A method that stimulates cholera toxin (CT) production by Vibrio cholerae Ol biotype El Tor (El Tor vibrios) to the level of several micrograms per ml in the culture fluid was established. Such a large amount of CT was obtained by the following method: El Tor vibrios were cultured in AKI medium (1.5% Bacto peptone, 0.4% yeast extract-Difco, 0.5% NaCl, 0.3% NaHCO₃) at 37C for 4hr in a stationary test tube and then for 16hr in a shaken flask, with inoculum sizes of 10⁵ to 10⁷/ml. With this method, 35 strains out of 60 examined produced 2 to 16μg/ml of CT as determined by the reversed passive latex agglutination test (RPLA). Thirty-three randomly selected strains out of the 60 produced reasonable amounts of rabbit skin vascular permeability factor, reflecting the amount of CT titrated with RPLA.

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In a biosynthetic study of the spore coat of Bacillus megaterium ATCC 12872 spore with galactosamine phosphate as a major component of the outer coat, high-performance liquid chromatography (HPLC) and enzyme immunoassay were applied for the measurement of UDP-N-acetylglucosamine-4-epimerase [EC 5.1.3.7] activity and the enzyme protein concentration, respectively. The new HPLC system using an ion-pair (or anion-exchange) column allowed us to determine successfully the enzyme activity and its application, proving that the specific activity of the enzyme in the cells increased at the later stage of sporulation. This increase in activity was parallel to the induction of enzyme protein synthesis, which was detected by sandwich enzyme immunoassay using antiserum to the purified enzyme. These results suggested that the regulation of this enzyme is at the genetic level and it plays an important role in the outer coat synthesis in the later sporulation stage of B. megaterium.

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In spite of the ability of the genetic determinants for enterotoxin production to be conjugally transferred, mobilized or transposed, enterotoxigenic Escherichia coli (ETEC) isolated from diarrheal patients is restricted to certain serotypes. Four conjugative enterotoxigenic plasmids (Ent plasmids) encoding either a heat-labile enterotoxin or a heat-stable enterotoxin or both and belonging to one of three incompatibility groups IncFI, IncHl, or IncX, were examined for their transferability to and stability in 157 nonenterotoxigenic Escherichia coli strains belonging to various serotypes and 89 clinical isolates nonenterotoxigenic but belonging to those serotypes in which ETEC from diarrheal patients are usually found. The serotypes of the strains to which Ent plasmids were efficiently transferred and in which they were maintained stably were not always the serotypes in which ETEC had usually been found and vice versa. The frequencies of transfer of four Ent and two R plasmids to each of the 157 recipients were correlated with each other, indicating that the frequency of transfer of the plasmid is not determined by a resident plasmid, if there is one, but by a recipient factor which commonly affects transferability to all donors. These results have led to the conclusion that the reason why only certain serotypes are found among ETEC isolated from diarrheal patients is not the ability of these strains specifically and preferentially to acquire and maintain the Ent plasmids.

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We examined Escherichia coli K-12 lipopolysaccharide (LPS), which is known to be an R-form LPS, for its ability to form a hexagonal lattice structure in vitro. The LPS from E. coli K-12 strain JE1011 did not form a hexagonal lattice structure when it was precipitated by addition of two volumes of 10mM MgCl₂-ethanol, but it did form such a structure when it was electrodialyzed and then converted to the magnesium or calcium salt form. The lattice constant of the magnesium salt form was 15.2±0.3nm and that of the calcium salt form 18.5±0.3nm. Since prior treatment of the LPS with proteinase K in the presence of sodium dodecyl sulfate did not affect its capability of hexagonal assembly, the lattice formation by the LPS does not require the presence of proteins.

