To investigate the modifying role of the intestinal microflora in the metabolism of 1-nitropyrene (1-NP) via enterohepatic circulation, we collected bile from male Wistar rats administered [³H]1-NP orally. The bile was mixed with the intestinal contents (IC) prepared from untreated rats and the mixture was incubated anaerobically under an atmosphere of nitrogen at 37C. Samples of the reaction mixture were removed at intervals to assay their mutagenic potential, to determine the radioactivity bound to the IC, and for analysis of the biliary metabolites. The binding of the radioactivity to the IC increased linearly as a function of time during the 1-hr incubation. The time-dependent binding does not occur with heat-treated IC and the binding was inhibited by addition of D-saccharic acid 1, 4-lacton, a β-glucuronidase inhibitor. The mutagenicity (for Salmonella typhimurium strain TA98 without S9 mix) of the bile increased early in the incubation period and then decreased very rapidly. The mutagenicity of the bile was also enhanced by treatment with a sonicated IC extract or β-glucuronidase, but not with a heat-treated IC or arylsulfatase. The metabolites produced after the bile was incubated for short periods with the IC were mainly nitrohydroxypyrenes; at later times nitroreduction occurred. The level of acetylaminohydroxypyrenes, which were formed by deconjugation, did not change during the incubation. To determine the degree of contribution of the IC to the total acetylating capacity, we measured acetyltransferase activity of the IC and various organs in Wistar rats. The liver had the highest N-acetyltransferase activity among the seventeen organs examined. Considerable activity was also detected in the kidney, small intestine, lung, and testis, but the IC showed very low activity. The acetylating capacity of the IC was 0.27% of the total capacity in rats, and that of the liver was more than 80%. These results suggest that the nitrohydroxypyrenes formed from 1-NP in the liver were conjugated to glucuronic acid and excreted via the bile duct into intestine. Hydrolysis of these glucuronide conjugates by bacterial β-glucuronidase liberated into intestine, free nitrohydroxypyrenes, which were direct-acting mutagens. The released aglycons were then rapidly nitro-reduced by intestinal microflora, but contribution of the intestinal microflora to acetylation of the reduced metabolites is very low.

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We obtained two monoclonal antibodies of IgM class and IgA class of immunoglobulin prepared from mouse spleen cells immunized with crude flagellar preparation, and a polyclonal antibody raised against purified flagellin monomer of Campylobacter jejuni in a rabbit. The specificity of the reaction of these antibodies for flagellar filament was confirmed by Western blotting and by immunoelectron microscopy. These antibodies caused agglutination of the bacteria and inhibited the motility of the bacteria. When a strain of C. jejuni was treated with IgM class monoclonal antibody before being inoculated into suckling mice, it reduced colonization of the intestinal tract by this bacteria. Inhibition of the colonization by IgA class monoclonal antibody was less effective than that of IgM class, and the polyclonal antibody consisting mostly of IgG class immunoglobulin was without effect.

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The distribution of the antibody to influenza C virus in the dogs and pigs in Yamagata prefecture, Japan was investigated by using three different serological methods: hemagglutination-inhibition (HI), radioimmunoprecipitation (RIP), and immunoblotting. The antibody against influenza C virus glycoprotein (gp88) was detected in 5 out of 112 sera collected from mongrel dogs, three by RIP test and two by any of the three methods, suggesting that the virus can cause natural infection in dogs. Significant levels of HI activity were found in 58 out of 269 sera collected from domestic pigs, but none of them showed positive reaction in the more sensitive method, RIP, which suggests that the inhibitors against the hemagglutination by influenza C virus rather than the antibody to gp88 are responsible for the observed HI activity. It appears, therefore, that at least in the Yamagata area, pigs do not play significant roles in the spread of influenza C virus in humans.

