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Two outbred mouse strains, ddY and CF1, were tested for their ability to be protected against infection with Salmonella typhimurium by several types of salmonella vaccines. These strains have the same levels of innate susceptibility to S. typhimurium, and also have the same capacity to develop delayed-type hypersensitivity (DTH) to salmonella antigens. Both the crude ribosomal fraction (CRF) and live-cell vaccines conferred acquired resistance on both strains, characterized by greater responses of T cells to salmonella antigens. Mice of the ddY strain were also protected by the purified transfer RNA (tRNA) vaccine, which was free of O antigens, but CF1 mice were not, despite the presence of T-cell reactivity with salmonella antigens. Neither strain was protected by the phenol-water-extracted lipopolysaccharide (LPS). The tRNA-immunized CF1 mice were protected by transfer of antiserum to CRF, but not by transfer of anti-LPS antibody. This antiserum to CRF, however, did not transfer acquired resistance into non-immune mice of either strain. These observations suggest that CF1 mice may require an antibody to another non-O antigen existing in CRF to develop acquired resistance, and that stimulation of the defence system by tRNA may be essential to the development of acquired resistance in CF1 mice.

Cleavage of C3 by CVF-B was demonstrated by hemolytic, immunoelectrophoretic and immune adherence reactions. No cleavage of C5 was detected by immunoelectrophoresis, but C5 hemolytic activity, assayed with EAC1423, decreased although less than C3 hemolytic activity. The co-existence of C3 with limiting amounts of C5 did not reduce the final degree of hemolysis of guinea pig erythrocytes (GPE) induced by late-acting components C6 through C9 and CVF-B. Thus, a CVF-B hemolytic system composed of GPE, C5 through C9 and CVF-B provided a method for titration of terminal components of human complement. CVF-B was able to generate hemolytically active sites of C567 on GPE by activation of C5, C6 and C7. The complex C567 in the fluid-phase decayed within 1 min but C567 on GPE was quite stable. Originally insensitive sheep erythrocytes became sensitive to the CVF-B hemolytic system if C3b sites were present, suggesting that cell-bound C3b played a role in orienting the positions of C567 to be fixed. CVF-B could be recovered quantitatively from the supernatant of the reaction mixture in which the hemolytically active intermediate GPEC-5678 had been formed through the interaction between C5 to C8 and CVF-B.

The effect of local injection of formalin-killed Corynebacterium kutscheri (FK.CK) on mouse survival after the intraperitoneal inoculation of Ehrlich ascites carcinoma in outbred ddY mice or P388 leukemia cells in inbred CDF1 mice was investigated. Treatment of mice in the dose range of greater than 10(6) organisms per mouse conferred the substantial protection on both mice. The initial phase of antitumor effect consisted of the marked increase in the number of peritoneal exudate cells and the enhanced cytotoxicity of peritoneal exudate cells. The Winn assay disclosed that antitumor effect by which tumor-burden mice could survive was attributable to nonadherent splenocytes whose activity was impaired by treatment with anti-T cell serum and complement. A single injection of FK.CK induced the cytotoxicity to three different murine tumor cells in serum of treated mice without a boosting injection of endotoxin. Furthermore, the generation of effector cells and serum cytotoxicity seemed to be paralleled by that of the delayed-type hypersensitivity to this organism. Thus, the antitumor resistance induced by C. kutscheri is considered to be in part T cell mediated.

We investigated mouse testicular extract (TE) to clarify its biological functions in reproductive immunity. TE, at concentrations of 50-300 micrograms/ml, enhanced macrophage activities of spreading, glucose consumption, and cytostasis against a susceptible tumor cell line. On the other hand, TE inhibited concanavalin A (Con A)-induced T-cell blastogenesis in the dose range of 10-600 micrograms/ml. To elucidate the origin of TE, W/Wv mice, which genetically lack germ cells, were used. TE obtained from W/Wv mice enhanced the spreadability of macrophages and inhibited Con A-induced blastogenesis of T cells. The enhancement of macrophage spreading was only achieved by the interstitial fluid (IF), while the suppression of Con A-induced T-cell responses was detected in seminiferous tubule fluid (STF) as well as in IF. TE did not affect listerial antigen-specific responses of lymphocytes in vitro. These results suggest that TE has the capacity to regulate the biological responses associated with reproduction.

