cells (range, 1, 654-3, 182), but that of Hib was markedly reduced to 147/100 host cells (range, 102-238). In this case, P value was<0.05 by using a paired Student t-test. The sonicated extract from HiN TMS11 organisms inhibited adherence of H. influenzae TMS11 organisms to monolayer at 76.3% and it inhibited adherence of Hib TMS24 organisms at 92.3%. This result indicates that a mediator existing on the surface of HiN organisms may be the same as that on type b organisms. The number of detected organisms in broncho and lung tissues 3 days after intranasal infection with HiN strains was significantly greater than that in infection with Hib strains. Therefore, in vitro adhesive capacity of H. influenzae organisms was correlative to infectivity by intranasal injection.

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Chromosomal DNAs were extracted from Clostridium butyricum strain BL6340 and Clostridium botulinum type E strain Mashike. The 6.0Kbp fragment coding for the entire light chain (L) component and the N-terminus of heavy chain (H) component of botulinum type E toxin was obtained from each extracted DNAs after digestion with HindIII. The entire nucleotide sequences for the light chain components of these cloned genes were determined, and the derived amino acid sequences were compared to each other, and with those of botulinum type A, C1, D, and tetanus toxins reported previously. The cleavage site of L and H components of type E toxin was presumed to be Arg-422. In a total of 422 amino acid residues of L component, 17 residues were different between butyricum and type E toxins, and all these differences were found within 200 residues of N-terminus of L component. On the contrary, five regions showing highly homologous sequences were found in L components among these six toxins, and one more region between botulinum type E and tetanus toxins.

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Botulinum C₂ toxin (C2T) elaborated by certain strains of Clostridium botulinum types C and D is composed of separate and dissimilar two proteins, components I and II. Previous studies have shown that these two components of C2T produced by type C and D strains were immunologically heterologous and that C2T-producers were classified into three groups depending on the difference in molecular characteristics of the components I and II. In the present study, the heterologous component IIs of C2T were purified from three representative strains of the groups and the molecular characteristics of the components were compared. Immunological analyses by agar gel double immunodiffusion test showed that the component IIs purified from the three strains have the specific epitope(s) in addition to the common one(s). The biological activity of C2Ts reconstituted with component I purified from a fixed strain and component II each from the three strains differed depending on the source of the component II. These results indicate that the component II of C2T produced by C. botulinum types C and D differs in molecular structure, which reflects on the difference in the biological activity of the toxin. The present study suggests that the pathophysiological activity of C2T, which possibly causes a necrotic enteritis, is dependent on the C2T-producing bacteria infected.

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The authors measured the osmotic stability of liposomes prepared with membrane lipids of bacteria, using the osmotic-shock release of entrapped carboxyfluorescein as an indicator. The sub-second physical changes of liposomes suspended in a solution of low osmotic pressure were examined by stopped flow spectrophotometry. The entrapped carboxyfluorescein was released when the liposomes burst on inflow of excess water. Liposomes prepared with the lipids of a stable Staphylococcus aureus L-form strain were more resistant to low osmotic pressure than those prepared from the wild strain of S. aureus, and liposomes prepared from Mycoplasma orale were even more resistant. Cardiolipin enhanced the lipid membrane stability in S. aureus and cholesterol in M. orale. The stability of lipid membranes to low osmotic pressure could be precisely determined by the present method.

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The role of type 1 fimbriae in promoting bladder colonization and the course of Escherichia coli cystitis were examined with type 1 fimbriated strains of clinically isolated E. coli. In the experiments of mice in vivo, intact bladder epithelium showed natural resistance to the adherence of type 1 fimbriated and nonfimbriated E. coli. However, the exfoliation of bladder superficial cells by trypsinization before the bacterial inoculation promoted the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium. After colonization of E. coli, maximum numbers of E. coli and leukocytes were observed 3 days after inoculation. Nine days after inoculation, both of E. coli and leukocytes disappeared and the regeneration of superficial cells was observed. On the other hand, superficial cells in mice injected with phosphate-buffered saline or non-fimbriated E. coli regenerated 5 days after trypsinization. The present study demonstrated that the removal of superficial cells is essential for the adhesion and colonization of type 1 fimbriated E. coli onto bladder epithelium in vivo and a new model of E. coli cystitis in mice was established. The model which we established is valuable for histopathological, immunological, and therapeutic studies.

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Two virus clones were isolated from a defective SSPE virus, the Yamagata-1 strain, and designated as the YA and YF clones. The YA clone-infected cells produced neither cell-free virus nor cell-associated virus, whereas the YF cloneinfected cells produced both cell-associated and cell-free virus. No difference of epitopes on structural proteins was observed between these two clones. Both clones had hemadsorption activity. Quantitation of structural protein by Western dot blots showed relatively a lower amount of M protein in the YA-infected cells than that in the YF-infected cells. The ratio, P plus M dicistronic/M monocistronic mRNA, in the YA-infected cells was about twice that in the YF-infected cells. Sequence analysis of cDNA corresponding to P plus M dicistronic mRNA revealed that the deduced M protein of the YF virus was smaller than that of the YA virus by five amino acids from the carboxy terminal. These results suggest that abundant production of P plus M dicistronic mRNA is responsible for the reduced amount of M protein in the non-productive YA clone.

