Specific antigens of the causative agents and their antibodies in the circulating immune complexes in acute dysentery

The level of circulating immune complexes has been determined in 53 patients in the dynamics of the disease. For the first time circulating immune complexes have been found to contain Shigella sonnei K-antigen and Shigella flexneri O-antigen, as well as IgA, IgG and IgM to Shigella. Shigella antigens can be detected from the first week of the disease, and their occurrence does not depend on the level of circulating complexes in patients blood serum.     

Depressed Cellular Formation of Antibody to Shigella Antigens In Vitro by Spleen Cell Cultures from Unresponsive Mice

Specific antibody plaque-forming cells (PFC) to Shigella-soluble antigen did not appear in spleen cell cultures from Shigella-tolerant mice, as occurred with similar cultures prepared from normal mice immunized with Shigella antigen prior to sacrifice. Cultures from tolerant mice also failed to form serologically detectable amounts of agglutinins in vitro. Exposure of cell cultures from tolerant mice in vitro to additional antigen had little or no effect on appearance of plaque-forming cells to Shigella. Spleen cells from normal control mice formed readily detectable levels of antibody, as well as specific antibody plaque-forming cells, after similar stimulation with antigen either in vivo or in vitro. The absence of antibody-forming cells in cultures prepared from spleens of tolerant mice was specific since such cultures, as well as those from normal control mice, formed numerous antibody plaques to unsensitized sheep erythrocytes in vitro after in vivo challenge of the mice with sheep erythrocytes. Tolerance to Shigella antigen, as assessed by absence of antibody-forming cells in vitro, persisted for several months. Spleen cell cultures from tolerant mice less than 3 to 4 months of age did not form significant numbers of antibody plaques, even after in vitro exposure to specific antigen. However, spleen cultures prepared from neonatally treated mice, approximately 6 to 8 months old, formed essentially normal numbers of specific PFC in vitro, indicating that the animals had "recovered" from tolerance and that their lymphoid cells were capable of responding to Shigella antigen in vitro. Absence of specific PFC in cell cultures from tolerant animals supports the concept that tolerance is due to a central failure of specific immunocompetent cells and not due to an inhibitory effect caused by either "excess" antigen or humoral antibody.     

Cathepsin D activity in splenocytes in relation to Shigella virulence in an experiment

The overtime study of changes in the activity of cathepsin D, a lysosomal enzyme, in the splenocytes of CBA mice after their infection with virulent and avirulent Shigella strains of the same origin and with the same antigenic structure has been made. As the result of two months of observations, changes in the activity of this enzyme in the cytoplasmic and lysosomal cell fractions have been found to occur in phases. The activity of cathepsin D has been shown to depend on the virulence of Shigella strains used for inoculation. Virulent Shigella strains induce the pronounced and prolonged activation of the enzyme in the lysosomes, as well as in the cytoplasm. The latter phenomenon is probably indicative of the pathological labilization of the lysosomal membranes, induced by the virulent culture. Avirulent Shigella strains induce only the transient activity of the enzyme in the lysosomes without any essential changes in the permeability of their membranes. These data point to the possibility of differentiating virulent and avirulent Shigella strains by the determination of the enzymatic activity of splenocytes in infected animals.     

The action of ribosomal preparations on nonspecific resistance to bacterial infection and on early tolerance to endotoxic shock

Ribosomal preparations from Shigella flexneri and Shigella sonnei, introduced parenterally into mice, enhance their resistance to infection with the causative agents of typhoid fever and staphylococci. This effect is considerably less pronounced than that produced by the preparation of homologous lipopolysaccharide isolated by Boivin's method. After the administration of ribosomes nonspecific resistance to bacterial infective agents lasts for a short time. Ribosomal preparations do not enhance the resistance of mice to the lethal action of endotoxin.     

