Protection from virulent Salmonella groups B and D after the peroral immunization of chicks with a hybrid of Salmonella typhimurium and Salmonella dublin]

Chickens over 10 days old, infected orally with virulent salmonellae, were found to remain alive. Histologic investigation showed the development of mild enteritis and more pronounced, lasting for more than two weeks, inflammation of the cecum, dissemination and focal lesions in the liver (granulomas, necrosis). In experiments on the oral immunization of 3-day old chickens the bivalent hybrid of S. typhimurium vaccine strain 274 and S. dublin induced only pronounced blast transformation in lymphatic follicles of the cecum, hyperplasia of activated macrophages and formation of granulomas from these macrophages and lymphocytes. After oral challenge of the immunized chickens with virulent salmonellae of group B (S. typhimurium) and group D (S. enteritidis, S. gallinarum-pullorum) the chickens exhibited sharply pronounced protection against adhesion, colonization and invasion, and a few penetrating bacteria were rapidly destroyed by immune macrophages. Hybrid strain 274/O9 proved to be suitable for use as oral bivalent vaccine against salmonellosis in chickens.     

Effect of rat polymorphonuclear leukocyte granule components on the growth and survival of Pseudomonas aeruginosa and Salmonella typhimurium

Granule contents from rat polymorphonuclear neutrophils were prepared by extraction with 0.2 M acetate buffer (pH 4.0), dialyzed against phosphate-buffered saline (pH 7.0), and tested for bactericidal activity against selected target bacteria. Salmonella typhimurium LT-2 and a series of progressively rough lipopolysaccharide outer membrane mutants derived from it were used to monitor antimicrobial activity. Although an antimicrobial potential was present in rat granule contents for S. typhimurium, the growth of Pseudomonas aeruginosa PAO-1 in antimicrobial assay mixtures containing rat granule contents was substantially enhanced over control values. The growth enhancement property of the granule protein was heat resistant and promoted increased oxygen consumption by whole cells.     

Protective effect of recombinant tumor necrosis factor-alpha in murine salmonellosis

The enhancement of resistance by i.p. administration of murine rTNF-alpha into mice against i.p. challenge with virulent Salmonella typhimurium was studied. Administration of TNF-alpha (5 x 10(4) U/mouse) into mice at 6 or 12 h before the challenge with S. typhimurium organisms enhanced the bactericidal capacity in the peritoneal cavities of the mice. The diminution of the infecting organism in the peritoneal cavities of the TNF-alpha-treated mice was not due to the systemic spread of the organism inasmuch as few organism were recovered from other areas such as the spleen and liver. The TNF-alpha treatment effected a slight increase of neutrophils in the peritoneal cavity, but did not effect an increase of macrophages, including Ia-bearing macrophages. The survival rate of mice infected with Salmonella was improved by the i.p. injection of TNF-alpha before infection. Co-administration of smaller doses of TNF-alpha (5 x 10(3) U) and murine rIFN-gamma (10(2) U) at 6 h before the challenge also effectively enhanced bactericidal activity and protectivity. The cooperative effect of TNF-alpha and IFN-gamma was only seen when these recombinant cytokines were injected together at the proper time before the challenge. Injection of rabbit anti-TNF-alpha serum abolished the effects of TNF-alpha and the cooperative effect of TNF-alpha and IFN-gamma. Furthermore, the serum could abolish the cooperative effect of IFN-gamma and LPS on bactericidal activity, suggesting participation of LPS-induced TNF-alpha in the cooperation.     

