Pathogen-associated porin turns IL-10 competent B-1a cells toward proinflammatory cytokine response.

Shigellosis is a major problem in the developing countries causing mortality and morbidity particularly among the children. Shigella spp. harbours the epithelial cells of the human colon to infect the host and spread the disease. We analyzed the response of B-1a cells, the major component of the mucosal immune system to porin of Shigella dysenteriae type 1. We show that porin while proliferating B-1a cells, deplete Siglec-G, the inhibitory molecule present on B-1a cells. Adjuvanticity of porin has been shown to govern innate signaling for promoting host adaptive immune response. Up-regulation of CD69 and CD40 denotes activation of the cells parallel to abrogation of Siglec-G. As a result of cell activation, porin stimulated the inflammatory cytokines of CD5+ B-1a cells, otherwise rich in IL-10. The work shows B-1a cell responses promote the immunopotentiating activity of porin.

Controlling the cytokine storm in severe bacterial diarrhoea with an oral Toll-like receptor 4 antagonist.

Shigella dysenteriae causes the most severe of all infectious diarrhoeas and colitis. We infected rhesus macaques orally and also treated them orally with a small and non-absorbable polypropyletherimine dendrimer glucosamine that is a Toll-like receptor-4 (TLR4) antagonist. Antibiotics were not given for this life-threatening infection. Six days later, the clinical score for diarrhoea, mucus and blood was 54% lower, colon interleukin-8 and interleukin-6 were both 77% lower, and colon neutrophil infiltration was 75% less. Strikingly, vasculitis did not occur and tissue fibrin thrombi were reduced by 67%. There was no clinical toxicity or adverse effect of dendrimer glucosamine on systemic immunity. This is the first report in non-human primates of the therapeutic efficacy of a small and orally bioavailable TLR antagonist in severe infection. Our results show that an oral TLR4 antagonist can enable controlled resolution of the infection-related-inflammatory response and can also prevent neutrophil-mediated gut wall necrosis in severe infectious diarrhoeas.

MAIT cells detect and efficiently lyse bacterially-infected epithelial cells.

Mucosal associated invariant T cells (MAIT) are innate T lymphocytes that detect a large variety of bacteria and yeasts. This recognition depends on the detection of microbial compounds presented by the evolutionarily conserved major-histocompatibility-complex (MHC) class I molecule, MR1. Here we show that MAIT cells display cytotoxic activity towards MR1 overexpressing non-hematopoietic cells cocultured with bacteria. The NK receptor, CD161, highly expressed by MAIT cells, modulated the cytokine but not the cytotoxic response triggered by bacteria infected cells. MAIT cells are also activated by and kill epithelial cells expressing endogenous levels of MRI after infection with the invasive bacteria Shigella flexneri. In contrast, MAIT cells were not activated by epithelial cells infected by Salmonella enterica Typhimurium. Finally, MAIT cells are activated in human volunteers receiving an attenuated strain of Shigella dysenteriae-1 tested as a potential vaccine. Thus, in humans, MAIT cells are the most abundant T cell subset able to detect and kill bacteria infected cells.

Characterization of a novel fusion protein from IpaB and IpaD of Shigella spp. and its potential as a pan-Shigella vaccine.

Shigellosis is an important disease in the developing world, where about 90 million people become infected with Shigella spp. each year. We previously demonstrated that the type three secretion apparatus (T3SA) proteins IpaB and IpaD are protective antigens in the mouse lethal pulmonary model. In order to simplify vaccine formulation and process development, we have evaluated a vaccine design that incorporates both of these previously tested Shigella antigens into a single polypeptide chain. To determine if this fusion protein (DB fusion) retains the antigenic and protective capacities of IpaB and IpaD, we immunized mice with the DB fusion and compared the immune response to that elicited by the IpaB/IpaD combination vaccine. Purification of the DB fusion required coexpression with IpgC, the IpaB chaperone, and after purification it maintained the highly α-helical characteristics of IpaB and IpaD. The DB fusion also induced comparable immune responses and retained the ability to protect mice against Shigella flexneri and S. sonnei in the lethal pulmonary challenge. It also offered limited protection against S. dysenteriae challenge. Our results show the feasibility of generating a protective Shigella vaccine comprised of the DB fusion.