Vaccination with a Shigella DNA vaccine vector induces antigen-specific CD8(+) T cells and antiviral protective immunity

A prototype Shigella human immunodeficiency virus type 1 (HIV-1) gp120 DNA vaccine vector was constructed and evaluated for immunogenicity in a murine model. For comparative purposes, mice were also vaccinated with a vaccinia virus-env (vaccinia-env) vector or the gp120 DNA vaccine alone. Enumeration of the CD8(+)-T-cell responses to gp120 after vaccination using a gamma interferon enzyme-linked spot assay revealed that a single intranasal dose of the Shigella HIV-1 gp120 DNA vaccine vector elicited a CD8(+) T-cell response to gp120, the magnitude of which was comparable to the sizes of the analogous responses to gp120 that developed in mice vaccinated intraperitoneally with the vaccinia-env vector or intramuscularly with the gp120 DNA vaccine. In addition, a single dose of the Shigella gp120 DNA vaccine vector afforded significant protection against a vaccinia-env challenge. Moreover, the number of vaccinia-env PFU recovered in mice vaccinated intranasally with the Shigella vector was about fivefold less than the number recovered from mice vaccinated intramuscularly with the gp120 DNA vaccine. Since the Shigella vector did not express detectable levels of gp120, this report confirms that Shigella vectors are capable of delivering passenger DNA vaccines to host cells and inducing robust CD8(+) T-cell responses to antigens expressed by the DNA vaccines. Furthermore, to our knowledge, this is the first documentation of antiviral protective immunity following vaccination with a live Shigella DNA vaccine vector.     

A newborn mouse model for the study of intestinal pathogenesis of shigellosis

Shigella infection is characterized by the induction of acute inflammation, which is responsible for the massive tissue destruction of the intestinal mucosa. A murine model would be a valuable tool for gaining a better understanding of the physiopathology of shigellosis and the host immune response to Shigella infection, but adult mice do not develop disease upon oral inoculation. We therefore attempted to develop a model of infection in newborn mice. Four-day-old mice inoculated with 50 microl of 5 x 10(9) invasive wild-type Shigella flexneri 5a were susceptible to bacterial infection, but mice inoculated with the non-invasive strain BS176 were not. Histologically, 4-day-old mice infected with the invasive strain presented intestinal lesions and inflammation similar to those described in patients with shigellosis. Moreover, cytokine and chemokine responses consistent with inflammation were observed. Lower bacterial inocula induced less severe intestinal damage. In contrast, 5-day-old mice inoculated with either the invasive or the non-invasive strain were not infected. We have thus established a mouse model that is suitable for the study of the pathogenesis of intestinal Shigella infection.     

Immunological tolerance to microbial antigens I. Absence of specific antibody-containing cells in lymphoid tissue of mice injected at birth with Shigella soluble antigen

Friedman, Herman (Albert Einstein Medical Center, Philadelphia, Pa.). Immunological tolerance to microbial antigens. I. Absence of specific antibody-containing cells in lymphoid tissue of mice injected at birth with Shigella soluble antigen. J. Bacteriol. 92:390-397. 1966.-Injection of a relatively large concentration of Shigella soluble antigen (SSA) into newborn mice results in specific immunological tolerance (paralysis) characterized by inability of the animals to form normal levels of anti-Shigella agglutinins upon subsequent challenge immunization with Shigella. Spleen and lymph nodes from Shigella-tolerant mice, as well as from normal and control immunized mice, were examined by the indirect immunofluorescence technique for evidence of cells containing anti-Shigella antibody. It was found that mice sacrificed at periodic intervals after neonatal administration of the tolerance-inducing inoculum of antigen and prior to and following challenge injection with a potential immunizing dose of SSA had only occasional specific fluorescing cells in spleens and lymph nodes. Tolerant mice also failed to develop significant levels of specific serum agglutinins after SSA challenge injection. In contrast, normal adult mice had a rapid appearance of numerous specific fluorescing cells in their spleens and lymph nodes, as well as a marked agglutinin response, after SSA immunization. Shigella-tolerant and normal control mice responded equally well with anti-Salmonella agglutinin formation and specific antibody-containing lymphoid cells after immunization with Salmonella antigen.     

