Alterations of host resistance to mouse typhoid infection by sex hormones.

The effect on mouse typhoid infection of a 3-day treatment of female virgin mice with 1 mg/day of female sex hormones (estrogen or progesterone), maintaining the same hormonal levels observed in pregnant mice for 30 days, was investigated in order to clarify the mechanisms of altered resistance during pregnancy. Estrogen-exposed mice were more susceptible to the intraperitoneal challenge with Salmonella typhimurium as compared with the vehicle control mice, while progesterone treatment increased the survival times of mice. Estrogen exposure increased the number of peritoneal cells after treatment, but the inflammatory cellular response after infection was significantly suppressed. Although the estrogen-treated and vehicle control mice had the same degrees of peritoneal cellular responses after infection, the death rates in the estrogen-treated mice were higher than those in the vehicle control mice against challenge with 1 LD50 of S. typhimurium. On the other hand, progesterone treatment resulted in the marked influx of peritoneal cells after treatment was terminated, and also it induced a significant increase in the number of peritoneal cells after infection. Although survival times in the progesterone group were higher than those in other groups, all progesterone-treated mice died after a challenge with 1,000 LD50 of S. typhimurium. These results suggest that progesterone enhances nonspecific resistance by increasing the influx of peritoneal cells after infection, while estrogen affects the acute inflammatory responses.

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 alpha 1-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 alpha (TNF-alpha) 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-alpha. Moreover, only DBA/2 splenocytes were able to produce interferon gamma (IFN-gamma) 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-gamma 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-alpha 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-gamma and ma IL-1 after infection.

Induction of hypersensitivity to endotoxin in C3H/HeJ mice by immunization with L-form Salmonella typhimurium.

When endotoxin low-responder C3H/HeJ mice were immunized with L-form Salmonella typhimurium, the mice were more susceptible to a lethal challenge with S. typhimurium 1 week after immunization (1-week mice) than were the unimmunized controls. One-week immune mice produced overwhelming amounts of tumor necrosis factor-alpha (TNF-alpha) in the blood after infection, while 4-week immune mice produced lesser amounts of this cytokine with a 75% survival rate at 60 days postinfection. Pretreatment with anti-TNF-alpha antibody prevented 1-week immune mice from succumbing to acute illness. Endotoxin-stimulated peritoneal macrophages from 1-week immune mice produced higher amounts of TNF-alpha in vitro than did those from 4-week immune mice and they expressed larger amounts of TNF-alpha mRNA on Northern blot. The capacity of macrophages to produce TNF-alpha in vitro was correlated with the degree of colonization by the L form in the cells. These results suggest that the colonization by L-form S. typhimurium in macrophages alters the susceptibility to S. typhimurium of C3H/HeJ mice and that TNF-alpha might play a major role in this alteration of host resistance.

Isolation of a cytotoxin from L-form Salmonella typhimurium.

A cytotoxic protein was isolated from the sodium dodecyl sulphate (SDS)-solubilized extract of the stable L forms of Salmonella typhimurium by ion-retardation chromatography, ion-exchange chromatography, isoelectric focusing and gel filtration. The purified toxin, with a molecular mass of 32 kDa and with isoelectric point of 6.4, was thermolabile and trypsin-sensitive. Against mouse macrophages, its cytolytic effect was detectable in vitro at concentrations higher than 0.7 micrograms/ml, with a complete lysis obtained at 5 micrograms/ml. In contrast, it stimulated C3H/HeJ macrophages in the dose range of 0.1-0.5 micrograms/ml to allow the cell to respond to endotoxin, resulting in the significant production of tumor necrosis factor alpha. By Northern blot analysis, this effect was detectable at a dose as low as 0.01 micrograms/ml. These findings suggest that the transformation of bacillary S. typhimurium into L forms in vivo may induce alterations in host resistance against murine typhoid.

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.

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 Reiter's syndrome associated with C. jejuni enteritis.

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.

[Effect of erythromycin on production of cytotoxin and attachment factors of bacteria].

We studied the effect of erythromycin (EM) on the attachment of Pseudomonas aeruginosa and Neisseria gonorrhoeae to HeLa and HT-177 cell and on cytotoxin production of P. aeruginosa. 1. EM inhibited attachment of these bacteria. 2. EM inhibited manifestation of the pili of these bacteria. 3. EM inhibited production of protein II, the second attachment factor of N. gonorrhoeae. 4. EM inhibited production of 66 K cytotoxin of P. aeruginosa. On the basis of these findings, it was suggested that EM might inhibit infection by repressing manifestation of the attachment factor and production of cytotoxin of the bacteria.

Immunogenicity of transfer RNA isolated from a two-heptose rough mutant of Salmonella typhimurium LT2 in mouse typhoid infection.