Multi-serotype outer membrane vesicles of Shigellae confer passive protection to the neonatal mice against shigellosis.

Recently, we have demonstrated, immunization of adult female mice with outer membrane vesicles (OMVs) of Shigella boydii type 4 protected their offspring passively from shigellosis. In our present study, we have advanced our research by formulating multi-serotype outer membrane vesicles (MOMVs), mixing the OMVs of Shigella dysenteriae 1 Δstx, Shigella flexneri 2a, 3a and 6, S. boydii type 4 and Shigella sonnei to achieve a broad spectrum protection against shigellosis. Adult mice were immunized orally with 50 µg of MOMVs, four times at weekly intervals. Immunological parameters were observed at various time points, before, during and after immunization, in adult mice. Passive protection was examined in their offspring by measuring protective efficacy and studying intestinal colonization, after challenging with various Shigella strains. Immunized dams exhibited a consistent broad spectrum antibody response. 3-4 day-old offspring of immunized dams showed significant long term passive protection against wild type S. flexneri 2a, 3a, and 6, S. boydii type 2 and S. dysenteriae 1. Their stomach extracts, essentially containing mother's milk, have also exhibited significant levels of anti-MOMVs immunoglobulins. In conclusion, MOMVs formulation represents an easy, safe immunization strategy that was found suitable to provide complete passive protection to the neonatal mice against all four serogroups of Shigellae. It could be exploited for the development of a novel non-living vaccine against human shigellosis in near future.

Shiga toxins expressed by human pathogenic bacteria induce immune responses in host cells.

Shiga toxins are a family of genetically and structurally related toxins that are the primary virulence factors produced by the bacterial pathogens Shigella dysenteriae serotype 1 and certain Escherichia coli strains. The toxins are multifunctional proteins inducing protein biosynthesis inhibition, ribotoxic and ER stress responses, apoptosis, autophagy, and inflammatory cytokine and chemokine production. The regulated induction of inflammatory responses is key to minimizing damage upon injury or pathogen-mediated infections, requiring the concerted activation of multiple signaling pathways to control cytokine/chemokine expression. Activation of host cell signaling cascades is essential for Shiga toxin-mediated proinflammatory responses and the contribution of the toxins to virulence. Many studies have been reported defining the inflammatory response to Shiga toxins in vivo and in vitro, including production and secretion of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), macrophage inflammatory protein-1α/ß (MIP-1α/ß), macrophage chemoattractant monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), interleukin 6 (IL-6), and Groß. These cytokines and chemokines may contribute to damage in the colon and development of life threatening conditions such as acute renal failure (hemolytic uremic syndrome) and neurological abnormalities. In this review, we summarize recent findings in Shiga toxin-mediated inflammatory responses by different types of cells in vitro and in animal models. Signaling pathways involved in the inflammatory responses are briefly reviewed.

Pathogenic bacteria prime the induction of Toll-like receptor signalling in human colonic cells by the Gal/GalNAc lectin Carbohydrate Recognition Domain of Entamoeba histolytica.

In mixed intestinal infections with Entamoeba histolytica trophozoites and enteropathogenic bacteria, which are wide-spread in areas of endemic amoebiasis, interaction between the pathogens could be an important factor in the occurrence of invasive disease. It has been reported that exposure of human colonic cells to enteropathogenic bacteria increased trophozoite adherence to the cells and their subsequent damage. We report here that the Carbohydrate Recognition Domain (CRD) of the amoebic Gal/GalNAc lectin binds to Toll-like receptors TLR-2 and TLR-4 in human colonic cells, activating the "classic" signalling pathway of these receptors. Activation induced expression of TLR-2 and TLR-4 mRNAs and the mRNAs of pro-inflammatory cytokines, as well as an increase in the corresponding proteins. Direct correlation was observed between the increased expression of TLRs and pro-inflammatory cytokines, the enhanced adhesion of trophozoites to the cells and the inflicted cell damage. When cells were exposed to pathogenic bacteria Staphylococcus aureus (Gram⁺) or Shigella dysenteriae (Gram⁻), elements of an innate immune response were induced. CRD by itself elicited a similar cell response, while exposure to a commensal Escherichia coli had a null effect. Pre-exposure of the cells to pathogenic bacteria and then to CRD rendered an inflammatory-like microenvironment that after addition of trophozoites facilitated greater cell destruction. Our results suggest that CRD is recognised by human colonic cells as a pathogen-associated-molecular-pattern-like molecule and as such can induce the expression of elements of an innate immune response. In the human host, an exacerbated inflammatory environment, derived from pathogen interplay, may be an important factor for development of invasive disease.