Interaction of T-activin with peritoneal macrophages

The action of T-activin on peritoneal macrophages of CBA mice after its introduction into the animals has been studied. In intact mice the phagocytic activity of macrophages and their resistance to the cytopathogenic action of Salmonella typhimurium live cells remains unchanged. The injection of corpuscular pertussis vaccine into mice leads to a decrease in the resistance of macrophages to the action of salmonellae. The simultaneous injection of T-activin into mice in doses of 0.1 and 1.0 microgram per animal abolishes the damaging action of the vaccine. The analysis of the in vitro action of T-activin on macrophages of intact mice revealed that the preliminary incubation of cells with the preparation sharply increases their resistance to the action of salmonellae, while its introduction simultaneously with bacteria or after them rapidly leads to the death of macrophages. The action of T-activin is supposed to be linked with triggering the biosynthetic processes mediating the resistance of macrophages to the cytopathogenic action of salmonellae.     

Effect of insulin and isoproterenol on macrophage phagocytic activity

To reveal the influence of cyclic adenosine monophosphate (cAMP) on the completion of the phagocytosis of salmonellae, the influence of insulin and isoproterenol on the phagocytic activity of peritoneal macrophages obtained from mice infected with S. typhimurium strains differing in virulence was studied in vitro. The study showed that isoproterenol, while increasing the intracellular content of cAMP, suppressed the bactericidal properties of macrophages with respect to salmonellae, whereas insulin decreased the level of cAMP in the cells and thus facilitated more rapid and complete digestion of ingested bacteria irrespective of their virulence.     

Neutrophil migration across monolayers of cytokine-prestimulated endothelial cells: a role for platelet-activating factor and IL-8

In a previous study we observed that neutrophils respond with a rapid rise in [Ca2+]i during adherence to cytokine-activated endothelial cells (EC), caused by EC membrane-associated platelet-activating factor (PAF). In the present study, we investigated whether this form of PAF was important in neutrophil adherence and migration across monolayers of rIL-1 beta- or rTNF alpha-prestimulated EC. PAF receptor antagonists prevented neutrophil migration across cytokine-pretreated EC by approximately 60% (P less than 0.005) without interfering with the process of adherence. The antagonists WEB 2086 and L-652,731 had no effect on neutrophil migration across resting EC induced by formylmethionyl-leucyl-phenylalanine (FMLP). A murine anti-IL-8 antiserum was found to also partially inhibit the neutrophil transmigration across cytokine-activated EC. When the anti-IL-8 antiserum was used in combination with a PAF receptor antagonist, neutrophil migration across cytokine-pretreated monolayers of EC was completely prevented. During transmigration, LAM-1 and CD44 on the neutrophils were down-modulated; both WEB 2086 and anti-IL-8 antiserum partially prevented this down-modulation caused by cytokine-prestimulated EC. Our results indicate that human neutrophils are activated and guided by EC-associated PAF and EC-derived IL-8 during the in vitro diapedesis in between cytokine-stimulated EC.     

The role of natural killer cells in experimental murine salmonellosis

This study was designed to determine if murine natural killer (NK) cells play a role in host protection against a Salmonella typhimurium challenge infection. Outbred ICR mice injected intravenously with either attenuated (RIA strain) or virulent (SR-11 strain) salmonellae elicited enhanced killing of YAC-1 targets, which was maximal at 24 h after challenging. When NK cells were depleted with antiasialo GM1 prior to challenging, the splenic bacterial numbers were significantly less in this group of mice compared to sham-injected and challenged animals. The rabbit antiasialo GM1 sera had no detectable direct or indirect effect on the salmonellae. Our results indicate that the NK or natural suppressor cells may be functioning as down-regulators.     