Mice orally infected with Campylobacter jejuni developed focal infiltrative necrotic lesions in the liver, as determined by both histology and liver function tests. The initial histopathological feature was a focal infiltrative lesion in the parenchyma and portal triads. Foci of infiltrative lesions became necrotic between days 30 and 60 post-inoculation (p.i.). During this period, portal infiltrates increased in severity. From month 4 p.i., focal areas of infiltrative necrosis in the liver parenchyma became extensive. Study of liver function demonstrated mild elevations of transaminases, alkaline phosphatase and lactic dehydrogenase, and also the presence of hypoalbuminaemia. Although histopathological changes of the liver became gradually more marked after day 30 p.i., liver functions of infected mice were most affected at 2 months p.i. The capacity of C. jejuni to induce hepatic lesions seemed to be related to that of organisms to persist in the gall bladder; there was no correlation between biliary carriage in infected mice and positive faecal culture.

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.

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.

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.

Dialyzable factors (DF) were prepared from ribosomal fractions of several organisms including rough mutants of Salmonella typhimurium LT2, salmonella species of different serogroups, other enteric bacteria and gram-positive organisms, and tested for their immunogenicity against S. typhimurium infection in mice. All of them conferred local resistance on mice challenged intramuscularly with S. typhimurium LT2 in the early stage of immunization before the establishment of delayed-type hypersensitivity (DTH) to salmonella antigens. Although DFs of enteric bacteria including rough mutants of S. typhimurium induced DTH to salmonella antigens, only DF of a two-heptose mutant of S. typhimurium LT2 afforded significant mouse protection but others only prolonged the mean time to death. DF of Listeria monocytogenes induced the cross-reacting immunity which afforded the low level of mouse protection as well as an increase in mean time to death without inducing DTH. Passive transfer of anti-O antibody did not enhance the mouse protection provided by each DF. Resistance conferred by DF of S. typhimurium LT2 consisted of two phases: (i) nonspecific macrophage activation resulting in reduction of organisms at the infected site, which became active in the early stage of immunization and (ii) salmonella-specific immunity capable of preventing systemic infection, which became active in the late stage of immunization.

Delayed-type hypersensitivity (DTH) induced with Salmonella typhimurium transfer factor (TF) contributed to an increase in mean survival days of mice challenged with homologous organisms and afforded only a low level of host protection as determined by survival rate, compared with that obtained by active immunization. TF of other enteric bacteria could transfer DTH which is cross-reactive to salmonella antigen but did not afford host protection. Although TF of Listeria monocytogenes did not transfer the cross-reactive DTH, it could confer the significant increase in mean survival days against the lethal challenge with S. typhimurium. Listerial ribosomal vaccine conferred the high level of mouse protection without inducing DTH to salmonella antigen. The resistance generated upon active immunization with listerial ribosomal vaccine could be enhanced by the injection of S. typhimurium TF to the same level as that obtained after immunization with homologous ribosomal vaccine. Among salmonella TF, there could be no cross-reactive immunity between S. typhimurium and S. choleraesuis, although the cross-reactive DTH was observed. The DTH transfer ability of TF was sensitive to Pronase which could not affect the ability to transfer host immunity, but RNase could abolish the ability to transfer host immunity without impairing DTH transfer activity. These results suggest that in mouse typhoid infection, DTH is not associated with host protection as determined by survival rate.