A protective factor of ribosomal shigellosis vaccine

Shigella ribosomal vaccine was shown previously to possess protective properties in the keratoconjunctival test on guinea pigs and to be capable of preventing experimental infection in 90% of challenged monkeys. The presence of the O-specific component (OSC) constituting about 0.5% of the ribosomal preparation by serological activity suggested its importance for the protective effect. This was studied in experiments with two O-specific immunosorbents prepared by coupling anti-O rabbit antibodies with Staphylococcus aureus cells or with CNBr-Sepharose. Ribosomes treated with immunosorbents proved to be lacking the serologically active OSC and lost their ability to induce O-antibody response in rabbits and mice. After the removal of this component ribosomal preparations were incapable of ensuring protection from Shigella kerato-conjunctival infection. The isolated OSC was also inactive in this test. The data obtained in this investigation confirm the hypothesis stating that the protective activity of Shigella ribosomal vaccine is based on the combined action of ribosomes and O-specific factor whose nature and properties require further study.     

Local immunity in the parenteral immunization of guinea pigs with a ribosomal dysentery vaccine

After immunization of guinea pigs with Shigella sonnei ribosomal vaccine O-antibodies appeared not only in the blood serum of the animals, but also in their lacrimal fluid. Since no correlation between the levels of serum and secretory antibodies was detected and since the time course of changes in these antibody levels was quite different (serum antibodies reached their peak on day 7 while secretory antibodies, on day 14 after vaccination), antibodies in lacrimal fluid were supposed to reflect local immune response induced by parenteral administration of ribosomal vaccine, irrespective of systemic immune response. The peak of secretory O-antibodies coincided in time with the period of the highest protection of guinea pigs from Shigella keratoconjunctivitis. The animals with a high level of secretory antibodies were better protected from Shigella infection than those with a low level of secretory antibodies. These data suggest that locally produced O-antibodies play an important role in protective immunity induced by parenteral administration of the ribosomal vaccine.     

Immune response to rotavirus VP4 expressed in an attenuated strain of Shigella flexneri

An attenuated strain of Shigella flexneri was utilised to express viral protein (VP) 4 of rotavirus and the immunogenicity of the recombinant constructs was studied in BALB/c mice. VP4 was expressed as a fusion with maltose binding protein (MBP) in both the cytoplasm and periplasm, with a much higher level of expression occurring in the former. While all constructs induced a Shigella-specific response in mice, only the construct expressing MBP-VP4 in the cytoplasm of Shigella stimulated an immune response specific to rotavirus. This study demonstrates that Shigella can be used to deliver rotavirus antigens and induces an immune response directed towards both rotavirus and Shigella.     

Immunochemical studies of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1 O-specific polysaccharide-core fragments and their protein conjugates as vaccine candidates

There is no licensed vaccine for the prevention of shigellosis. Our approach to the development of a Shigella vaccines is based on inducing serum IgG antibodies to the O-specific polysaccharide (O-SP) domain of their lipopolysaccharides (LPS). We have shown that low molecular mass O-SP-core (O-SPC) fragments isolated from Shigella sonnei LPS conjugated to proteins induced significantly higher antibody levels in mice than the full length O-SP conjugates. This finding is now extended to the O-SPC of Shigella flexneri 2a and 6, and Shigella dysenteriae type 1. The structures of O-SPC, containing core plus 1-4 O-SP repeat units (RUs), were analyzed by NMR and mass spectroscopy. The first RUs attached to the cores of S. flexneri 2a and 6 LPS were different from the following RUs in their O-acetylation and/or glucosylation. Conjugates of core plus more than 1 RU were necessary to induce LPS antibodies in mice. The resulting antibody levels were comparable to those induced by the full length O-SP conjugates. In S. dysenteriae type 1, the first RU was identical to the following RUs, with the exception that the GlcNAc was bound to the core in the β-configuration, while in all other RUs the GlcNAc was present in the α-configuration. In spite of this difference, conjugates of S. dysenteriae type 1 core with 1, 2, or 3 RUs induced LPS antibodies in mice with levels statistically higher than those of the full size O-SP conjugates. O-SPC conjugates are easy to prepare, characterize, and standardize, and their clinical evaluation is planned.     

Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors

Shigella possess 220 kb plasmid, and the major virulence determinants, called effectors, and the type III secretion system (TTSS) are exclusively encoded by the plasmid. The genome sequences of S. flexneri strains indicate that several ipaH family genes are located on both the plasmid and the chromosome, but whether their chromosomal IpaH cognates can be secreted from Shigella remains unknown. Here we report that S. flexneri strain, YSH6000 encodes seven ipaH cognate genes on the chromosome and that the IpaH proteins are secreted via the TTSS. The secretion kinetics of IpaH proteins by bacteria, however, showed delay compared with those of IpaB, IpaC and IpaD. Expression of the each mRNA of ipaH in Shigella was increased after bacterial entry into epithelial cells, and the IpaH proteins were secreted by intracellular bacteria. Although individual chromosomal ipaH deletion mutants showed no appreciable changes in the pathogenesis in a mouse pulmonary infection model, the DeltaipaH-null mutant, whose chromosome lacks all ipaH genes, was attenuated to mice lethality. Indeed, the histological examination for mouse lungs infected with the DeltaipaH-null showed a greater inflammatory response than induced by wild-type Shigella, suggesting that the chromosomal IpaH proteins act synergistically as effectors to modulate the host inflammatory responses.     

Intravenous infection of virulent shigellae causes fulminant hepatitis in mice

Shigella spp. are pathogenic bacteria responsible for bacillary dysentery in humans. The major lesions in colonic mucosa are intense inflammation with apoptosis of macrophages and release of pro-inflammatory cytokines. The study of shigellosis is hindered by the natural resistance of rodents to oral infection with Shigella. Therefore, animal models exploit other routes of infection. Here, we describe a novel murine model in which animals receive shigellae via the caudal vein. Mice infected with 5 x 10(6) (LD(50)) virulent shigellae died at 48 h post infection, whereas animals receiving non-invasive mutants survived. The liver is the main target of infection, where shigellae induce microgranuloma formation. In mice infected with invasive bacteria, high frequency of apoptotic cells is observed within hepatic microgranulomas along with significant levels of mRNA for pro-inflammatory cytokines such as IL-1beta, IL-18, IL-12 and IFN-gamma. Moreover, in the blood of these animals high levels of IL-6 and transaminases are detected. Our results demonstrate the intravenous model is suitable for pathogenicity studies and useful to explore the immune response after Shigella infection.     

Animal toxicity of Shigella dysenteriae cytotoxin: evidence that the neurotoxic, enterotoxic, and cytotoxic activities are due to one toxin

The lethal effect to rabbits and mice of Shigella dysenteriae toxin and the ability of the toxin to induce fluid accumulation in rabbit ileal loops were studied in relation to the cytotoxic activity. The relative concentrations of the three activities were approximately the same in a crude toxin preparation and in purified, electrophoretically homogenous toxin. The cytotoxic and lethal activities eluted identically from a high pressure liquid chromatography column and migrated at the same rate in polyacrylamide gel electrophoresis under nondenaturing conditions. The cytotoxic, lethal, and enterotoxic activities were inactivated to essentially the same extent upon incubation for few minutes at 80 degrees C and upon treatment with urea. Graded precipitation of Shigella toxin with different amounts of an antiserum to Shigella toxin in each case removed essentially the same fraction of the cytotoxic, the lethal, and the enterotoxic activity. The data indicate that one molecular entity is responsible for the three biologic effects of Shigella toxin studied. After i.v. injection, the LD50 dose was estimated to be 2.2 ng/kg in rabbits and 450 ng/kg in mice.     

