Transmission intensity determines lymphocyte responsiveness and cytokine bias in human lymphatic filariasis

Humans living in areas where filariasis is endemic vary greatly in their exposure to mosquito-borne infective third-stage larvae (L3) of these parasitic helminths. Because the intensity of exposure to Ags affects T cell differentiation and susceptibility to parasitic infections in murine models, we compared T cell and cytokine responses in 97 residents of two villages in Papua New Guinea, where transmission intensity of Wuchereria bancrofti differed by 63-fold (37 vs 2355 L3 per person per year). Residents of the high transmission village had 4- to 11-fold lower proliferation and IFN-gamma responses to filarial Ags, nonparasite Ag, and PHA by PBMC compared with the low transmission village (p < 0.01) even when subjects were matched for intensity of infection. In contrast, filarial Ag-driven IL-5 production was 5.5-fold greater (p < 0.001), and plasma IL-4 and TGF-beta levels were 4-fold and 34% higher, respectively, in residents of the high transmission village. IL-4 and IL-10 responses by PBMC differed little according to village, and increased production of the counterregulatory cytokines IL-10 or TGF-beta by PBMC did not correlate with weak proliferation and IFN-gamma responses. Plasma IL-5, IFN-gamma, and IL-10 levels were similar in the two villages. These data demonstrate that the intensity of exposure to L3 affects lymphocyte responsiveness and cytokine bias possibly by a mechanism that alters APC function.     

Concomitant induction of CD4+ and CD8+ T cell responses in volunteers immunized with Salmonella enterica serovar typhi strain CVD 908-htrA

Type 1 cell-mediated immunity might play an important role in protection from typhoid fever. We evaluated whether immunization with Salmonella enterica serovar Typhi (S. Typhi) strain CVD 908-htrA (a Delta aroC Delta aroD Delta htrA mutant), a leading live oral typhoid vaccine candidate, elicits specific CD4(+) and CD8(+) S. Typhi immune responses. Potent CTL responses and IFN-gamma secretion by CD8(+) T cells were detected following immunization with CVD 908-htrA in high (4.5 x 10(8) CFU) and low (5 x 10(7) CFU) dosages. S. Typhi-specific CTL were observed in six of eight vaccinees (four high and two low dose) after immunization. Mean increases in the frequency of IFN-gamma spot-forming cells (SFC) in the presence of S. Typhi-infected targets were 221 +/- 41 SFC/10(6) PBMC and 233 +/- 87 SFC/10(6) PBMC, in the high and low dose groups, respectively. Strong CD4(+) T cell responses were also observed. Increases in the IFN-gamma production to soluble S. Typhi flagella (STF) occurred in 82 and 38% of the volunteers who received the high and low doses, respectively. Robust correlations were observed between volunteers that responded with IFN-gamma SFC to stimulation with S. Typhi-infected cells and IFN-gamma released in response to stimulation with STF Ags (r = 0.822, p < 0.001) and between CTL and IFN-gamma production to STF (r = 0.818, p = 0.013). These data demonstrating the concomitant induction of both CD4- and CD8-mediated CMI are consistent with a significant role for type 1 immunity in controlling typhoid infection and support the continuing evaluation of CVD 908-htrA as a typhoid vaccine candidate.     

Circular deoxyribonucleic acid from Shigella dysenteriae Y6R

Circular deoxyribonucleic acid was isolated from Shigella dysenteriae Y6R and was found to consist of six species having molecular weights of 10(6), 1.3 x 10(6), 2.6 x 10(6), 3.8 x 10(6), 20 x 10(6), and 24 x 10(6) daltons. These size classes were partially resolved by sucrose density gradient centrifugation. The minicircles (10(6) and 1.3 x 10(6)) were found to have a buoyant density in CsCl of 1.710 g/ml. The 3.8 x 10(6) dalton class had a density of 1.707 g/ml. The two largest species had a density of 1.702 g/ml. Two other strains, S. sonnei II and S. dysenteriae 60, also contained circular deoxyribonucleic acid.     