An immunogenic dialyzable factor was obtained by dialysis of the freeze-thawed ribosomal fraction derived from a smooth virulent strain (LT2) of Salmonella typhimurium. Ion exchange chromatography of the dialyzable factor on Dowex 1-X2 (Cl- form) demonstrated the presence of four peaks and the fourth peak eluted with 0.4 M NaCl in 0.005 N HCl was found to be necessary for protection. This effective peak was not obtained by chromatography of nonprotective dialyzable factors such as an RNase digest. Dowex chromatography of the dialyzable factors isolated from rough mutants of strain LT2 revealed that the dialyzable factor of strain SL1004 whose live vaccine is capable of inducing protective immunity contained fairly large amounts of peak IV. DEAE-cellulose for two-dimensional thin layer chromatography was used to identify the composition of the dialyzable factor and peak IV. Eight spots were located under ultraviolet light and seven spots were characterized by their absorption ratios. In peak IV, four nucleotides were located and identified by comparison with a map of the original dialyzable factor. The data show that the effective components of the dialyzable factor are mixed nucleotides and may be unique to ribonucleic acids of strains of S. typhimurium in which live vaccines are capable of affording mouse protection.

The preparation, properties, and immunogenicity of the dialyzable factor from a ribosomal fraction of Salmonella typhimurium are described. The ribosomal fraction was purified to eliminate O-antigenic components, by affinity chromatography (Sepharose-anti-O antibody conjugates used as immunoadsorbent). The dialyzable factor was obtained in the concentrated dialysate of the purified ribosomal fraction which was alternately frozen in dry-ice acetone and thawed in an 80 C water bath, for a total of five or six cycles. When this preparation was tested for its ability to protect mice against challenge with 1,000 LD50 of the homologous bacteria, it afforded 100% protection at a dose equivalent to 5.0 micrograms of RNA. The protection conferred by this factor was mainly cell mediated but immune serum enhanced this immunity despite the fact that no antibodies were detected in it. The protective activity of this factor was sensitive to RNase digestion but resistant to proteolytic enzymes. Ion exchange chromatography of this factor with DEAE-Sephadex A-25 (in 7 M Urea-0.02 M Tris-HCl buffer, pH 7.5) resulted in a single A260 peak which was found to be immunogenic. Chemical analysis of this peak after it was concentrated and desalted revealed that this immunogenic fraction was composed mainly of mixed nucleotides. The data indicate that protective immunity conferred by a ribosomal vaccine is associated with RNA but may not require the intact RNA molecule.

Various small fragments of (see article) which is one of the immunodominant groups of hen egg-white lysozyme (HL), were tested for macrophage migration inhibition (MMI) of peritoneal exudate cells (PEC) from guinea pigs immunized with HL. P17 was split in the middle with cyanogen bromide. The terminal portion of (see article) showed positive MMI, whereas the non-terminal half of P17, P17i (sequence 13-27) only showed very weak MMI activity. A fragment derived from the middle portion of P17, P17m (sequence 11-22), was inactive. When P17 were reduced and alkylated, one of the resultant peptides, P17N (sequence 1-[CM-Cys-6]-27) still has MMI activity with PEC taken from guinea pigs immunized with HL, although no antibody reacting with it was detected, but P17C (sequence 123-[CM-Cys-127]-129) was inactive. The peptides P17 and P17N were both immunogenic in guinea pigs in respect to the delayed hypersensitivity response. Again P17t and P17N were immunodominant groups, but the reactivity of P17i in MMI assay of this group of animals was greater than that in guinea pigs immunized with HL. The reactivities of HL with PEC taken from guinea pigs immunized with P17 or P17N were generally weaker than those of the antigens used for immunization.

A complex, CVF-B, between cobra venom factor (CVF) and human factor B(B) showed weak, short-lived enzymatic activity against the third component of human complement (C3). Once activated with trypsin, it showed strong, stable activity against C3 and C5. CVF-B, an activated form of CVF-B complex, was not affected by the trypsin inhibitor, diisopropylfluorophosphate and neuraminidase. Heating at 56 C for 30 min completely destroyed its activity and heating at 50 C for 30 min destroyed approximately half its activity. The activity of DVF-B decrease markedly at pH 6.0 but was stable at pH 6.5 to 8.5. CVF-B lost 90% of its activity on reduction with 1 mM dithiothreitol, and was completely adsorbed on a cellulose acetate membrane. CVF-B was found to be a complex of CVF and glycine-rich gamma-glycoprotein, with a molecular weight of 340,000. The CVF-B molecule consisted of 4-polypeptide chains, 3 of which were derived from CVF and one from GGG. Hemolytically active CVF-B may be formed from 2 molecules with four-polypeptide chains linked by unknown bonds. Human, rat and guinea pig sera could react with CVF-B to generate a lytic factor. Human and sheep erythrocytes were not sensitive to the lytic factor generated by CVF-B, whereas liposomes prepared from their membrane lipids were equally sensitive to the lytic factor.