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Dialyzable low molecular weight antibody-augmenting factors (LMAAF) were found in the culture supernatant of human tonsillar lymphocytes which were not stimulated by antigen and/or mitogen in vitro. Phagocyte-depleted nylon wool-adherent lymphocytes (M^-Ny⁺ cells) were responsible for the release of the LMAAF. Marbrook's culture system was adopted to assay for the LMAAF. The M^-Ny⁺ cells, which were cultured without antigen and/or without mitogen in the reservoir of Marbrook's diffusion culture vessel, released the LMAAF, which diffused across a dialysis membrane and significantly augmented the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) response of phagocyte-depleted lymphocytes (M^- cells) cultured in the inner vessel. Phagocyte-depleted nylon wool-passed lymphocytes (M^-Ny^- cells) cultured in the reservoir could not augment the PWM-induced PFC response of the M^- cells cultured in the inner vessel. The exuded fluid, which was the dialysate of the culture supernatant of the M^-Ny⁺ cells ultrafiltrated with dialysis tubing, also enhanced the PFC response of M^- cells cultured in 24-well multi plates. The exuded fluid also augmented the total IgM and IgG production of human tonsillar and peripheral blood lymphocytes measured by enzyme-linked immunosorbent assay (ELISA) systems. Gel filtration chromatography on Sephadex G-25 Superfine column showed that the LMAAF activity was demonstrated in the fractions corresponding to a molecular weight (m.w.) of 362 to 1, 355 and a m.w. of 3, 560 to 5, 700, with a peak activity at about 4, 500 dalton. The LMAAF were inactivated by treatment with proteinase K, but not by trypsin, α-chymotrypsin, RNase, and DNase, and were stable when treated at 56C for 60min. The dialysates of culture supernatants from two out of seven Epstein-Barr virus (EBV)-transformed M^-Ny⁺ cell lines showed LMAAF-like activity. These results indicate that phagocyte-depleted nylon wool-adherent lymphocytes, possibly B cells, release low molecular weight factors displaying augmenting activity for human antibody production in vitro.

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Antiserum against a strain of the rice blast fungus Pyricularia oryzae was elicited in rabbits immunized with its cell fragments emulsified with incomplete Freund's adjuvant. The fragments were also used as solid-phase antigens. A highly sensitive, competitive type enzyme-linked immunosorbent assay for P. oryzae was developed by using these two preparations as the immune reagents together with the use of β-D-galactosidase-labeled anti-rabbit IgG as the tracer. Cross-reactivity of nine different strains of P. oryzae were measured by the assay. Sensitivity and accuracy of the assay was improved by choosing the cell fragments of the least cross-reactive strain as the solid-phase antigen. The improved method was successfully applied for sensitive and accurate assay of all ten strains of P. oryzae with the common measuring range between 1 and 100ng per tube. Other species of microorganisms had little reactivity in this immunoassay indicating that the assay is specific to P. oryzae group microorganisms.

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We compared the expressions of class I and class II major histocompatibility antigen complex (MHC) on the surface of Jijoye and P3HR-1 cells of Burkitt's lymphoma sublines. Jijoye cells had a large amount of class I and class II MHC antigens, whereas these antigens were less expressed on P3HR-1 cells. On a subline of P3HR-1 K cells the expression of class I antigen markedly diminished and class II antigen was undetectable. On the other hand, Jijoye, P3HR-1, and P3HR-1 K cell lines were confirmed to be Epstein-Barr virus (EBV) nonproducer, low producer, and high producer, respectively. The chemical activation of EBV genome by treating P3HR-1 cells with 12-O-tetradecanyl phorbol-13 acetate (TPA) and n-butyrate resulted in inhibition of the expression of class I and II antigens, while the addition of retinoic acid, an inhibitor of virus replication, blocked the decrease in the MHC antigen expression. These findings suggested that there might be an inverse correlation between the virus production and the expression of class I and II MHC antigens.

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This study was carried out on the effect of histamine hydrochloride and its antagonists on the production of interleukin-1 (IL-1) by lipopolysaccharide (LPS)-stimulated adherent human monocytes (AHM) from normal healthy blood donors. IL-1 activity was evaluated by incorporation of [³H]-thymidine in mouse thymocytes in samples of 1:3 dilution. The result indicated that histamine hydrochloride significantly suppressed IL-1 production by AHM at 10^-3M and 10^-10M in 14 donors with maximal suppression observed at 10^-3M. A 1-hr incubation with histamine hydrochloride (10^-3M) before addition of LPS was found to be appropriate. Cimetidine, an H₂-antagonist at 10^-3M, 10^-5M, and 10^-7M significantly inhibited the effect of histamine hydrochloride (10^-3M) and gave maximum inhibition at 10^-5M, whereas chlorpheniramine maleate, and H₁-antagonist had no significant inhibitory effect at the concentrations studied (10^-4M, 10^-5M, and 10^-7 M). Histamine hydrochloride (10^-3M) added alone had no significant suppressive effect, while cimetidine (10^-5M) alone had a significant stimulatory effect on IL-1 production by AHM.