Cutting edge: the SLAM family receptor Ly108 controls T cell and neutrophil functions

Ly108, a glycoprotein of the signaling lymphocytic activation molecule family of cell surface receptors expressed by T, B, NK, and APCs has been shown to have a role in NK cell cytotoxicity and T cell cytokine responses. In this study, we describe that CD4(+) T cells from mice with a targeted disruption of exons 2 and 3 of Ly108 (Ly108(DeltaE2+3)) produce significantly less IL-4 than wild-type CD4(+) cells, as judged by in vitro assays and by in vivo responses to cutaneous infection with Leishmania mexicana. Surprisingly, neutrophil functions are controlled by Ly108. Ly108(DeltaE2+3) mice are highly susceptible to infection with Salmonella typhimurium, bactericidal activity of Ly108(DeltaE2+3) neutrophils is defective, and their production of IL-6, IL-12, and TNF-alpha is increased. The aberrant bactericidal activity by Ly108(DeltaE2+3) neutrophils is a consequence of severely reduced production of reactive oxygen species following phagocytosis of bacteria. Thus, Ly108 serves as a regulator of both innate and adaptive immune responses.     

Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity

Neutrophil granules contain proteins important in host defense against bacterial pathogens. Granule proteins released from activated neutrophils facilitate opsonization, phagocytosis, tissue digestion, and antimicrobial activity. Three similar, if not identical, neutrophil proteins, bactericidal/permeability-increasing protein (BPI), 57,000 m.w. cationic antimicrobial protein, and bactericidal protein have been described that specifically kill gram negative bacteria. Since LPS is a structure common to all gram-negative bacteria, we investigated whether the microbicidal protein BPI affects biologic activity of LPS in vitro. Human neutrophils can be activated both in vitro and in vivo by LPS. Upon stimulation, surface expression of CR1 and CR3 increases markedly. Using flow microfluorimetry, we analyzed surface expression of CR1 and CR3 as a measure of neutrophil stimulation in response to LPS. CR up-regulation on neutrophils was TNF independent, suggesting direct LPS stimulation of neutrophils in this system. Purified BPI completely inhibited CR up-regulation on neutrophils stimulated with both rough and smooth LPS chemotypes at 1.8 to 3.6 nM (100 to 200 ng/ml). By comparison, the polypeptide antibiotic polymyxin B completely inhibited the same dose of LPS at 0.4 nM. The inhibitory activity of BPI appeared to be specific for LPS because neutrophil stimulation by formylated peptide or TNF was unaffected. The specificity of BPI for LPS was further demonstrated by inhibition of LPS activity in the limulus amebocyte lysate assay. Therefore, the role of BPI in infection may not be limited to its microbicidal activity, but it may also regulate the neutrophil response to LPS.     

Staphylococcus aureus resists human defensins by production of staphylokinase, a novel bacterial evasion mechanism

Alpha-defensins are peptides secreted by polymorphonuclear cells and provide antimicrobial protection mediated by disruption of the integrity of bacterial cell walls. Staphylokinase is an exoprotein produced by Staphylococcus aureus, which activates host plasminogen. In this study, we analyzed the impact of interaction between alpha-defensins and staphylokinase on staphylococcal growth. We observed that staphylokinase induced extracellular release of alpha-defensins from polymorphonuclear cells. Moreover, a direct binding between alpha-defensins and staphylokinase was shown to result in a complex formation. The biological consequence of this interaction was an almost complete inhibition of the bactericidal effect of alpha-defensins. Notably, staphylokinase with blocked plasminogen binding site still retained its ability to neutralize the bactericidal effect of alpha-defensins. In contrast, a single mutation of a staphylokinase molecule at position 74, substituting lysine for alanine, resulted in a 50% reduction of its alpha-defensin-neutralizing properties. The bactericidal properties of alpha-defensins were tested in 19 S. aureus strains in vitro and in a murine model of S. aureus arthritis. Staphylococcal strains producing staphylokinase were protected against the bactericidal effect of alpha-defensins. When staphylokinase was added to staphylokinase-negative S. aureus cultures, it almost totally abrogated the effect of alpha-defensins. Finally, human neutrophil peptide 2 injected intra-articularly along with bacteria alleviated joint destruction. In this study, we report a new property of staphylokinase, its ability to induce secretion of defensins, to complex bind them and to neutralize their bactericidal effect. Staphylokinase production may therefore be responsible in vivo for defensin resistance during S. aureus infections.     