A complex formed between cobra venom factor (CVF) and isolated human factor B (B) was found to be converted by trypsin to a stable enzyme, CVF-B which cleaved the third component (C3) and the fifth component (C5) of human complement. The formation of CVF-B by trypsin required divalent cations, whereas the formation of the lytic factor from human serum occurred even in the presence of EDTA. CVF-B purified by gel filtration could initiate the hemolysis of unsensitized guinea pig erythrocytes when incubated with human complement components C5 to C9 in 0.01 M EDTA buffer. C3 was not required for the lysis of guinea pig erythrocytes initiated by CVF-B because of the beta1C precipitation line formed between human serum and anti-beta1C antibody did not inhibit the hemolysis by CVF-B in agarose gel. Treatment of beta1C and beta1F globulins in whole human serum with CVF-B in the presence of 0.01 M EDTA converted them to components with higher mobilities on immunoelectrophoresis.

The active component in crude ribosomal fraction (CRF) of Salmonella typhimurium, capable of inducing protective antibody, was partially purified by two series of chromatography (Sephadex G-150 and DEAE-Sepharose CL6B) after sodium dodecyl sulphate (SDS)-treated CRF was precipitated with ammonium sulphate. The major active component was eluted by 0.4-0.45 M NaCl from DEAE-Sepharose CL6B, and its molecular weight was 43,000 as determined by SDS-polyacrylamide gel electrophoresis. Immunization with the fraction containing 43,000 component alone did not always confer protection on CF1 mice, but its administration together with either the purified transfer RNA (tRNA) or Freund's complete adjuvant (FCA) was much more effective against infection with S. typhimurium. Antibody to the fraction containing 43,000 component was not only free in serum but also associated with peritoneal cells. Macrophages that had been exposed to the antibody had enhanced anti-bacterial activity. Western blot analysis showed that 43,000 component did not react to antiserum to lipopolysaccharide (LPS), but to antiserum to CRF. The antibody elicited by non-O antigenic component and the cell-mediated resistance stimulated by the adjuvant effect of RNA together confer effective protection on CF1 mice.

In order to compare the potential of salmonella vaccines prepared from Salmonella typhimurium to provide the longer-lasting protection from the aspects of cell-mediated immunity, groups of mice were immunized with optimal doses of the following preparations: live cells, ribosome-rich extract, acetone-killed cells, and heat-killed cells. At various intervals post-immunization, mouse peritoneal macrophages and splenic T cells were tested for biological activities. The capacity of each vaccine to confer mouse protection against a lethal challenge with S. typhimurium correlated with the degree of macrophage activation engendered by each of them in the early stage of immunization. In the late stage of immunization, the level of mouse protection conferred by each vaccine was found to be based on the capacity of T cells to respond to salmonella antigens, which correlated with the degree of adoptive immunity by T cells. The live-cell and ribosomal vaccines were superior to killed-cell vaccines in inducing the cell-mediated protection. Thus, the longer-lasting immunity provided by the live-cell and ribosomal vaccines can be accounted for by the fact that T cells of mice immunized with both vaccines have the persistent reactivity to salmonella antigens.

Five kinds of fragments of hen egg-white lysozyme (HL) were tested by macrophage migration inhibition (MMI) assay using peritoneal exudate cells (PEC) of guinea pigs immunized with HL in complete Freund's adjuvant. (See article). These four kinds of HL fragments were also shown to be composed of the immunodominant groups of the HL molecule for circulating antibody against HL in guinea pigs. The relationships between the antigenic sites related to circulating antibody and the cellular recognition sites are discussed.