A piglet model of acute gastroenteritis induced by Shigella dysenteriae Type 1.

BACKGROUND: The lack of a standardized laboratory animal model that mimics key aspects of human shigellosis remains a major obstacle to addressing questions about pathogenesis, screening therapeutics, and evaluation of vaccines. METHODS: We characterized a piglet model for Shigella dysenteriae type 1. RESULTS: Piglets developed acute diarrhea, anorexia, and dehydration, which could often be fatal, with symptom severity depending on age and dose. Bacteria were apparent in the lumen and on the surface epithelium throughout the gut initially, but severe mucosal damage and bacterial cellular invasion were most profound in the colon. Detached necrotic colonocytes were present in the lumen, with inflammatory cells outpouring from damaged mucosa. High levels of interleukin (IL)-8 and IL-12 were followed by high levels of other proinflammatory cytokines. Elevated levels of tumor necrosis factor-alpha, IL-1beta, IL-6, and IL-10 were detected in feces and in gut segments from infected animals. Bacteria were present inside epithelial cells and within colonic lamina propria. In contrast, an isogenic strain lacking Shiga toxin induced similar but milder symptoms, with moderate mucosal damage and lower cytokine levels. CONCLUSION: We conclude that piglets are highly susceptible to shigellosis, providing a useful tool with which to compare vaccine candidates for immunogenicity, reactogenicity, and response to challenge; investigate the role of virulence factors; and test the efficacy of microbial agents.

Selective deletion of CD8(+) cells upregulated by caspases-1 via IL-18 in mice immunized with major outer membrane protein of Shigella dysenteriae 1 following infection.

INTRODUCTION: Mucosal lymphoid changes were observed in cryopreserved rectal tissues obtained from BALB/c mice infected with Shigella dysenteriae 1, immunized with 57-kDa major antigenic outer membrane protein, and infection after immunization. DISCUSSION: Our data suggested that caspase-3 is downregulated in CD4(+) cells of immunized BALB/c mice following infection with substantial increased expression of interleukin (IL)-2 and interferon (IFN)-gamma, while caspase-1 is upregulated in CD8(+) cells with decreased expression of IL-4 and IL-10. This indicated an involvement of Fas-mediated lytic pathway for selective deletion of CD8(+) cells out of CD3(+) T cells. IL-18 promotes inflammation and induces IFN-gamma and tumor necrosis factor (TNF)-alpha as the expression of IFN-gamma and TNF-alpha cytokines was evident in this study. It is assumed that the role of caspase-1 in inducing the CD4+ T cell activity increased with IL-18 rather than CD8+ suppressor cell activity. Bcl-2 is capable of inhibiting the Fas/Fas-L-mediated cell death for helper cells. Overall, the findings indicate that majority of the apoptotic cells were CD8(+) T cells in the groups of infection following immunization, and there might be a selective deletion of T lymphocytes mediated by caspase-1 via IL-18.

Lactobacilli inhibit Shigella dysenteriae 1 induced pro-inflammatory response and cytotoxicity in host cells via impediment of Shigella-host interactions.

OBJECTIVE: Shigella dysenteriae Type 1 dysentery is a major cause of morbidity and mortality in children from less developed and developing countries. The present study explores the hypothesis that lactobacilli protect the host cell during S. dysenteriae Type 1 infection and its mechanism of action. METHODS: Caco-2 cells incubated for 1h with Lactobacillus rhamnosus or Lactobacillus acidophilus at the multiplicity of infection of 100, either alone or in combination followed by addition of Shigella at the same multiplicity of infection for 5h served as treatment groups. Cells incubated with Shigella without lactobacilli addition served as infected cells. At the end of experimental period, cells were processed suitably to enumerate adherent and internalized Shigella. Reverse transcription-polymerase chain reaction was performed to assess mRNA expression of interleukin-8 and tumour necrosis factor-alpha. Immunoblot for heat shock protein-70 and cytotoxicity assay were performed. RESULTS: Pretreatment with the combination of lactobacilli significantly (p<0.05) prevented adherence and internalization of Shigella coupled with reduced expression of tumour necrosis factor-alpha and interleukin-8 in host cells. CONCLUSION: L. rhamnosus and L. acidophilus, synergistically offered better protection during S. dysenteriae Type 1 infection by efficiently inhibiting adherence and internalization of Shigella coupled with inhibition of pro-inflammatory response.