Modulation of immune system cells by lactobacilli

The influence of lactobacilli on the proliferative potential of immune system cells after the intragastral administration of viable microbial cells and their native filtrates to mice CBA is evaluated. The data have been obtained on the modulating influence of lactobacilli on the formation of T- and B-cell immune response--their role in maintaining homeostatsis and specific features of cell mediated immune reactions after the intragastral administration of virulent Shigella dysenteriae for modeling experimental infection in CBA mice. The mechanisms of the immunomodulating action of lactobacilli on local and systemic reactions of the host as well as realization of the protective properties of lactobacilli against the causative agents of acute enteric infections are discussed.     

Shigella flexneri infection is dependent on villin in the mouse intestine and in primary cultures of intestinal epithelial cells

Villin is an actin-binding protein present in intestinal and kidney brush borders. Villin has been shown to present in vitro Ca(2+)-dependent bundling and severing F-actin properties. The study of villin knock-out mice allowed us to show that while bundling of F-actin microfilaments is unaffected, this protein is important for the reorganization of the actin cytoskeleton elicited by various signals during both physiological and pathological conditions. Here, we studied the role of villin during infection by Shigella flexneri, the causative agent of bacillary dysentery. This bacterium induces the reorganization of the host actin cytoskeleton to penetrate into epithelial cells and spread from cell to cell. In vivo, we show that unlike newborn vil+/+ mice, which are sensitive to Shigella invasion, resulting in a destructive inflammatory response of the intestinal mucosa following intragastric inoculation, newborn vil-/- mice appear fully resistant to infection. Using primary cultures of intestinal epithelial cells derived from vil+/+ or vil -/- mice, we demonstrate that villin plays an essential role in S. flexneri entry and cell-to-cell dissemination. Villin expression is thus critical for Shigella infection through its ability to remodel the actin cytoskeleton.     

Structure and genetics of Shigella O antigens

This review covers the O antigens of the 46 serotypes of Shigella, but those of most Shigella flexneri are variants of one basic structure, leaving 34 Shigella distinct O antigens to review, together with their gene clusters. Several of the structures and gene clusters are reported for the first time and this is the first such group for which structures and DNA sequences have been determined for all O antigens. Shigella strains are in effect Escherichia coli with a specific mode of pathogenicity, and 18 of the 34 O antigens are also found in traditional E. coli. Three are very similar to E. coli O antigens and 13 are unique to Shigella strains. The O antigen of Shigella sonnei is quite atypical for E. coli and is thought to have transferred from Plesiomonas. The other 12 O antigens unique to Shigella strains have structures that are typical of E. coli, but there are considerably more anomalies in their gene clusters, probably reflecting recent modification of the structures. Having the complete set of structures and genes opens the way for experimental studies on the role of this diversity in pathogenicity.     

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

SUMMER DIARRHEA IN THE SAN JOAQUIN VALLEY

In laboratory, epidemiologic and clinical studies of 85 patients with diarrhea admitted to the General Hospital of Fresno County and the San Joaquin County Hospital during part of the summer of 1949 the following features were noted:1. Cultures were positive for Shigella in about 45 per cent of the cases in the San Joaquin County Hospital and in about 15 per cent of those in the Fresno County Hospital.2. In 80 per cent of cases in which there was gross blood in the stools before the third day of hospitalization, Shigella grew on cultures. No gross blood was noted in 27 per cent of cases in which Shigella was demonstrated.3. The cases in which Shigella was demonstrated did not differ greatly from the others with regard to fever, leukocytosis, or response to therapy.4. Salmonella organisms were not grown on cultures in any case.5. Most of the patients were infants younger than one year of age.6. Poor socio-economic and hygienic conditions characterized the households from which these county hospital patients came.7. Household follow-up studies indicated that there had been one or more instances of diarrhea among household associates of approximately half the hospitalized patients at some time during the month prior to admission.