Transfer ribonucleic acid (tRNA) was isolated from a two-heptose mutant of Salmonella typhimurium LT2 (strain SL1004) and was found to afford 100% mouse protection against challenge with 1000 LD50 of strain LT2. The intraperitoneal minimum effective dose of tRNA was 5 micrograms RNA per mouse and this dose was significantly lower than that of ribosomal RNA for ddY mouse strain. The protective immunity was independent of the presence of antibodies to cell-surface antigens, and was transferred mainly by T cells. The protective moiety of tRNA was sensitive to ribonuclease digestion which resulted in 85% reduction in the mouse survival rate, but was completely resistant to protease digestion. The present study demonstrates that the immunogenic activity of salmonella RNA is present in both ribosomal RNA and tRNA.

Immunogenicity of the ribosomal fraction of Salmonella typhimurium: analysis of humoral immunity.

The ribosomal fraction prepared from Salmonella typhimurium LT2 was further purified by gel filtration of Sepharose 4B and afforded excellent protection against homologous challenge. The highly effective immunogens were composed of several fractions which could give different types of protection to mice. The first type of protection was heat-labile antigens which could induce humoral immunity, and the second type of protection was heat-stable antigens capable of evoking cellular resistance in mice. The former were different from O-antigens and the latter were free of endotoxin and rich in ribonucleic acid. The third type of protection was heat-resistant substances of cell wall components, which were mainly composed of O-antigens. The high immunogenicity observed in this study could be obtained only by the heat-stable antigens rich in ribonucleic acid, and the immunity conferred by this kind of antigen was due to the cellular type of protection.

Passive hemagglutination test for detection of antibody to gonococcal ribosomal antigen in sera from patients with asymptomatic gonorrhea.

Ribosomal fractions were obtained from a culture of type 2 Neisseria gonorrhoeae strain P-17 which was isolated from a patient with an acute gonococcal infection; these fractions were purified to eliminate the components of the outer membrane complex by affinity chromatography (Sepharose-anti-outer membrane complex antibody conjugates were used as the solid immunosorbent), and the resulting preparation was designated the purified ribosomal fraction, The purified ribosomal fraction was used to detect antibody activity in sera obtained from culture-positive asymptomatic carriers and healthy controls by a passive hemagglutination test. This passive hemagglutination test had a specificity of 100% for both sexes and sensitivities of 99.4 and 88.2% for female and male carriers, respectively, when an antibody titer of more than 1:3 was defined as abnormal. Absorption of the sera with nongonococcal organisms did not affect the antibody activity, and no significant difference in antigenicity among various N. gonorrhoeae strains was observed in ribosomal fractions. An enzyme-linked immunosorbent assay was also used to measure the relative amounts of specific antibodies to the purified ribosomal fraction, and this assay revealed that the anti-purified ribosomal fraction antibodies were immunoglobulin G.

Analysis of immune responses in genital tracts of mice immunised with purified ribosomal fractions of Neisseria gonorrhoeae.

Immunisation of ddY mice with the purified ribosomal fraction of Neisseria gonorrhoeae was found to protect against intravaginal challenge with homologous organisms. This protection correlated with the presence of bactericidal antibody to purified ribosomal fraction in serum as well as in vaginal secretions. Analysis of the vaginal fluids from control mice and those immunised with purified ribosomal fraction showed that the enhanced elimination of gonococci in immune mice might be because of an early response of leucocytes generated by the reaction mediated by antibody and complement. Absorption studies showed that there was at least one major protective antigen in purified ribosomal fraction, other than cell surface substances such as lipopolysaccharide, outer membrane proteins, and pili. Bactericidal assays mediated by antibody and complement showed that matched samples of serum and vaginal fluid from immune mice had comparable gonococcidal activity, which was augmented by the effect of progesterone. Although delayed hypersensitivity was produced in immune mice that were resistant to N gonorrhoeae, the exact role of cellular immunity could not be clarified in this study. These results suggest that antibody to purified ribosomal fraction plays a major part in protection against gonococcal infection in the genital tract, and that such protection may entail both cellular immunity and hormonal changes.

A mouse model for the study of gonococcal genital infection.

Intravaginal inoculation of approximately 10(6) piliated gonococci into female mice at different stages of the estrous cycle without any antibiotic pretreatment resulted in gonococcal endometritis. The percentage of mice with positive cultures for Neisseria gonorrhoeae one week after challenge was at least 80%, regardless of at which estrous stage the mice were inoculated. Recovery of gonococci from the uterus continued for more than one month, and the recovery rate appeared to depend on the estrous stage at inoculation. Serum levels of antibodies to crude outer membrane complex began rising one week after inoculation with gonococci and reached a maximum three week after challenge. This animal model is proposed for the study of gonococcal genital infection.

Different sensitivity of complement to Salmonella typhimurium accounts for the difference in natural resistance to murine typhoid between A/J and C57BL/6 mice.