Porin of Shigella dysenteriae directly promotes toll-like receptor 2-mediated CD4+ T cell survival and effector function.

Porin of Shigella dysenteriae type 1 up-regulated Toll-like receptor (TLR)2 on CD3-stimulated CD4(+) T cells but could not induce the expression of other TLRs. TLR2 in association with myeloid differentiating factor 88 (MyD88) triggered the downstream signal transduction pathway leading to activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB), and degradation of IkappaB, the NF-kappaB inhibitor. TLR2 co-stimulation by porin resulted in T cell expansion by inducing both proliferation and survival of the CD4(+) T cells. Extracellular signal-regulated kinase (ERK)1/2 activation inhibitor U0126 and NF-kappaB translocation inhibitor SN-50 significantly inhibited proliferation of T cells, highlighting a direct role of ERK and NF-kappaB in the process. However, cell survival involving Bcl-X(L) induction was found to be regulated essentially by ERK with no significant role of NF-kappaB. Porin-induced proliferation was supported by induction of IL-2 and CD25 that are known to play a pivotal role in T cell expansion. Apart from inducing T cell proliferation, porin triggered effector functions of the cells, evident from TLR2- and MyD88-dependent release of type 1 cytokines tumor necrosis factor (TNF) and interferon (IFN)-gamma along with the induction of type 1 chemokines macrophage-inflammatory protein (MIP)-1alpha and MIP-1beta and their receptor CCR5. The proliferation, survival and effector function of CD4(+) T cells through TLR2 co-stimulation show the capability of porin to directly turn adaptive immunity into action.

Serogrupos y susceptibilidad antimicrobiana en cepas de Shigella / Serogroups and antimicrobial susceptibility in Shigella strains

Shigella spp es uno de los agentes causales más importantes de diarrea aguda en los niños. La presente investigación tuvo como objetivo conocer la frecuencia de serogrupos y la susceptibilidad antimicrobiana a los fármacos de elección y a los alternativos. MÉTODOS. Se realizó un estudio descriptivo y retrospectivo entre enero de 2004 y diciembre de 2006 a partir de 34 cepas de Shigella spp. aisladas en heces de niños menores de 5 años ingresados en el Hospital Aleida Fernández Chardiet (Municipio Güines) a causa de enfermedad diarreica aguda. RESULTADOS. Los serogrupos encontrados fueron S sonnei (70,5 por ciento) y S flexneri (29,5 por ciento). Ambos serogrupos mostraron altos niveles de resistencia al trimetoprim-sulfametoxazol y a la ampicilina, ademas en las cepas de S sonnei se encontró resistencia al ácido nalidíxico y en las de S flexneri al cloranfenicol. Todas las cepas mostraron altos porcentajes de sensibilidad a la ceftriaxona, norfloxacina y ciprofloxacina. El 70 por ciento de las cepas de S sonnei fueron multirresistentes. El patrón de multirresistencia (ampicilina, trimetoprim-sulfamtetoxazol y Acido nalidíxico) se encontró en ambos serogrupos. CONCLUSIONES. La determinación y vigilancia de los patrones de resistencia facilita el control de la política de uso de antibióticos en la región estudiada y previene el surgimiento de cepas resistentes a fármacos de nueva generación.

Shigella ssp. is one of the more important causal agents of acute diarrhea in children. Present research has as aim to know serogroups frequency and antimicrobial susceptibility to choice drugs, and to its alternatives. METHODS: A descriptive retrospective study was carried out between January 2004 and December 2006 of 34 strains of Shigella isolated from children lower than 5 years admitted in Aleida Fernßndez Chardiet Hospital in Güines Municipality by acute diarrheic disease. RESULTS: Serogroups included S sonnei (70,5 percent), and S flexeneri (29,5 percent). Both serogroups showed high levels of resistance to Trimethoprim-Sulfamethoxaxole and to Ampicillin and to in strains of S sonnei there was resistance to nalidixic acid, and in that of S flexeneri, to Chloramphenicol. All strains showed high percentages of sensibility to Cephtriaxone, Norphloxacine, and to Cyprophloxacine. The 70 percent of strains of S sonnei were multi-resistant. Multiresistance pattern (Ampicillin, Trimetroprim-Sulfamethoxaxole and nalidixic acid) was present in both serogroups. Assessment and surveillance of resistance patterns allow control of policy on use of antibiotics in study region, and to foresee rise of strains resistant to new generation-drugs.