Killing of yeast, germ-tube and mycelial forms of Candida albicans by murine effectors as measured by a radiolabel release microassay

Candida albicans undergoes yeast to mycelial conversion under both in vivo and in vitro conditions but the relative pathogenicity of the two forms of growth is still unknown. By adapting a recently developed 51Cr radiolabel release assay, we have quantified the killing ability of different murine effector cell populations for the hyphal form of C. albicans. Up to 50% of specific 51Cr release from the mycelial form could be detected after incubation for only 1 h, with no requirement for opsonization, provided that appropriate effector: target cell ratios were used. The specific 51Cr release correlated well with viability, as assessed by dye exclusion tests, and with pathogenicity potential in cyclophosphamide-immunodepressed mice. Comparison of the activity of different murine effectors against yeast and hyphal forms showed that hyphal forms were killed by murine effectors to a similar, if not greater, extent than yeast forms. In particular, thioglycollate-induced murine polymorphonuclear neutrophils were able to kill hyphal cells extracellularly and without an opsonic requirement.     

An experimental study of a lactoglobulin preparation against opportunistic bacteria and salmonellae]

A preclinical study of seven batches of lactoglobulin, a new biological preparation against opportunistic bacteria and salmonellae, has been carried out. High antibacterial activity of the preparation with respect to the virulent forms of Salmonella typhimurium, Salmonella enteritidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris, Proteus mirabilis has been established. The preparation has been shown to be safe and nontoxic. The 4-year term of its storage at a temperature of 6 degrees +/- 4 degrees C has been substantiated.     

Adoptive transfer of natural killer cell activity in B6D2F1 mice challenged with Salmonella typhimurium

The purpose of this study was to extend our previous findings as to the role of murine NK cells in host protection to a challenge infection with virulent Salmonella typhimurium SR-11. B6D2F1 mice were depleted of NK cells with anti-asialo GM1 or a monoclonal antibody, anti-NK 1.1, followed by a salmonellae challenge. Significantly decreased numbers of splenic bacteria (P less than 0.005) in the NK cell-depleted mice were note at 12, 24, and 48 hr postchallenging, compared to the sham-injected control animals. When Percoll gradient-enriched large granular lymphocytes (NK cells) were adoptively transferred to NK cell-depleted mice followed by challenging, the splenic bacterial numbers were comparable to those present in NK cell-intact, control mice. These data indicate that large granular lymphocytes (NK cells) are responsible for the down-regulation of the protective host response in mice challenged with the facultative intracellular parasite. S. typhimurium.     

Outer membrane protein A deficient Escherichia coli activates neutrophils to produce superoxide and shows increased susceptibility to antibacterial peptides

The outer membrane protein A (OmpA) of Gram-negative bacteria has been ascribed multiple functions including maintenance of structural membrane integrity and porin activity. OmpA has also been implicated in various host defense processes in that it contributes to bacterial serum resistance and activates certain immune cells. Recently, OmpA was shown to be the molecular target for neutrophil elastase (NE), and Escherichia coli mutants lacking OmpA were resistant to the bactericidal effects of NE. In addition to NE, neutrophils use a variety of other antibacterial effector molecules such as oxygen radicals and bactericidal peptides or proteins. The aim of this study was to investigate the role of E. coli OmpA regarding susceptibility to other neutrophil-derived defense systems. We found that OmpA-deficient (OmpA(-)), but not wild-type isogenic, E. coli activated human neutrophils to produce oxygen radicals intracellularly. This activation was found to require an intact neutrophil cytoskeleton but was independent of bacterial phagocytosis. Furthermore, we found that the OmpA(-) strain was more susceptible to membrane-acting bactericidal peptides than the wild-type strain, although the susceptibility to different oxygen radicals was independent of the presence of OmpA. Taken together, these data suggest an important role for OmpA in the context of bacteria vs. host interactions.     

