An oral killed cholera vaccine containing 1×10¹¹ cells of Vibrio cholerae O1 (heat- or formalin-killed) representing the Ogawa and Inaba biotypes and containing 1mg of B-subunit of cholera toxin (CTB) produced by recombinant DNA technology (the WC/rCTB vaccine) was subjected to temperatures of 4C, 30C or 42C for up to 6 months time. Lipopolysaccharide antigen (LPS) and CTB content of the vaccine samples determined at various times remained unchanged during the study except for the CTB component which decreased by about 50% after 6 months of storage at 42C. Immunogenicity determined by immunization of rabbits with the vaccine in Freund's complete adjuvant and measuring anti-LPS and anti-CTB antibody titers in the serum by an ELISA was also found to be unaltered. Lyophilization of the vaccine and storage at room temperature for 7 days also did not have any adverse effect on antigen content or immunogenicity as tested above. There was up to one log reduction in serum antibody titers after immunization without using any adjuvant or using Freund's incomplete adjuvant, and up to two logs following oral immunization. Immunization by oral feeding of the vaccine followed by RITARD challenge with a virulent V. cholerae O1 strain showed evidence of protection against severe or lethal diarrhea. The results suggest that the vaccine retains its antigen content and ability to induce antibodies unchanged when maintained at elevated temperatures for relatively long periods of time.

View full abstract

An experimental rodent model was used to demonstrate the viability of the coccoid form of Helicobacter pylori. Concentrated suspensions were prepared for the two different morphologies: at 2 days incubation for the bacillary forms and at 20 days incubation for the“dormant”forms. The strains used for incubation were two fresh isolates from humans with duodenal ulceration, and two collection strains. Five hundred microliters of culture (OD₅₅₀=5Mc Farland) of Helicobacter pylori with bacillary (2-5×10⁹CFU/ml) and coccoid (0CFU/ml) morphology were inoculated intragastrically in BALB/c mice. The gastric mucosa of the mice was colonized by Helicobacter pylori with the administration of fresh bacillary and coccoid cultures and not with the established cultures. Helicobacter pylori was isolated at 1 week after inoculation with the administration of fresh bacillary cultures, while fresh coccoid Helicobacter pylori was recovered in mice stomachs after 2 weeks of inoculation. After colonization, histopathologic changes occurred after 1 month from inoculation; all colonized mice showed a systemic antibody response to Helicobacter pylori. These results support the thesis of the viability of coccoid Helicobacter pylori non-culturable in vitro and confirm that concentrated bacterial suspensions are able to colonize and to produce gastric alterations in this suitable animal model.

View full abstract

The role of capsular polysaccharides (CPS) of Cryptococcus neoformans in phagocytosis by murine alveolar macrophages was investigated in four strains of C. neoformans serotype A, YC-11, YC-5, YC-27 and YC-13. Phagocytosis rates increased markedly after adding 10% mouse serum, compared to fetal calf serum. The reverse relation between capsular thickness of C. neoformans and phagocytosis by alveolar macrophages was observed except in YC-27, which had thin capsules and high virulence. The phagocytosis rate in mice serum was 17.3% in YC-11 (capsule thickness 2.8-3.5μm), 39.8% in YC-5 (capsule size 0.8-1.5μm), 20.3% in YC-27 (capsule size 0.6-1.1μm), and 62.8% in YC-13 (capsule not detected microscopically). The CPS of YC-11, YC-5, and YC-27 analyzed by gel-filtration using CL-2B showed high molecular fractions near the void volume. However, the CPS of YC-13 showed only low molecular fractions. The widely eluted CPS of YC-11 was separated into 3 fractions and each fraction was added in the phagocytosis assay of YC-13. Phagocytosis was markedly suppressed particularly by the addition of a higher molecular fraction. These results suggest that phagocytosis of C. neoformans by alveolar macrophages is influenced by the molecular sizes of the CPS.