Priming of CD4+ T cells with porin of Shigella dysenteriae activates the cells toward type 1 polarization.

Macrophages treated with porin of Shigella dysenteriae were potent stimulators of naive T(h) cells since the porin-pulsed macrophages strongly proliferated the T cells and up-regulated the activation molecules CD69 and CD25 on CD4(+) T cells in allogeneic mixed leukocyte reaction. Immunization of C57BL/6 mice with porin selectively induced the intracellular expression and release of IFN-gamma and had no effect on IL-4 expression. In parallel to the predominant release of the T(h)1 cytokine IFN-gamma, up-regulation of CCR5 on the immune CD4(+) T cells and messenger ribonucleic acid (mRNA) expression of the T cell chemokines macrophage inflammatory protein-1 alpha and regulated on activation normal T cell expressed and secreted showed T(h)1 bias. The porin-primed CD4(+) T cells expressed the mRNA for T-box expressed in T cells, a T(h)1-specific transcription factor confirming the adjuvant-induced transition of T cells to polarized effector T(h)1 cells. The immune CD4(+) T cells proliferated and released IL-2 and IFN-gamma profoundly in response to re-challenge with porin-pulsed macrophages but not to BSA-pulsed macrophages in vitro, which demonstrated the presence of porin-specific CD4(+) T cells of T(h)1 phenotype. The study highlights that porin has the capacity of an adjuvant to unfold and maintain the T cell function and thereby to activate adaptive immunity.

[Effects of Huoxiang Zhengqi liquid on enteric mucosal immune responses in mice with Bacillus dysenteriae and Salmonella typhimurium induced diarrhea].

OBJECTIVE: To explore the effects of Huoxiang Zhengqi liquid (HXZQ) on enteric mucosal immune responses in mice with Bacillus dysenteriae and Salmonella typhimurium induced diarrhea (BSD). METHOD: Mice were randomly divided into four groups with 10 mice in each group: control group (control), BSD group, Huoxiang Zhengqi liquid treated BSD groups at high dosage and low dosage (HXZQ high, HXZQ low). HXZQ was administrated from the day of diarrhea induction at dosage of 5.21 g kg(-1) and 0.52 g kg (-1) respectively. Peyer's patch and periphery lymphocytes were prepared for flow cytometry, and level of TNF-alpha in periphery and enteric tissue homogenate were determined with ELISA. Student's t-test was used for statistics. RESULT: Mice in BSD group started showing continuous diarrhea at the day of induction till the fourth day when the mice were sacrificed. Diarrhea in the mice of HXZQ high and low groups lasted for 36 and 54 h respectively. There were more CD4+ and CD8+ cells in periphery, less CD4+ cells in peyer's patch in BSD mice comparing to normal mice. In peyer's patch, there were more CD8+ cells in mice in HXZQ high and low groups and more CD4+ in mice in HXZQ high group. Higher level TNF-alpha in periphery and intestinal tissue homogenate in BSD group were observed. Mice in HXZQ high group showed the decreased level TNF-alpha in periphery and enteric tissue homogenate. CONCLUSION: The immune regulation on peyer's patch CD4+ and CD8+ cells and suppression on TNF-alpha level in enteric homogenate might partially explain the effect of HXZQ on improvement of BSD.

Porin of Shigella dysenteriae enhances Toll-like receptors 2 and 6 of mouse peritoneal B-2 cells and induces the expression of immunoglobulin M, immunoglobulin G2a and immunoglobulin A.