The effect of oxidant stress on diamide-treated human granulocytes

The role of sulfhydryls in the protection of human polymorphonuclear neutrophils against extracellular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant-generating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xanthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants.     

The effect of oxidant stress on diamide-treated human granulocytes.

The role of sulfhydryls in the protection of human polymorphonuclear neutrophils against extracellular oxidant attack was investigated by simultaneously exposing polymorphonuclear neutrophils to the thiol-oxidizing agent diamide and the oxidant-generating system xanthine-xanthine oxidase. Neither diamide nor the oxidants generated by the xanthine-xanthine oxidase system alone impaired the burst in chemiluminescence, hexose monophosphate shunt activity or formate oxidation normally seen during polymorphonuclear neutrophil phagocytosis. Incubation of the polymorphonuclear neutrophils simultaneously with diamide and xanthine-xanthine oxidase markedly impaired polymorphonuclear neutrophil phagocytosis, hexose monophosphate shunt activity, chemiluminescence and formate oxidation. Although the polymorphonuclear neutrophils exposed to diamide and xanthine-xanthine oxidase did not respond to a variety of phagocytizable stimuli, trypan blue exclusion was normal and hexose monophosphate shunt activity could be stimulated by diamide. The damaging effect of the diamide xanthine-xamthine oxidase system could be blocked by the addition of superoxide dismutase or catalase, but not by hydroxyl radical or singlet oxygen scavengers. We hypothesize that an unidentified population of thiols may play a role in protecting the polymorphonuclear neutrophil from endogenously derived oxidants.     

Genetic control of the innate resistance of mice to Salmonella typhimurium: expression of the Ity gene in peritoneal and splenic macrophages isolated in vitro

The mouse Chromosome 1 locus Ity regulates the extent to which Salmonella typhimurium replicates within the reticuloendothelial cell system (RES) during the first days of infection. If animals are homozygous for the Itys susceptibility allele, the Gram-negative bacterium undergoes rapid net multiplication, and mice die of a typhoid fever-like disease by day 10 of infection. Animals that are homozygous or heterozygous for the resistance allele, Ityr, control net bacterial replication and survive the first phase of salmonellosis. Indirect studies have implicated the resident macrophage as the effector cell for regulation of early in vivo salmonellae growth. To verify this supposition and to evaluate the phenotypic expression of Ity, we developed an in vitro assay to compare kinetics of S. typhimurium growth within Ityr and Itys macrophages. Resident peritoneal and splenic macrophages were used from inbred Ityr and Itys mice and from Ity congeneic mice. With these mice and through the use of radiolabeled S. typhimurium and an avirulent temperature-sensitive mutant of the bacterium, we found that: phagocytosis of S. typhimurium by Ityr and by Itys macrophages was the same; S. typhimurium grew to a greater extent in Itys peritoneal and splenic macrophages than in Ityr cells; Ityr macrophages killed intracellular salmonellae more efficiently than did Itys macrophages. Thus, we have demonstrated directly that Ity is expressed by the macrophage and have shown for the first time with Ity congeneic mice that the basis for differential net growth of virulent S. typhimurium in Ityr and Itys macrophages is a variation in the degree of bacterial kill.     

Opsonizing and bactericidal activity of antibodies against common antigen of Enterobacteriaceae

Domingue, Gerald J. (State University of New York at Buffalo, Buffalo, N.Y.), and Erwin Neter. Opsonizing and bactericidal activity of antibodies against common antigen of Enterobacteriaceae. J. Bacteriol. 91:129-133. 1966.-In addition to the well-known O, H, K, and Vi antigens, Enterobacteriaceae produce a common antigen, which was first identified by hemagglutination tests with Escherichia coli O14 antiserum. Studies on the biological significance of this antigen by in vitro phagocytic experiments have revealed that opsonization is markedly enhanced in the presence of the corresponding antibody, rabbit polymorphonuclear leukocytes, and normal rabbit serum. This effect is specific, because Pseudomonas aeruginosa, which is devoid of this antigen, is not opsonized under these conditions, and removal of the antibody by absorption markedly reduces the uptake of enteric bacteria containing the antigen. Opsonization thus represents another method for the study of this antigen-antibody system. Bactericidal tests have revealed that antibodies against this antigen, engendered in rabbits by different strains of enteric bacteria and various procedures, are bactericidal for E. coli O14 but not for other enteric bacteria, possibly due to previously demonstrated differences between the antigen moieties obtained from these microorganisms.     