Cytokine release induced by killed bacteria associated with anti-IFN-gamma antibody in Shigella infection

An effort was made to analyze the effect of in vitro stimulation on macrophages using killed Shigella dysenteriae type-1 (KSD1) coupled with anti-Interferon Gamma (anti-IFN-gamma) antibody. The stimulated macrophages were co-cultured with primed or non-primed T-cells from Shigella infected patients. T-cell cultures were also established by co-culturing KSD1 coupled with or without PHA stimulated macrophages. Emulsified KSD1 coupled with anti-IFN-gamma antibody was found to act as a potent immunogen, inducing the release of Th1 cytokine from primed T-cells cultured in acute stage of the disease. It was observed that the levels of IFN-gamma and IL-2 production rather than IL-4 and IL-6 were increased as the disease became more severe. On comparison, the subsequent values of IL-6, IFN-gamma, IL-4 and IL-2 were found to be less significant in healthy primed T-cell cultures. This was also associated with a substantial production of superoxide ions (O2-), which probably inhibits the colonization of intracellular Shigella due to the presence of anti-IFN-gamma antibodies. On the other hand KSD1 with or without PHA failed to induce such responses. The above findings reflect that in the presence of anti-IFN-gamma antibody, KSD1 acts as a potential immunogen for eliciting cellular immunity against shigellosis.     

Porin of Shigella dysenteriae enhances mRNA levels for Toll-like receptor 2 and MyD88, up-regulates CD80 of murine macrophage, and induces the release of interleukin-12

Sera of patients convalescing from shigellosis reacted strongly and specifically with the 38,000 Da monomer of porin of Shigella dysenteriae type 1. Since human, the only natural host of S. dysenteriae type 1, recognized the protein through humoral immune response, it is of great significance to study the surface-exposed outer membrane antigen as an adjuvant. Porin treatment of CD11b+ peritoneal cavity (PerC) MPhi of BALB/c mouse was found to up-regulate CD80 on cell surface and had no effect on CD86 expression. The surface expression of CD80 got increased by 1.6-fold in the presence of gamma interferon (IFN-gamma) supporting selective regulation of the B7-1 (CD80) member of the B7 family. MPhi released 7.25 pg of interleukin-12 (IL-12) in the presence of porin. The protein in combination with IFN-gamma augmented profoundly the release of IL-12 by 2.6-fold. Porin-mediated induction of IL-12 release would therefore influence Th1-type response, known to be preferentially triggered due to up-regulation of CD80 expression. Treatment of PerC MPhi by the protein showed an increase of mRNA for both Toll-like receptor (TLR)2 and myeloid differentiation factor 88 (MyD88) by 2- and 2.3-fold respectively, emphasizing that TLR2 is essential for recognition of S. dysenteriae type 1 porin. Understanding the mechanism of adjuvanticity of porin of S. dysenteriae type 1 is a necessary step towards the development of a better adjuvant against shigellosis.     

Production of Shiga toxin and a cytolethal distending toxin (CLDT) by serogroups of Shigella spp.

Abstract 56 strains of Shigella including 12 Shigella dysenteriae (serotypes 1, 2, 9, 11 and 12), 23 Shigella flexneri (serotypes 1, 2, 3, 4, 6, var. X and var. Y), 19 Shigella boydii (serotypes 1, 2, 4, 5, 7, 11, 13, 14, 15 and 18), and 2 Shigella sonnei were screened for their ability to produce both classic Shiga toxin and a new heat-labile cytolethal distending toxin (CLDT). Whereas extracellular Shiga toxin was only detectable in filtrates of five S. dysenteriae type 1 strains, CLDT was produced by four strains of S. dysenteriae type 2 and an isolate of S. boydii type 7. No cytotonic enterotoxins similar to Escherichia coli LT were observed in this study. None of the S. flexneri or S. sonnei isolates tested were found to produce extracellular cytotoxic factors. The Shiga toxin produced by the S. dysenteriae type 1 was neutralizable by anti-toxin to verotoxin 1 of E. coli O157 : H7. The Shigella CLDT was neutralizable by antisera prepared to a CLDT-producing E. coli O55 : H4.     

Gene cloning and characterization of alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine racemases reported.     

Monoclonal antibodies against the surface antigens of Shigella flexneri serotype 1b and Shigella dysenteriae serotype 1

Monoclonal antibodies against the surface antigens of Shigella flexneri 1b and S. dysenteriae 1 were prepared. The specificities of the antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), and quantitative agglutination using microtiter plate. Monoclonal antibodies against S. flexneri 1b, designated Sf2B2 and Sf2G4, belonged to IgG2a and IgG1 subclass, respectively. The former was specific for S. flexneri 1b, whereas the latter was reactive not only to S. flexneri 1b, but also weakly to 3a and 4b. Monoclonal antibody against S. dysenteriae 1, Sd5E1 (IgM), reacted with S. dysenteriae 1, 3, 6, 7, and S. boydii 2.     