Porin of Shigella dysenteriae type 1 increased the mRNA levels for Toll-like receptors TLR2 and TLR6, by 1.8-fold and twofold, respectively, in peritoneal cavity B-2 cells from C57BL/6 mice, implicating that the co-expression of TLR2 and TLR6 occurs as a combinatorial repertoire in response to porin. Among the two key TLRs, TLR2 and TLR4, which are primarily responsible for recognizing the majority of bacterial products, TLR2 alone participates in porin recognition. TLR2 expression was increased on B-2 cells, whereas the expression of TLR4 remained unaffected. Besides TLRs, mRNA for myeloid differentiation factor 88 (MyD88), an effector molecule associated with the TLR-mediated response, was enhanced by twofold, suggesting its involvement in the activity of porin. The B-2 cells showed a 1.8-fold increase in mRNA expression of the signalling molecule, nuclear factor-kappa B (NF-kappaB), in the presence of porin. Porin treatment of B-2 cells selectively up-regulated the expression of the costimulatory molecule, CD86, by 4.4-fold. Porin induced the cell-surface expression of immunoglobulin (Ig)M, of IgG2a preferentially among the IgG subclasses, and of IgA, on B-2 cells. The porin-mediated inductions of IgG2a and IgA were augmented by interleukin-6 on B-2 cells, by 2.7- and 1.6-fold, respectively.

Role of 57 kDa major antigenic component of Shigella dysenteriae outer membrane proteins in induction of major histocompatibility complex II-restricted T-cell response.

BACKGROUND: In the past, many Shigella surface antigens were used to activate both T and B lymphocytes but failed to induce antigen-specific responses in Shigellosis. Our objective was to identify in vitro T-cell components using 57 kDa major antigenic fraction of Shigella dysenteriae 1 (IPC-31) outer membrane proteins (OMPs) in modulating specific T-cell subset responses against Shigellosis. METHODS: Antigen-specific T- and B-cell activation was studied in immunized Balb/c mice against 57 kDa antigen by proliferative responses using [3H]-thymidine incorporation and avidin-biotin complex (ABC) peroxidase staining for CD4, CD8, CD3, CD22, and CD25 followed by IL-2 and IL-4 estimation. Macrophage functional assays for migration inhibition factors (MIF) and superoxide (O2-) anions were also performed against 57 kDa antigen, whole OMPs, and phytohemagglutinin (PHA) stimulation. RESULTS: Greater increase of lymphocyte proliferation was observed after 57 kDa antigen stimulation than post-OMP and -PHA stimulation. Proportionately, CD4+ and CD25+ expression of total CD3+ T-cells was significantly dominant (p >0.05) over CD8+ T-cells. On day 7 of this stimulation, it was found to increase % MIF and O2- anions with decrease of IL-2 leading to activation of MHC-II antigens. Later, on day 28 of immunization, IL-2 levels were more increased than on days 7 and 14 but insignificant with non-immunized mice stimulated with 57 kDa. Levels of IL-2 were also noted with low degree of internalization to its IL-2R receptors rather than to IL-4 receptors. In parallel, expression of CD22 was also recorded higher in this stimulation than in PHA, indicating a T-cell-dependent humoral response. CONCLUSIONS: Our results suggested that 57 kDa major antigenic OMP is immunogenic for MHC II-restricted T-cell response to acquire host defense against Shigella infection.

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.

Apoptosis in acute shigellosis is associated with increased production of Fas/Fas ligand, perforin, caspase-1, and caspase-3 but reduced production of Bcl-2 and interleukin-2.

Shigella dysenteriae type 1-induced apoptotic cell death in rectal tissues from patients infected with Shigella dysenteriae type 1 was studied by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique and annexin V staining. Expression of proteins and cytokines participating in the apoptotic process (caspase-1, caspase-3, Fas [CD95], Fas ligand [Fas-L], perforin, granzyme A, Bax, WAF-1, Bcl-2, interleukin-2 [IL-2], IL-18, and granulocyte-macrophage colony-stimulating factor) in tissue in the acute and convalescent stages of dysentery was quantified at the single-cell level by in situ immunostaining. Apoptotic cell death in the lamina propria was markedly up-regulated at the acute stage (P < 0.05), where an increased number of necrotic cells were also seen. Phenotypic analysis of apoptotic cells revealed that 43% of T cells (CD3), 10% of granulocytes (CD15), and 5% of macrophages (CD56) underwent apoptosis. Increased activity of caspase-1 persisted in the rectum up to 1 month after onset. More-extensive expression of Fas, Fas-L, perforin, caspase-3, and IL-18, but not IL-2, at the acute stage than at the convalescent stage was observed. Increased expression of caspase-3 and IL-18 in tissues with severe inflammation compared to expression in those with mild inflammation was evident, implying a possible role in the perpetuation of inflammation. Significantly reduced cell death during convalescence was associated with a significant up-regulation of Bcl-2, Bax, and WAF-1 expression in the rectum compared to that in the acute phase of infection. Thus, induction of apoptosis at the local site in the early phase of S. dysenteriae type 1 infection was associated with a significant up-regulation of Fas/Fas-L and perforin and granzyme A expression and a down-regulation of Bcl-2 and IL-2, which promote cell survival.