Genetic control of the innate resistance of mice to Salmonella typhimurium: Ity gene is expressed in vivo by 24 hours after infection

The early response of inbred mice to infection with S. typhimurium is controlled by the mouse Chromosome 1 locus, Ity. To better understand the expression of this gene, the initial interactions between the reticuloendothelial system (RES) and i.v. injected salmonellae were compared in resistant (Ityr) and susceptible (Itys) mice. In both mouse strains 99% of the bacteria was cleared from the blood within 2 hr, and uptake of S. typhimurium by splenic and hepatic macrophages was similar regardless of Ity genotype. In vivo phagocytosis of bacteria was followed by a 30 to 60% decline in viable bacteria, which was attributed to the bactericidal activity of RES macrophages. Experiments with radiolabeled S. typhimurium strains TML and TML/TS27 (a temperature-sensitive mutant) confirmed that the efficiency of this early phase killing was not under Ity control. Despite the equivalent uptake and initial bactericidal activity by resident macrophages, bacterial numbers in the RES organs of Itys mice were significantly greater than in Ityr mice by approximately 24 hr after infection. These data suggest that Ity regulates the level of surviving intracellular bacteria that accumulate within resident macrophages of the liver and spleen.     

Inability of recombinant interferon-gamma to activate the antibacterial activity of mouse peritoneal macrophages against Listeria monocytogenes and Salmonella typhimurium

The effect of recombinant murine interferon-gamma (rIFN-gamma) as single stimulus for the activation of antibacterial activity of macrophages was investigated on the basis of the rate of intracellular killing of Listeria monocytogenes and Salmonella typhimurium by normal and rIFN gamma-activated peritoneal macrophages of CBA and C57BL/10 mice, which differ in natural resistance to infection by these bacteria. Eighteen hours after i.p. injection of 10 to 1 X 10(4) U rIFN-gamma, resident and exudate peritoneal macrophages which had phagocytosed L. monocytogenes or S. typhimurium in vivo, killed both species in vitro just as efficiently as did resident macrophages of normal mice. Similar results were obtained after 18 hr of in vitro incubation of resident or exudate peritoneal macrophages with 0.1 to 1 X 10(4) U/ml rIFN-gamma. Consistent with the in vitro findings, two i.v. injections of 5 X 10(4) U rIFN-gamma did not affect the rate of in vivo proliferation of L. monocytogenes or S. typhimurium in the spleens of mice during the first 2 days after i.v. injection of the bacteria. Compared with the effect on the controls, two i.p. injections of 5 X 10(2) to 5 X 10(4) U rIFN-gamma did not decrease the numbers of viable S. typhimurium in either the peritoneal cell suspension or the spleen 24 hr after i.p. injection of the bacteria. Checking the state of activation of rIFN-gamma-activated macrophages on the basis of two commonly used criteria for macrophage activation showed that rIFN-gamma-activated macrophages inhibited the intracellular replication of Toxoplasma gondii and displayed enhanced O2 consumption and H2O2 release after stimulation with phorbol myristate acetate compared with macrophages from normal CBA and C57BL/10 mice. The present findings show that as single activating stimulus, rIFN-gamma is not capable of activating the antibacterial effector functions of peritoneal macrophages against facultative intracellular pathogens such as L. monocytogenes and S. typhimurium.