Characterization of cryptic flagellin genes in Shigella boydii and Shigella dysenteriae.

Flagellin (fliC) genes of 12 Shigella boydii and five Shigella dysenteriae strains were characterized. Though these strains are nonmotile, the cryptic fliCSB gene, cloned from S. boydii strain C3, is functional for expression of flagellin. It consists of 1,704 bp, and encodes 568 amino acid residues (57,918 Da). The fliCSD gene from S. dysenteriae strain 16 consists of 1,650 bp encoding 549 amino acid residues (57,591 Da) and contains an IS1 element inserted in its 3' end. The two genes are composed of the 5'-constant, central variable and 3'-constant sequences, like other known fliC genes. The two genes share high homology in nucleotide and amino acid sequences with each other and also with the Escherichia coli fliCE gene, indicating that both genes are closely related to the fliCE gene. Comparison of the central variable sequences of six different fliC genes showed that the fliCSB and fliCSD genes share low homology in amino acid sequence with the other fliC genes, suggesting that they encode antigenic determinants intrinsic to respective subgroups. However, Southern blotting using as probes the central variable sequences of several fliC genes showed that four of 12 S. boydii strains have a fliC gene similar to that of Shigella flexneri, and that among five fliC genes from S. dysenteriae strains, one is similar to that of S. flexneri, two are similar to that of S. boydii, and only one is unique to S. dysenteriae. Some of these variant alleles were verified by immunoblotting with flagellins produced from cloned fliC genes. The presence of variant fliC alleles in S. boydii and S. dysenteriae indicates that subdivision into subgroups does not reflect the ancestral flagella H antigenic relationships. These data will be useful in considering the evolutionary divergence of the Shigella spp..     

Use of coagglutination for serotyping Shigella dysenteriae and Shigella boydii

The coagglutination test was used to identify Shigella boydii and Shigella dysenteriae. A trial was carried out with 13 native rabbit antisera to S. boydii and 10 antisera to S. dysenteriae, as well as with coagglutinating reagents prepared from these antisera. The use of coagglutinating reagents was shown to ensure the complete specificity of the results, to prevent the adsorption of diagnostic antisera and to decrease their consumption 50 times. The importance of the coagglutination test for the identification of shigellae is discussed.     

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.     

The stability of O-antigen plasmid is determined by a chromosomal region of Shigella dysenteriae 1

It is well established that plasmids are involved in the expression of lipopolysaccharide in certain species of Shigella. In Shigella sonnei, both the biosynthesis of oligosaccharide side chains (O antigen), and cell invasiveness are controlled exclusively by a 120 megadalton (MDa) plasmid. In Shigella dysenteriae 1, a 10 kilobase (kb) plasmid is required for O-antigen production. Shigella dysenteriae 1 strains devoid of this plasmid lose the ability to synthesize O antigen. Interestingly, this 10-kb plasmid is not stably maintained in Escherichia coli K-12 strains, where it is lost spontaneously at a high frequency. Our genetic analyses of Shigella dysenteriae 1 strain IDBM11 and its derivatives indicate that the stability of this plasmid is associated with the histidine region of the chromosome which is unique to Shigella dysenteriae. Furthermore, the 10-kb plasmid is stably maintained in wild-type IDBM11 with an intact histidine locus. However, this plasmid is not stable in IDBM11 derivatives (e.g., IDBM11-1 and IDBM11-2), in which the his locus has been substituted with the histidine region of an E. coli K-12 chromosome. The S. dysenteriae IDBM11 strain, and its derivatives (lacking a 10-kb plasmid), displayed an invasive property as demonstrated by their internalization by HeLa cells in an in vitro assay. Thus the 10-kb plasmid of Shigella dysenteriae 1 is required for O-antigen synthesis but not for cell invasion.     

Differences among Shigella spp. in susceptibility to the bactericidal activity of human serum

Clinical isolates of Shigella spp. were examined for their susceptibility to human serum. The susceptibility of the strains to immune and nonimmune human serum was dependent upon the size of the bacterial inoculum and the concentration of serum. There were differences among Shigella spp. in susceptibility to human serum: S. sonnei strains were the least susceptible, strains of S. boydii and S. flexneri serotype 6 were intermediate, and those of S. flexneri other than serotype 6 and S. dysenteriae were the most susceptible. Experiments in which heat-treated (56 degrees C for 30 min, or 50 degrees C for 20 min) serum was used, and analysis of activation of complement by lipopolysaccharides (LPS) from each Shigella sp., suggested that LPS composition, especially the O antigen polysaccharide chains, contributes to the differences among Shigella spp. in susceptibility to human serum.     