The difference in natural resistance to Salmonella typhimurium between S. typhimurium-resistant A/J mice and S. typhimurium-susceptible C57BL/6 mice was analyzed. In both strains, the growth of S. typhimurium was controlled in the spleen until 48 hr of infection, while serum C3b levels were increased in A/J mice immediately after infection but not in C57BL/6 mice. Incubation of A/J mouse serum with S. typhimurium or its lipopolysaccharide (LPS) generated sufficient amounts of C3b, but that of C57BL/6 mouse serum with them did not. A/J macrophages had higher intracellular killing activity in vitro than did C57BL/6 cells against S. typhimurium pre-opsonized with each corresponding fresh serum. However, the cells from both mice exhibited a similar level of killing activity against S. typhimurium pre-opsonized with fresh A/J serum or rabbit complement. The resistance of C57BL/6 mice was significantly increased by opsonizing S. typhimurium with fresh A/J serum or rabbit complement before inoculation. The serum level of interferon-gamma (IFN-gamma) in A/J mice was 2.7 times as high as in C57BL/6 mice at 48 hr post-infection. Recombinant murine IFN-gamma enhanced the intracellular killing activity of macrophages from both mice when S. typhimurium was pre-opsonized with fresh A/J serum but not with fresh C57BL/6 serum. These findings suggest that A/J macrophages exhibit maximal killing activity against A/J serum-opsonized S. typhimurium in vivo when the cells are activated with IFN-gamma. Therefore, the rapid and sufficient activation of complement by Salmonella LPS may render A/J mice more resistant against murine typhoid.

Expression of clumping and fibrinogen-binding activities of Staphylococcus aureus at various growth stages.

Clumping and fibrinogen-binding activities of 4 Staphylococcus aureus strains (Cowan I, Newman D2C, Wood 46 and NCTC 5655) were assayed with a semiquantitative clumping test and an enzyme-linked immunosorbent assay (ELISA), respectively. Distinct positive clumping was detected with whole cells of the 3 strains except Wood 46. Amounts of fibrinogen required for a definite clumping depended greatly on strains as well as on their growth phases. On the other hand, fibrinogen-binding activities were detected both in culture supernatants and in cell lysates of all the 4 strains, and the levels were rather comparable with one another and relatively steady through their growth cycles. No significant correlation was thus found among expression behavior of clumping and fibrinogen-binding activities.

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.

Effect of estrogen (17 beta-estradiol) on the susceptibility of mice to disseminated gonococcal infection.

Studies of the effect of sex hormones on the susceptibility of mice to the disseminated gonococcal infection demonstrated significantly enhanced susceptibility of mice injected with estrogen (17 beta-estradiol). In mice treated with estradiol, bacteremia progressively developed within 12 h postinoculation and mice died within the next 6 h, whereas bacteremia in mice treated with progesterone was completely cleared within 3 h postinoculation. The administration of estradiol affected the function of polymorphonuclear leukocytes (PMN) responsible for eliminating gonococci, but the administration of progesterone did not. The bactericidal activity of PMN mediated by myeloperoxidase was affected by estradiol, but the capacity of PMN to release superoxide anion was not. Furthermore, peritoneal cell analysis demonstrated that the infiltration of PMN in the peritoneal cavity of estradiol-treated mice significantly decreased when mice were injected intraperitoneally with gonococci. These effects on PMN by estradiol may play an important role in the enhanced susceptibility of estradiol-treated mice to gonococcal infection.

Stimulation of locomotion of peripheral blood monocytes by human plasma fibronectin.

The motility of human peripheral blood granulocytes and monocytes in response to human plasma fibronectin was quantified by an in vitro assay using blind-well chemotaxis chambers. Purified fibronectin under nondenaturing conditions produced increased migration of granulocytes only at concentrations higher than 100 nM, and induced increased chemotactic and random locomotion of monocytes at concentrations higher than 0.1 nM. The monocyte migration-inducing activity of fibronectin was concentration dependent, and was strongly inhibited by low concentrations of colchicine (100 nM-100 microM). These findings suggest the possibility that plasma fibronectin serves as a chemotactic stimulus for monocytes in vivo and attracts these cells to sites of microscopic tissue injury where plasma fibronectin is deposited.

Opsonic effect of fibronectin on staphylococcal phagocytosis by human polymorphonuclear leukocytes: its relative inefficiency in post-phagocytic metabolic activities and in intracellular killing.

The binding of 125I-labeled human plasma fibronectin (FN) to two strains of live Staphylococcus aureus (S. aureus) (a coagulase-positive Cowan I and a coagulase-negative Newman D2C) and the opsonic effect of FN on phagocytosis of these bacteria by human polymorphonuclear leukocytes (PMN) have been studied. 125I-FN bound to a similar extent in both staphylococcal strains. The 125I-FN-binding was significantly inhibited by human fibrinogen as well as unlabeled FN. The FN-binding was also reduced markedly by trypsinization of these bacteria, but the extent of its decrease did not correlate with their tryptic susceptibility of protein A and clumping factor. FN enhanced the uptake of these bacteria by PMN. However, its binding had no effect on superoxide anion (O2-) generation. The FN-binding definitely stimulated staphylococcal ingestion and intracellular killing by PMN, but the extent of such promotion was dissimilar between these two strains of bacteria. These results suggest that post-phagocytic metabolic activities as well as intracellular killing of these Staphylococci may also be greatly influenced by FN-unrelated factors as are other bacteria having no FN-receptors.