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A murine killer T cell line, G-CTLL 1, whose proliferation depends on the presence of interleukin 2 (IL-2), was used to analyze the mechanism of IL-2 action with respect to sterol synthesis and arachidonate metabolism. De novo sterol synthesis was substantially enhanced much earlier than DNA synthesis, and the rate reached a maximum at 13hr after the addition of IL-2. Compactin, which is a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase, the enzyme in the rate-limiting step of the sterol synthesis), inhibited the IL-2-induced DNA synthesis. The addition of mevalonate, the product of HMG CoA reductase, prevented the inhibition of DNA synthesis by compactin, suggesting that the supply of a sufficient amount of sterol is an essential prerequisite for IL-2 action. The IL-2-induced DNA synthesis was also inhibited by AA861, a specific inhibitor of arachidonate 5-lipoxygenase, and by other lipoxygenase inhibitors such as nordihydroguaiaretic acid and esculetin. In contrast, indomethacin, an inhibitor of arachidonate cyclooxygenase, had no effect. These findings suggest that synthesis of 5-lipoxygenase products is also a prerequisite. The inhibition of DNA synthesis was effectively inhibited only when compactin or lipoxygenase inhibitors were added early enough to block the synthesis of sterols or 5-lipoxygenase products; addition of the reagents after 3hr decreased the inhibition with time. Therefore, about 3hr after the addition of IL-2, several drastic intracellular changes are assumed to begin and to lead to DNA synthesis.

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After C57BL/6 (B6) mice were inoculated with BALB/c spleen cells via tail vein, kinetics of cytotoxic activities in the B6 mice against sensitizing alloantigens (H-2^d) and against syngeneic antigens were investigated using, as target cells, P815 mastocytoma cells (H-2^d) and B16 melanoma cells (H-2^b). Cytotoxic activity against P815 in the B6 spleen cells reached a peak 3 days after alloantigen inoculation, decreased drastically on day 5 and rose again thereafter. The profile of anti-B16 cytotoxic activity was similar to that of anti-P815 activity. The cytotoxic activity against P815 was inhibited partially by cold B16, but that against B16 was not inhibited by cold P815. Surface phenotype of cytotoxic cells against P815 was Lyt2⁺, Thyl⁺, Asialo GM1⁺ and that of cytotoxic cells against B16 was Lyt2^-, Thyl^+/-, and Asialo GM1⁺. The results indicate that inoculation of B6 mice with allogeneic BALB/c spleen cells induce two types of cytotoxic cells; one is similar to lymphokine-activated killer (LAK) cells and the other is activated natural killer cells.

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The hemagglutination (HA) by pilus antigen 987P of an enterotoxigenic Escherichia coli strain 987 was examined using fresh and glutaraldehyde (GA)-fixed erythrocytes (RBC) of various animals. Only when GA-fixed RBC was employed, a strain 987 exhibited striking HA activities. This was also demonstrated by using latex heads sensitized with the 987P antigen. The 987P-specific antiserum inhibited HA of strain 987 and 987P sensitized latex beads against GA-fixed RBC. We concluded that HA of strain 987 against GA-fixed RBC was specifically associated with the presence of 987P pilus antigen but do not exclude a possibility that adhesin is distinct from pili antigen.

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Temperature-sensitive growth mutants of Salmonella enteritidis, NUB 209 and NUB 323, characterized as protein synthesis mutants, were unable to proliferate at 37C and lost the parent's virulence. In mice, these mutants conferred high levels of protection as live vaccines. Although the vaccination effect of NUB 323 was not so good as that of NUB 209, NUB 323 was preferred as a safer live vaccine because this mutant was completely avirulent and no back mutation appeared.

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