Dissociation between cytokine mRNA expression and protein production in shigellosis.

In our study, infection with Shigella dysenteriae type 1 (n = 16) or Shigella flexneri in adults (n = 5) was associated with a gradual accumulation of mRNA for interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, IL-6, transforming growth factor-beta, IL-10, IL-4, TNF-beta, interferon (IFN)-gamma and perforin in the rectal biopsy samples during the convalescent stage of the disease demonstrated by in situ hybridization. In contrast, immunohistochemical staining in rectal tissues of cytokine protein-producing cells at the single-cell level exhibited a steady-state expression during 2-36 days after the onset of the disease. The frequency of cytokine mRNA-expressing cells varied in the range of 3-100-fold higher than that of the corresponding protein-synthesizing cells. The accumulation of cytokine mRNA in vivo during shigellosis represented a long-lasting phenomenon throughout the disease course, and may be linked to its immunopathogenesis. The results also indicate that assessment of both protein and mRNA in vivo may provide complementary information. Stimulation in vitro of peripheral blood mononuclear cells from normal healthy donors with Shigella-derived lipopolysaccharide or shiga toxin was carried out to elucidate the role of Shigella antigens in the regulation of translation of cytokine-specific mRNA. The incidence of cytokine (IFN-gamma, IL-6 and TNF-alpha) mRNA- and cytokine protein-expressing cells was very similar and congruent after both these Shigella-derived stimuli. We could, thus, not find evidence for shiga toxin-induced down-regulation of cytokine mRNA translation as the explanation for the observed discrepancy between cytokine mRNA and protein levels in the tissue biopsies.

Shigella infection induces cellular activation of T and B cells and distinct species-related changes in peripheral blood lymphocyte subsets during the course of the disease.

Immunophenotypic changes in peripheral blood lymphocytes (T, B, and NK cells) in patients during shigellosis was characterized by using triple-color flow cytometry. Eleven Shigella dysenteriae 1-infected adult patients (SDIP), 11 Shigella flexneri-infected adult patients (SFIP), 15 age- and sex-matched healthy controls from Bangladesh (C-B), and 15 healthy volunteers from Sweden (V-S) were studied. In SDIP and SFIP, a significant increase in the CD45RO+ cells in both CD4+ and CD8+ T cells were seen. We found evidence for sequential T-cell activation, as shown by increased proportions of CD25 and CD4+ cells; HLA-DR and CD38 on CD8+ cells, and CD54 on CD4+ and CD8+ cells. We found differences in the lymphocyte activation and subset patterns related to the infecting Shigella species. Thus, a decrease in CD45 expression was seen in SFIP; this decrease progressed further during the disease. The proportions of NK cells (CD56+ cells) and CD3- CD8+ cells out of the total CD8+ cells were increased in SFIP but not in SDIP. The CD3+ CD8+ CD57+ T-cell subset was significantly lower in SDIP than in C-B. The proportion of B-lymphocyte-expressing activation markers CD80 and CD23 was higher in patients than in C-B. There was a significant increase in the proportion of CD4+ T cells and a significant decrease in the percentages of total B cells, the CD3+ CD8+ CD57+ T-cell subset, and the CD56+ CD16+ NK-cell subset for V-S compared with C-B. Our results indicate that distinct subset changes and activation patterns are elicited in SDIP compared with SFIP and also that the degree of activation is related to disease severity. In addition, a common sequence of cell activation was seen during the disease course. The difference in the subset patterns seen in C-B and V-S may be related to differences in the levels or spectra of infectious agents in the environment.