View full abstract

The isolation of Chlamydia pneumoniae, especially from elderly persons, is generally not easy. Recently, we succeeded in isolating a chlamydial strain, which was designated KKpn-15, from a 57-year-old man suffering from acute bronchitis. It was compared with well established strains of C. pneumoniae, C. trachomatis and C. psittaci, and its biological properties, such as the morphology of elementary bodies (EBs) and inclusions, and the immunochemistry of EB proteins, were investigated. Based on the results obtained in the present study, it was confirmed that the new chlamydial strain, KKpn-15, is a member of the C. pneumoniae strain and that the organisms of KKpn-15 are useful as an antigen for the serodiagnosis and epidemiology of C. pneumoniae infection.

View full abstract

The PCR product amplified from Rickettsia japonica with the primer pair Rr 190.70p and Rr 190.602n of R. rickettsii 190-kDa antigen gene was cloned into M13mp19 RF DNA at the EcoRI site and sequenced by chemiluminescent DNA sequencing. The sequence revealed a molecular size of 533 base pairs (bp). The primer-flanking region of 491 bp, an open reading frame, was compared with the corresponding region of R. rickettsii, demonstrating 35 nucleotide substitutions in R. japonica. The sequence of primer portions in R. japonica DNA was also analyzed, revealing one nucleotide substitution in the Rr 190.70p and two in the Rr 190.602n portion. The homology in the overall sequence of PCR-amplified regions between R. japonica and R. rickettsii was 93% in nucleotide and 85% in putative amino acid structure. The sequence contains no cleavage site for the restriction endonuclease AfaI but two PstI sites giving three fragments of 121, 159, and 253bp, which differentiated R. japonica from other spotted fever group rickettsiae in addition to R. rickettsii. The cleavage sites for endonucleases AluI, HinfI, and MunI that disappeared or appeared in the sequence by nucleotide substitution differentiated R. japonica from others, as did PstI. The estimation of molecular size of DNA fragments on polyacrylamide gel electrophoresis is discussed.

View full abstract

Functional importance of Vpx protein of human immunodeficiency virus type 2 was evaluated in various types of cells. In 8 lymphocytic or monocytic cell lines tested, vpx mutant virus grew as well as wild-type virus. Only in primary peripheral blood mononuclear cell cultures, severely retarded growth of mutant virus was observed. No replication of vpx-minus virus was detected in primary macrophage cells. A highly sensitive single-round replication assay system was used to determine the defective replication phase in primary mononuclear cells of vpx mutant virus. In all cell lines examined, vpx mutant displayed no abnormality. In contrast, the vpx mutant was demonstrated to be defective at an early stage of the infection cycle in primary cell cultures. No evidence of a replication-defect at a late phase in primary cells of the vpx mutant was obtained by a transfection-coculture method. These results indicate that the virion-associated Vpx protein is essential for early viral replication process in natural target cells such as primary macrophages.

View full abstract

Thymocytes which have developed in the C3H thymus showed depressed proliferative responses to stimulation with anti-CD3 antibody as compared with those which have developed in the thymus of other strains of mice (i.e. AKR). The present study was conducted to analyze immunological functions of the thymic stromal cell population (low-density adherent cells, LDAC) in the C3H mice using allogeneic bone marrow (BM) chimeras established by BM transplantation in the reciprocal combination of AKR and C3H mice as donor or recipient. The thymic LDAC from C3H mice or the [AKR(donor)→C3H(recipient)] chimeras contained a high proportion of Mac1⁺ cells as compared to AKR mice or the [C3H→AKR] chimeras. The proportion of Mac-1⁺ cells paralleled the IL-1- and PGE₂-secreting ability of the LDAC cultured either in the presence or absence of LPS and also paralleled the antigen-presenting cell functions of the LDAC. Furthermore, after anti-CD3 stimulation the PGE₂ inhibited more profoundly proliferative responses of [AKR→C3H] or normal C3H thymocytes than those of the [C3H→AKR] chimera or normal AKR thymocytes. A PGE₂ inhibitor, indomethacin, reversed the depressed responses of the thymocytes which had developed in the C3H thymus. These findings suggest that the lower responsiveness of thymocytes from [AKR→C3H] chimeras to anti-CD3 stimulation may be attributable to large amounts of PGE₂ secreted by LDAC and/or to increased sensitivity of thymocytes themselves to PGE₂.

