Fumiko Nishikawa

Hepatotoxic activity of Campylobacter jejuni.

Hepatotoxic factor(s) were isolated from whole-cell lysates of Campylobacter jejuni GIFU 8734 and purified by chromatography. A single intravenous injection of 10 micrograms of this factor reproducibly produced hepatitis in mice, as determined by histology and liver function tests. The hepatic lesions were very similar to those evoked by C. jejuni infection. Tissue-culture studies with mouse hepatocytes demonstrated that low concentrations of the factor caused release of hepatic enzymes into the medium without appreciable cytolysis. High concentrations of the factor induced cytolysis. These effects were neutralised by antiserum to the factor, but not by antisera to the lipopolysaccharide of C. jejuni or to the heat-labile enterotoxin of Escherichia coli. Among 20 clinical isolates of C. jejuni, only four evoked hepatitis in mice and produced the hepatotoxic factor.

Contribution of interferon gamma and membrane-associated interleukin 1 to the resistance to murine typhoid of Ityr mice.

Resistance of mice to Salmonella, typhimurium in the early phase of infection is known to be controlled by the expression of chromosome 1 locus Ity. To clarify the mechanism by which the genetically resistant (Ityr) mice can overcome the first phase of salmonellosis, the early response in DBA/2 (Ityr) and BALB/c (Itys) mice was compared after a subcutaneous injection of S. typhimurium. In both strains, the growth of S. typhimurium was controlled in livers and Kupffer cells until day 3, but thereafter the bacteria multiplied rapidly in BALB/c mice. Over the first 2 days nonspecific responses (changes in levels of blood leukocytes, plasma iron, and α1‐antitrypsin) were not significantly different between the strains, and the capacity of Kupffer cells isolated from infected mice of both strains to produce interleukin 1 (IL‐1) and tumor necrosis factor α (TNF‐α) was of the same degree. Thereafter, only DBA/2 Kupffer cells were able to produce membrane‐associated IL‐1 (ma IL‐1) as well as TNF‐α. Moreover, only DBA/2 splenocytes were able to produce interferon γ (IFN‐γ) upon stimulation with Salmonella antigens, although concanavalin A‐stimulated splenocytes of both strains produced the same level of interleukin 2. Furthermore, administration of recombinant murine IFN‐γ and DBA/2 Kupffer cells of day 6 to BALB/c mice 3 days after infection resulted in a significant level of protection, whereas neither of these materials alone induced protection. Injection of anti‐TNF‐α antibodies did not affect the resistance of DBA/2 mice. Thus, these findings suggest that the early resistance of Ityr mice is partly attributable to their capacity to produce IFN‐γ and ma IL‐1 after infection.

Mononuclear cell response in the liver of mice infected with hepatotoxigenic Campylobacter jejuni.

Intragastric inoculation with hepatotoxigenic strains of Campylobacter jejuni led to the death of mice during the late phase of infection. Histological study disclosed a massive infiltration of mononuclear cells in the liver, mimicking intrahepatic hypersensitivity. Neither enterotoxigenic nor enteroinvasive Escherichia coli induced such a lesion. However, the same histopathological change was induced by injecting the hepatotoxic factor of hepatotoxigenic C. jejuni intravenously on two occasions separated by 14 days. Neither a single injection of an increased dose of the hepatotoxic factor nor two injections, the second of which was heat-inactivated, induced this change. Pre-treatment with rabbit antibody to the hepatotoxic factor inhibited the development of the hepatic lesion. These results suggest that C. jejuni-induced hepatic lesions in mice may be caused, at least in part, by the active moiety of the hepatotoxic factor. The possible mechanisms by which the toxic factor induces hepatitis as a consequence of hypersensitivity are discussed in relation to Guillain-Barré syndrome and Reiters syndrome associated with C. jejuni enteritis.

Development of a Mucosal Complex Vaccine against Oral Salmonella Infection in Mice

We examined the immunogenicity of a Salmonella enterica complex vaccine (CV), consisting of flagellin and polysome purified from serotype Typhimurium LT2. CV plus cholera toxin (CT), in three oral doses given at 7‐day intervals, conferred complete protection on C57BL/6 mice against lethal oral infection with a wild‐type strain. It elicited mucosal IgA>IgG2a>IgG1 and systemic IgG2a>IgG1>IgA antibodies to flagellin and polysome, and delayed footpad response (DFR) to both antigens. In Peyers patches (PPs) and lamina propria (LP), IgA was produced under a Th1‐dominant environment; CD4T cells from produced interleukin (IL)‐2, interferon (IFN)‐γ, and IL‐10 by stimulation with salmonella extract. On the same protocol, flagellin plus CT induced flagellin‐specific mucosal and systemic IgA and IgG1 antibodies, CD4T cells producing IL‐10 and IFN‐γ in PPs and LP, and only minimal levels of flagellin‐specific DFR. Polysome plus CT induced polysome‐specific mucosal and systemic IgG2a in addition to IgG1 and IgA antibodies, CD4+T cells producing IFN‐γ and IL‐2 in PPs and LP, and polysome‐specific DFR. These two vaccines, however, conferred at most 50–60% survival rates. Our results suggest that polysomes in CV provide effective adjuvant activity for the induction of both mucosal and systemic Th1‐biased responses toward flagellin.