A case of shigellosis caused by Shigella dysenteriae type 1 and Shigella flexneri type 5B

Shigella dysenteriae type 1 and Shigella flexneri type 5b strains were isolated as causative agents of bacterial dysentery in a patient having visited South-East Asia. Both strains are a rare finding for Bulgaria. S. dysenteriae 1 strains have not been isolated since 1962, and there were only single isolates of S. flexneri 5b. The strains were of the same antibiotic resistance patterns. Conjugation experiments showed that resistance is determined by transferrable R-plasmids having identical characteristics. It is assumed that in the patient's gut transfer of an R-plasmid occurs from E. coli of the normal flora to the pathogenic shigellae.     

Lipopolysaccharide core structures and their correlation with genetic groupings of Shigella strains. A novel core variant in Shigella boydii type 16

Bacteria Shigella, the cause of shigellosis, evolved from the intestinal bacteria Escherichia coli. Based on structurally diverse O-specific polysaccharide chains of the lipopolysaccharides (LPSs; O-antigens), three from four Shigella species are subdivided into multiple serotypes. The central oligosaccharide of the LPS called core is usually conserved within genus but five core types called R1-R4 and K-12 have been recognized in E. coli. Structural data on the Shigella core are limited to S. sonnei, S. flexneri and one S. dysenteriae strain, which all share E. coli core types. In this work, we elucidated the core structure in 14 reference strains of S. dysenteriae and S. boydii. Core oligosaccharides were obtained by mild acid hydrolysis of the LPSs and studied using sugar analysis, high-resolution mass spectrometry and two-dimensional NMR spectroscopy. The R1, R3 and R4 E. coli core types were identified in 8, 3 and 2 Shigella strains, respectively. A novel core variant found in S. boydii type 16 differs from the R3 core in the lack of GlcNAc and the presence of a D-glycero-D-manno-heptose disaccharide extension. In addition, the structure of an oligosaccharide consisting of the core and one O-antigen repeat was determined in S. dysenteriae type 8. A clear correlation of the core type was observed with genetic grouping of Shigella strains but not with their traditional division to four species. This finding supports a notion on the existing Shigella species as invalid taxa and a suggestion of multiple independent origins of Shigella from E. coli clones.     

Mechanisms of the natural reactivity of lymphocytes from noninfected individuals to membrane-associated Leishmania infantum antigens

Membrane-associated Leishmania Ags (MLA) or soluble Leishmania Ags were used in vitro to stimulate cord blood or PBMC from healthy donors noninfected by Leishmania parasites. MLA, but not soluble Leishmania Ags, constantly induce strong proliferation of cord blood mononuclear cells and PBMC from noninfected individuals. Responding cells are CD3+, CD4+, TCRalphabeta+, CD45RO+, and CD45RA+ and secrete IFN-gamma and IL-10, but not IL-4. MLA do not activate NK cells nor NKT cells. Membrane Ags also induce purified macrophages from noninfected individuals to secrete IL-10 and TNF-alpha, but have no effect on IL-1alpha or IL-12 secretion. The effects of MLA are proteinase K-sensitive and resistant to lipid extraction. The lymphoproliferative responses are inhibited by anti-HLA-DR Abs and require Ag processing by APCs, excluding that the biological effect of MLA could be attributed to a superantigen. Finally, TCR repertoire analysis shows that the T cell expansion induced by MLA uses TCR with various variable beta segment rearrangements and CDR3 lengths, features much more characteristic to those observed with a polyclonal activator than with a conventional Ag. These results suggest a particular mechanism developed during the host's natural response to Leishmania parasites that allows direct activation of naive CD4 lymphocytes by parasite membrane-associated Ags.     