View full abstract

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1, 000kb to 30kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.

View full abstract

The growth of Helicobacter pylori, a Gram-negative microaerophilic bacterium, is often difficult and requires complex media with the supplementation of 5% to 10% blood or blood derivatives. We have found that Brucella broth supplemented with 1% heated horse serum and 0.1% β-cyclodextrin supports the good growth of H. pylori. The degree of growth and production of urease and vacuolating cytotoxin in this medium were equal to those in the medium supplemented with 5% horse serum. This medium was found to be suitable for both the routine laboratory culture and primary isolation of H. pylori from biopsy samples.

View full abstract

A polymyxin B (PXB)-resistant mutant of Klebsiella pneumoniae O3 was isolated. Lipopolysaccharide (LPS) extracted from the PXB-resistant isolate bound little PXB, although LPS from the parental strain did. The ³¹P nuclear magnetic resonance (NMR) spectrum of PXB-resistant type LPS showed that it contained much less of the phosphomonoesters and the pyrophosphate esters, and an increased amount of the phosphodiesters, compared to the parental type LPS. The decrease in the binding of PXB might be due to altered phosphate groups on the PXB-resistant type LPS, suggesting that it might explain the PXB-resistance of the mutant.

View full abstract

Hantaan virus often causes a fatal human disease, hemorrhagic fever with renal syndrome (HFRS). An assay for strand-specific detection and quantitation of Hantaan virus RNA was developed based on the polymerase chain reaction (PCR). A protocol for the efficient detection of both genomic RNA and its transcript is presented, in which a reverse transcriptase reaction (RTR) followed by PCR with two common primers was used to distinguish between positive-and negative-stranded RNA. Using this method, the growth characteristics of Hantaan virus, strain 76-118, were studied in Vero E6 cells. Positive-stranded RNA was detected on the next day after infection, and the amount increased remarkably beginning from 3 days after infection. The negative-stranded RNA (genomic), was detected at 2 days after infection which predominated over the transcript RNA. Three days after infection, the amount of viral RNA attained 80% of the maximum amount which was detected by 6 days after infection, while, 4 days after infection the amount of the positive-stranded RNA became over 80% of the maximum amount of 6 days after infection and reached the amount of viral RNA. Both RNAs reached plateaus at 6 days after infection and after that the amounts of synthesized viral RNA and its transcripts were constant.

View full abstract

This study was carried out to evaluate the effect of excreted/secreted antigens on macrophages infected by Toxoplasma gondii. Six proteins 28, 30, 45, 58, 63 and 145kDa were separated by different chromatographic techniques Mouse peritoneal macrophages were treated in vitro and in vivo with these purified fractions. Penetration and proliferation assays of T. gondii in the macrophages were performed in vitro. The different antigens used did not change the rate of penetration and proliferation of the parasites. Therefore, the secreted products, which are capable of provoking an immune response, could not directly activate the macrophages. Furthermore, the secreted products were not cytotoxic and neither did they possess a visible phospholipasic activity which would have increased penetration.

View full abstract

In a previous study, we observed that a cell-free Salmonella typhimurium extract induced suppression of mitogen-induced T-cell proliferation and that this suppression involved non-responsiveness of T-cells to interleukin-2 (IL-2) and augmentation of IL-2 receptor (IL-2R) expression. In this study, we found that inhibition of phytohemagglutinin (PHA)-stimulated murine spleen cell proliferation induced by a cell-free S. typhimurium extract was reversed by treatment with an anti-interferon-γ monoclonal antibody (anti-IFN-γAb), but not by interleukin-4 or N^G-monomethyl-L-arginine, which is known to inhibit nitric oxide (NO)-secretion from spleen cells in culture. However, IL-2R expression was augmented by treatment with the extract, although this was independent of an NO-mediated mechanism. Only anti-IFN-γ Ab treatment reduced the augmented IL-2R expression to a normal level. These results suggest that the suppression of T-cell proliferation induced by the Salmonella cell-free extract is associated with augmentation of IL-2R expression in an NO production-independent manner.

View full abstract