Virulence of transparent and opaque colony types of Neisseria gonorrhoeae for the genital tract of mice

The virulence of transparent (Tr) and opaque (Op) colony types of Neisseria gonorrhoeae in the genital tract of female mice was evaluated at two stages of oestrous. Isogenic pairs of Tr and Op variants were isolated from N. gonorrhoeae strain 57-120. Both variants exhibited a T2 morphology, but only the Op variant possessed protein II (P.II) in outer-membrane fractions. When administered by intravaginal inoculation Op gonococci were highly infective only for mice in late pro-oestrous, whereas Tr gonococci were virulent for mice at both late pro-oestrous and dioestrous. Gonococci recovered from the uterus were of both Tr and Op phenotypes in equal proportions when mice were infected at dioestrous with Tr cells. In contrast, greater than 90% of recovered colonies were of Op phenotype when mice were infected at late pro-oestrous with either Op or Tr cells. These results indicate that the virulence of gonococci for the genital tract of female mice differs from that for the chicken embryo. Furthermore, gonococcal survival in the female genital tract might be attributable to phase variation from Tr to Op phenotypes.

THE FULL EXPRESSION OF THE ITY PHENOTYPE IN ITYR MICE REQUIRES C3 ACTIVATION BY SALMONELLA LIPOPOLYSACCHARIDE

Our previous study has shown that the rapid and sufficient activation of complement by Salmonella lipopolysaccharide occurs in genetically resistant (Ityr) A/J mice. To assess whether the level of complement activation by a virulent strain of Salmonella typhimurium regulates the level of murine natural resistance, we compared levels of serum complement activation by S. typhimurium and kinetics of serum‐opsonized S. typhimurium grown in macrophages using several strains of resistant (Ityr) and susceptible (Itys) mice. Itys macrophages killed intracellular S. typhimurium to the same extent as did Ityr macrophages when the pathogen was opsonized with Ityr serum. Opsonization of S. typhimurium with Itys serum reduced intracellular killing activity in Ityr macrophages to the same level as seen with Itys macrophages. Incubation of S. typhimurium with 25% Mg2+ EGTA (5 mm MgCl2–3 mm ethylene glycol‐bis (β‐aminotheyl either)‐N,N,N′,N′‐tetraacetic acid)‐chelated Ityr serum resulted in higher levels of C3 deposition onto the surface of this bacteria, C3b generation and also C3 consumption, compared with that with Mg2+ EGTA‐chelated Itys serum. Opsonization of S. typhimurium with A/J serum prior to infection increased early resistance in Itys mice. Infection with a virulent strain of S. typhimurium induced the expression of interleukin‐10 (IL‐10) mRNA at higher levels in C57BL/6 mice than in A/J mice. However, opsonization of S. typhimurium with A/J serum decreased bacterial growth in the spleen of C57BL/6 mice to the same level as observed for A/J mice in association with decreased expression levels of IL‐10 mRNA. Moreover, administration of anti‐C3 antibodies reduced the resistance of A/J mice in association with a decrease in serum levels of C3. These results indicate that the high level of complement activation via the alternative pathway in Ityr serum by a virulent strain of S. typhimurium reduces the virulence of this pathogen, which may contribute to the full expression of Ity phenotype in Ityr mice.

Biological functions of mouse seminal vesicle fluid. II, Role of water-soluble fraction of seminal vesicle fluid as a nonspecific immunomodulator

The suppressive mechanisms of T cells induced by water-soluble fraction of mouse seminal vesicle fluid (WSF-SVF) were investigated to clarify its immunological roles in the reproductive immunity. WSF-SVF inhibited the blastogenic responses to concanavalin A (Con A) or phytohemagglutinin (PHA) of T cells. Pretreatment of splenocytes with WSF-SVF did not suppress the blastogenesis of splenocytes to Con A when treated cells were washed before cultures. WSF-SVF did not inhibit the proliferation of Con A-activated splenocytes, that of listeria-immune splenocytes to listeral antigen and growth of tumor cells (Yac 1 cells, Ehrlich ascites carcinoma cells, EL 4 cells). Listerial antigen-specific immune response was not observed when mice were immunized with both listerial antigen and WSF-SVF, whereas it was observed when mice were immunized with only listerial antigen. WSF-SVF also significantly inhibited allogenic MLR. WSF-SVF did not adsorb Con A, and its suppressive activity was rather enhanced by heating at 56 degrees C for 30 min. These results suggest that WSF-SVF inhibits the stage of sensitization of T cells with antigen or stimulant, such as mitogen nonspecifically, without adsorption to antigen or mitogen, and its substance is stable.