Ex vivo cytokine and memory T cell responses to the 42-kDa fragment of Plasmodium falciparum merozoite surface protein-1 in vaccinated volunteers

A number of blood-stage malaria Ags are under development as vaccine candidates, but knowledge of the cellular responses to these vaccines in humans is limited. We evaluated the nature and specificity of cellular responses in healthy American volunteers vaccinated with a portion of the major merozoite surface protein-1 (MSP1) of Plasmodium falciparum, MSP1(42), formulated on Alhydrogel. Volunteers were vaccinated three times with 80 microg of either MSP1(42)-FVO/Alhydrogel or MSP1(42)-3D7/Alhydrogel. Cells collected 2 wk after the third vaccination produced Th1 cytokines, including IFN-gamma and IL-2 following Ag stimulation, and greater levels of the Th2 cytokines IL-5 and IL-13; the anti-inflammatory cytokine IL-10 and the molecule CD25 (IL-2Ralpha) were also detected. The volunteers were evaluated for the MSP1(42)-FVO or MSP1(42)-3D7 specificity of their T cell responses. Comparison of their responses to homologous and heterologous Ags showed ex vivo IFN-gamma and IL-5 levels that were significantly higher to homologous rather than to heterologous Ags. The epitopes involved in this stimulation were shown to be present in the dimorphic MSP1(33) portion of the larger MSP1(42)-3D7 polypeptide, and indirect experiment suggests the same for the MSP1(42)-FVO polypeptide. This contrasts with B cell responses, which were primarily directed to the conserved MSP1(19) portion. Furthermore, we explored the maturation of memory T cells and found that 46% of vaccinees showed specific memory T cells defined as CD4(+)CD45RO(+)CD40L(+) after long-term in vitro culture. The identification of human-specific CD4(+) memory T cells provides the foundation for future studies of these cells both after vaccination and in field studies.     

Studies on transformation in Shigella

Transformation of pBR322 DNA into Shigella occurred at a low frequency. The efficiency of transformation was highest in S. dysenteriae 1 and lowest in S. flexneri. Treatment of cells with CaCl2 for a prolonged period (24h) increased the efficiency of transformation in all strains, except in S. flexneri, where transformation efficiency could not be improved by a variety of manipulations. Transformation efficiency did not increase in any of the strains when transformation was carried out with plasmid DNA obtained from a transformant (homologous transformation), suggesting the absence of a strong restriction-modification system. Extracellular deoxyribonuclease (DNase) levels were low in all the strains tested, but the levels of endogenous DNAse, released after CaCl2 treatment or sonication of the cells, were high. Washing the cells with a solution of CaCl2 did not enhance transformation, suggesting that endogenous DNase could be a significant factor affecting transformation efficiency in species of Shigella.     

Frequency of serovars and antimicrobial resistance in Shigella spp. from Brazil

A total of 296 Shigella spp. were received from State Public Health Laboratories, during the period from 1999 to 2004, by National Reference Laboratory for Cholera and Enteric Diseases (NRLCED)--IOC/Fiocruz, Rio de Janeiro, Brazil. The frequency of Shigella spp. was: S. flexneri (52.7%), S. sonnei (44.2%), S. boydii (2.3%), and S. dysenteriae (0.6%). The most frequent S. flexneri serovars were 2a and 1b. The highest incidence rates of Shigella isolation were observed in the Southeast (39%) and Northeast (34%) regions and the lowest rate in the South (3%) of Brazil. Strains were further analyzed for antimicrobial susceptibility by disk diffusion method as part of a surveillance program on antimicrobial resistance. The highest rates of antimicrobial resistance were to trimethoprim-sulfamethozaxole (90%), tetracycline (88%), ampicillin (56%), and chloramphenicol (35%). The patterns of antimicrobial resistance among Shigella isolates pose a major difficulty in the determination of an appropriate drug for shigellosis treatment. Continuous monitoring of antimicrobial susceptibilities of Shigella spp. through a surveillance system is thus essential for effective therapy and control measures against shigellosis.     

Differentiation of Shigella by esterase electrophoretic polymorphism

The electrophoretic mobilities of four esterases (A, B, C, and I) of 182 strains of Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei were compared to those of 636 strains of Escherichia coli from various origins, including the Alkalescens Dispar group and enteroinvasive strains. Discriminant analysis of the distribution of esterases among the strains revealed that Shigella could be distinguished from E. coli by differences in the distribution of allozymes of esterases C and I. Principal components analysis distinguished four major clusters of Shigella strains corresponding to the following: S. dysenteriae serotype 1; S. flexneri serotypes 1 to 5; S. flexneri serotype 6 and S. boydii serotypes 2 and 4; and S. sonnei. The last three were characterized by distinct electrophoretic variants of carboxylesterase B, as judged by the two-dimensional electrophoretic profile and titration curves. The distinct esterase pattern obtained for the strains of S. boydii serotype 13 substantiates the view that this serotype may constitute a new species.