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Shiga toxin was purified from the culture supernatant of Shigella dysenteriae 1 by ammonium sulfate fractionation, DEAE-cellulose column chromatography and repeated chromatofocusing column chromatography. About 1.6mg of purified Shiga toxin was obtained from 15 liters of culture with a yield of about 27%. The molecular weight of purified Shiga toxin was estimated to be 62, 000. The toxin consisted of A and B subunits with molecular weights of about 30, 000 and 5, 000-6, 000, respectively. The isoelectric point of purified Shiga toxin was 7.0. Purified Shiga toxin showed the following biological activities: lethal toxicity to mice when injected intraperitoneally with an LD₅₀ of 28ng per mouse; cytotoxicity to Vero cells, killing about 50% of the cells at 1pg and all of the cells at 10pg; and fluid accumulation in rabbit ileal loops at a concentration of more than 1μg.

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A monoclonal antibody to human interferon-α, termed HT-1 antibody, with a broad reactivity to various subtypes of interferon-α was prepared. It bound and neutralized all of the four subtypes of E. coli-derived human recombinant interferon-α (α1, α2, α4, and α6) tested; it also neutralized human natural leukocyte interferon but only partially. Human interferon-β and -γ were not bound. The antibody conjugated to Sepharose beads retained over 90% of human leukocyte interferon induced by Sendai virus. The bound interferon was recovered by acid elution in good yields and in almost pure form (specific activity was about 2×10⁸ international units/mg protein). The purified interferon showed, in SDS-polyacrylamide gel electrophoresis, an activity profile with major peaks in a mol. wt. range of 17, 000-22, 000, which completely agreed with the profile shown by polyclonal antibody-purified interferon. Such purified leukocyte interferon-α preparations containing most of the naturally occurring subtypes can be useful for clinical and other purposes.

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The reactogenicity and immunogenicity of Tween-ether split trivalent A(H1N1), A(H3N2), and B influenza vaccine in primary school children aged seven to 12 years, and the persistence of antibodies following two doses of vaccine were studied during 1980-1984. Adverse reactions were infrequent, and, even when reported, were chiefly local ones, mild in nature and of short duration. Most of the reactions were less frequent after the second dose than after the first dose. Most of the systemic reactions occurred during the intervaccination period with almost equal frequency, indicating that careful consideration is required to judge whether they were induced by vaccination or not. This vaccine had induced adequate hemagglutination inhibiting (HAI) antibody because the geometric mean titers (GMTs) of the vaccinees were two- to eightfold higher than those of the nonvaccinees to any of the vaccine antigens following two doses of vaccine. In general, the responses to A(H3N2) virus were the best among the vaccine antigens through the four vaccination seasons, but there was a tendency to show a poorer response to the same type (or subtype) of virus antigen as the causative one during a protracted epidemic. The antibodies induced by either vaccination or natural infection were shown to persist for less than a year, supporting the recommendation for annual vaccination.

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A total of 1, 995 primary school children (1, 464 vaccinees and 531 nonvaccinees) were studied to evaluate the protective efficacy of Tween-ether split trivalent A(H1N1), A(H3N2), and B influenza vaccines by comparison of the incidence of confirmed infection in two groups during 1980 to 1984. During the study period, epidemics caused by antigenically different influenza viruses, that is A(H1N1) epidemics in 1981 and 1984, a B epidemic in 1982 and an A(H3N2) epidemic in 1983, were experienced, and at the same time strains changed by antigenic drift were frequently isolated. In these epidemics, 61% to 87% of the children reported respiratory illnesses and 18% to 48% of the illnesses were influenza confirmed by seroconversion. Throughout these four epidemics, the incidence of confirmed infection among the vaccinees (7.8% to 33.8%) was 6.5% to 34.8% lower than that among the nonvaccinees (35.4% to 51.6%, ), demonstrating that the vaccine was effective (χ²=76.34, P<0.001). However, this effectiveness was not seen in an epidemic in one of the entrant schools in 1984, possibly caused by a strain with intense antigenic drift. On the basis of data on incidence of various symptoms, duration of fever and the number of days of absence from class, it was considered that clinical symptoms in the vaccinees were milder than those in the nonvaccinees. When the titers of hemagglutination-inhibiting (HAI) antibody against the vaccine strains were measured, the protective level of HAI antibody giving _??_50% incidence of infection was 1:64, but it increased to 1:256 in the 1984 epidemic, reflecting the high rate of isolates with intense antigenic drift.