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A marked level of cell-mediated immunity (CMI) to Salmonella typhimurium-infection in mice, as determined by acquired resistance, delayed-type hypersensitivity, interleukin-2 production and interferon-γ production, was induced by immunization with porin or viable cells but not with killed cells of S. typhimurium LT2. When the up-regulation of immune system to each immunogen was studied by comparing increases of Ia-bearing macrophages, the immunization with porin or viable cells, but not killed cells, could stimulate the immune system for more than 14 days. Interleukin-1 (IL-1) production of macrophages to each immunogen was also examined; the result showed that immunization with porin or viable cells could induce a notable level of IL-1 production, while killed cells could not. However, when the abilities to induce these immune responses were compared between UV-killed and heat-killed cells, UV-killed cells were superior to heat-killed cells. These results suggested that the ineffectiveness of immunogen that lacked CMI-inducing ability might be ascribed to the denaturation of antigen and the insufficient inductions of Ia-bearing macrophages and IL-1 production.

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T cells that recognize the cross-reactive idiotype expressed on the heavy (H) chain of M104E (IgM, λ₁) were induced in BALB/c mice by immunization with Dextran B-1355. T cells derived from mice immunized with 1mg of Dextran B-1355 showed a marked proliferative response against M104E, whereas T cells from mice immunized with Ficoll or lesser amounts of Dextran B-1355 did not. BCL₁Id, which had an immunoglobulin istype identical to M104E, did not induce proliferation of the T cells. These T cells also proliferated against J558 (IgA, λ₁) which shared the cross-reactive idiotype of the anti-α (1→3) glucosidic linkage antibody with M104E on the H chain. The T cells proliferated more efficiently against F(ab')₂-104E, Fab-104E and H^104E, the H chain of M104E, than against intact M104E. The T cell proliferative response against the idiotype on M104E or even H^104E required macrophages as antigen-presenting cells (APC) and the response was inhibited when APC were treated with NH₄Cl or chloroquine, inhibitors of antigen processing. Moreover, anti-CD4 antibody or anti-Ia antibody inhibited the proliferative response. These results indicated that anti-idiotypic T cells of the helper type, which recognized a cross-reactive idiotype associated with Ia molecules in processed form, could be induced physiologically through a network mechanism.

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An S-layer (surface regular array) was found in the cell wall from six out of ten strains of Lactobacillus acidophilus examined by electron microscopic observations. All of the six strains which were shown to carry the S-layers belonged to the deoxyribonucleic acid (DNA) homology group A, but not to B, which had been classified by Johnson et al (Int. J. Syst. Bacteriol. 30: 53-68, 1980). On the other hand, the other four strains which possessed no S-layers were in the homology group B. The apparent molecular weights of the S-layer proteins ranged from 41 to 49 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the S-layer proteins from the six strains, three were susceptible to chemical cleavage with N-chlorosuccinimide, giving different peptide maps. All of the six S-layer proteins were fragmented by limited proteolysis with Staphylococcus aureus V8 protease, and gave markedly different peptide patterns by the subsequent peptide mapping analysis, except that the peptide maps of the S-layer proteins from the two strains which were in the same subgroup were identical.

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Lateral (L-) flagella-having vibrios were classified into 13 H-serogroups (flagellar antigen serogroups) by means of H-agglutination test. Vibrio parahaemolyticus was classified into 3 serogroups, HL1 to 3. V. alginolyticus and V. harveyi were classified into 5 and 3 serogroups, respectively, but 2 of those were serogroups common to the both species. V. fluvialis and V. furnissii constituted a same serogroup, HL8. Cross-reactivity between each serogroup was not observed in H-agglutination test, although some cross-reactivity was observed in gel diffusion test. Furthermore, similarity of DNA sequence of L-flagellar structure gene was demonstrated by dot blot hybridization test with a DNA probe of HL2 L-flagellar gene fragment. These results suggest conservation of DNA sequence of the L-flagellar gene of vibrios.

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Induction of interferon for a kind of dairy lactic acid bacteria, Lactobacillus acidophilus (L. acidophilus), was investigated in murine peritoneal macrophage (Mφ) cultures. Lactobacillus acidophilus JCM 1034, 1132^T, 1229 and 2125 induced IFN (12-34 I.U./ml) in Mφ cultures in vitro. Strain 1132^T- and 2125-induced IFNs were characterized as IFNα/β by treatment with anti-IFNs serum. The results indicate that the inducing activity of IFNs may be one of the available biological parameters for designating the dairy products containing L. acidophilus as “physiologically functional foods.”

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The Treponema pallidum hemagglutination assay (TPHA) is a widely used method for screening syphilis. We report our experience with six elderly (age 72.4±8.3 years) patients with syphilis, whose TPHA was negative. Their cardiolipin (RPR) and absorbed fluorescence treponemal tests (FTA-ABS) were positive. TPHA-negative patients with syphilis were compared with TPHA-positive syphilitics by immunological analysis. We found that both the numbers and the percentage of CD4 cells in TPHA-negative syphilitics were significantly lower than those in TPHA-positive syphilitics (722±142 vs. 1, 064±141/mm³, P<0.01:35.1±6.9 vs. 48.4±6.4%, P<0.01) and that the ratio of CD4 to CD8 was also lower in TPHA-negative syphilitics compared with TPHA-positive syphilitics (1.08±0.46 vs. 2.24±1.07, P<0.05). These data suggest that the TPHA is insufficient for excluding elderly syphilitics because of immunological impairment seen in aged patients.

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A gelatin particle agglutination (PA) test for Japanese spotted fever has been developed. Gelatin particles were sensitized with a sonicated causative rickettsia and used as antigens. The antibodies by PA test were detected as early as days 4-7 after the onset, whereas those by indirect immunoperoxidase (IP) test were detected after days 8-11. In addition, PA titers were higher than IP titers before days 20-23. The agglutinins detected by PA test were proven to be IgM because they were all sensitive to dithiothreitol. PA test was, however, less specific than IP test, giving a little nonspecific reaction to the sera from patients with scrub typhus and from individuals unrelated to those two rickettsioses. Nevertheless, PA test, which is simple, rapid, and easy to interpret the results, is useful for the early serodiagnosis of Japanese spotted fever.

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