Hormonal regulation of soluble immune response suppressor (SIRS): A possible role of SIRS in the maintenance of pregnancy

This study was conducted to investigate the effects of sex hormones upon the nature of soluble immune response suppressor (SIRS) produced by concanavalin A-stimulated Lyt-2+ T cells. Conventional SIRS affected IgM PFC only. However, SIRS made with progesterone (20-400 ng/ml or Prog-SIRS) suppressed IgM PFC, one-way MLR, and generation CTL; and SIRS made with estrogen (0.2-50 ng/ml or Est-SIRS) enhanced these responses. The factor(s) (MW 40,000-55,000) to stimulate macrophages to produce the second soluble factor (M phi-SF) was isolated from all preparations by gel filtration. Furthermore, Est-SIRS contained a factor(s) (MW 10,000-30,000) to enhance IgM PFC, MLR, and mitogen-induced blastogenesis of both T and B cells; and Prog-SIRS possessed the suppressive factor(s) to IgM PFC, MLR, and mitogen-induced T-cell proliferation. These activities were not impaired by 2-mercaptoethanol. Moreover, the suppressive activity of Prog-SIRS was completely absorbed by T cells only, but the enhancing activity of Est-SIRS was not completely absorbed by a single-cell population. These data suggest that progesterone can contribute to the suppression of allograft rejection through soluble factors, and estrogen can enhance host responses which may be affected by several soluble factors during pregnancy.

Transfer of Protection to Murine Typhoid Conferred by L-Form Salmonella typhimurium in Dependence of Cooperation between L Form-Adopted Macrophages and L Form-Induced Lyt-2+ T Cells

The effector cells responsible for protection to Salmonella typhimurium in C3H/HeJ mice, conferred by L‐form S. typhimurium, were determined by cell transfer test. Nonfractionated spleen cells from 6‐week immune mice but not from 24‐week immune animals transferred anti‐S. typhimurium immunity. Treatment with anti‐macrophage antiserum and complement most effectively abolished protective capacity in 6‐week immune cells, while anti‐T cell monoclonal antibody plus complement reduced it to a lesser extent. However, adoptive protection was achieved only by transfer of immune macrophages along with Lyt‐2+ T cells selected from 6‐week immune spleen cells. These Lyt‐2+ T cells were cytotoxic to Kupffer cells from C3H/HeJ mice which had been infected 48 hr previously and from the mice which had been immunized 1 week previously, but not to the cells from 6‐week immune mice and from normal animals. Moreover, protective capacity in immune macrophages seemed to be correlated to the degree of colonization by the L forms, and the inability to transfer immunity of 24‐week immune spleen cells may be due to the decrease in the L form‐colonization. These results suggest that cooperation between the L form‐colonized macrophages and L form‐induced cytotoxic Lyt‐2+ T cells contributes to anti‐S. typhimurium immunity, and might imply the immunological difference between the 6‐week immune phagocytes and the cells at an early stage of infection or immunization.

Protective Capacity of L-Form Salmonella typhimurium against Murine Typhoid in C3H/HeJ Mice

L forms of Salmonella typhimurium LT2 conferred strong protection to a lethal challenge with its parental bacterium on innately hypersusceptible C3H/HeJ mice, and its minimal protective dose was approximately 150 L‐forming units. Although L‐form S. typhimurium was avirulent for C3H/HeJ mice, it multiplied slowly in both the liver and spleen with the maximal growth 2–3 weeks after immunization and thereafter it persisted in the liver until 24 weeks. Protective immunity began to work between 4 and 6 weeks after immunization, and it remained active as long as the L forms colonized the liver (until 24 weeks after immunization). Vaccination with the L form induced a population of T cells responding to L‐form whole‐cell lysate (WCL), while delayed‐type hypersensitivity (DTH) to the extract of S. typhimurium was induced after the establishment of solid immunity. Moreover, neither T‐cell responses nor DTH to heat‐killed S. typhimurium was generated. In addition, antibody responses were elicited to WCL but not to heat‐killed S. typhimurium. These results indicate that protection conferred by the L forms is attributable to the persistent colonization of the L forms rather than the presence of DTH, and also that Salmonella cytoplasmic antigens are involved in induction of immunological responses by vaccination with the L forms.