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Microimmunofluorescence using Terasaki plates and a direct plate freezing method were combined for effective screening of hybridoma supernatants. The microplates, in which the fused cells (myeloma and spleen cells) were cultured and hybridoma colonies were growing, were frozen after harvest of supernatants and saved at -80C for several weeks without affecting antibody production ability of hybridomas. Microimmunofluorescence was performed in Terasaki plates on which target cells were attached by poly-L-lysine and glutaraldehyde or by short time culture of the cells in Terasaki plates. The direct plate freezing method prevented initial hybridoma cells from changes or disappearance of antibody productions during screening of hybridoma supernatants; the microimmunofluorescence staining method permits fast and detailed estimation of specificity of antibodies of hybridomas by saving time and minimal consumption of supernatant for checking. The combination of these two methods is a powerful tool for obtaining desired monoclonal antibodies.

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An investigation was undertaken to determine the components of mycobacteria responsible for pulmonary cavity formation in tuberculosis. Rabbits received an intrapulmonary injection through the chest wall, of mycobacterial protein, II-p, mixed with either mycobacterial lipids, synthetic adjuvants or Nocardia cell wall skeleton as adjuvant. Six weeks later, they were killed and the lung lesions were examined. Cavities and necrosis were produced by the injection of II-p mixed with cord factor, Nocardia cell wall skeleton or N-acetylmuramyl dipeptide conjugated with long-chain branched fatty acids. Cavities were not produced by either the injection of II-p together with phospholipid, N-acetylmuramyl dipeptide (MDP), MDP-derivatives having no long-chain branched fatty acid, or by the injection of individual components of the mixtures. The results suggest that in tuberculosis a mycobacterial component with a long-chain branched fatty acid such as mycolic acid plays an important role in pulmonary cavity formation by enhancing the antigenicity of mycobacterial protein and helping it induce cell-mediated immunity at the site of the lesion. Passive transfer with sera from rabbits highly sensitized with tubercle bacilli failed to enhance cavity formation in the recipient animals.

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Inhalation exposure to silica dust enhanced interleukin 1 (IL-1) production by alveolar macrophages (AM), which is attributable to an increase in Ia-positive lung cells. While the proportion of Ia-positive cells in lavaged bronchoalveolar cells (BAC) was much lower (0-3%) in unexposed control rats, about a third of the rats that inhaled silica showed higher proportions (8.0-18.5%); these were designated “Ia-high” exposed animals. The number of total cells, Ia-positive cells and lymphocytes in BAC was significantly increased (P<0.05, P<0.001, and P<0.001, respectively) in these “Ia-high” exposed animals, compared to the control animals. Adherent AM populations obtained from BAC preparations also contained significantly higher (P<0.001) proportions of Ia-positive cells in the “Ia-high” exposed animals. When these adherent AM cultures were stimulated with lipopolysaccharide, IL-1 activity of the culture supernatants was enhanced and was significantly higher (P<0.001) in the “Ia-high” exposed rats, compared to the control animals. These results indicate that silica-exposure can induce populational changes in lung cells and also activation of AM associated with the increase in Ia-positive cells.

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The effects of disodium ethane-1-hydroxy-1, 1-diphosphonate (EHDP) on the in vitro functions of guinea pig macrophages were studied. A high dose (1mg/ml) of EHDP inhibited interleukin 1 (IL 1) production by oil-induced peritoneal macrophages stimulated with muramyl dipeptide (MDP), lipopolysaccharide (LPS), phorbol myristic acetate (PMA), heat-aggregated IgG2 or calcium ionophore A23187. On the other hand, low doses (<0.125mg/ml) of EHDP augmented the MDP induced IL 1 production by macrophages. This biphasic effect was also observed when macrophages were exposed to EHDP at 37C for 24hr and then stimulated with IL 1 inducers. Superoxide anion generation induced by formyl peptide or PMA was not affected by preincubation of the macrophages with doses of EHDP up to 1mg/ml. Adherence and spreading of macrophages was inhibited by EHDP in a dose dependent manner without affecting cell viability. These results demonstrated that EHDP acted on macrophages directly and modulated IL 1 production in vitro.

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