Shigella antigen-specific B memory cells are associated with decreased disease severity in subjects challenged with wild-type Shigella flexneri 2a.

The role of Shigella-specific B memory (BM) in protection has not been evaluated in human challenge studies. We utilized cryopreserved pre- and post-challenge peripheral blood mononuclear cells and sera from wild-type Shigella flexneri 2a (wt-2457T) challenges. Challenged volunteers were either naïve or subjects who had previously ingested wt-2457T or been immunized with hybrid Escherichia coli-Shigella live oral candidate vaccine (EcSf2a-2). BM and antibody titers were measured against lipopolysaccharide (LPS) and recombinant invasion plasmid antigen B (IpaB); results were correlated with disease severity following challenge. Pre-challenge IgA IpaB-BM and post-challenge IgA LPS-BM in the previously exposed subjects negatively correlated with disease severity upon challenge. Similar results were observed with pre-challenge IgG anti-LPS and anti-IpaB titers in vaccinated volunteers. Inverse correlations between magnitude of pre-challenge IgG antibodies to LPS and IpaB, as well as IgA IpaB-BM and post-challenge IgA LPS-BM with disease severity suggest a role for antigen-specific BM in protection.

The identification of novel immunogenic antigens as potential Shigella vaccine components.

BACKGROUND: Shigella is a major diarrheal pathogen for which there is presently no vaccine. Whole genome sequencing provides the ability to predict and derive novel antigens for use as vaccines. Here, we aimed to identify novel immunogenic Shigella antigens that could serve as Shigella vaccine candidates, either alone, or when conjugated to Shigella O-antigen. METHODS: Using a reverse vaccinology approach, where genomic analysis informed the Shigella immunome via an antigen microarray, we aimed to identify novel immunogenic Shigella antigens. A core genome analysis of Shigella species, pathogenic and non-pathogenic Escherichia coli, led to the selection of 234 predicted immunogenic Shigella antigens. These antigens were expressed and probed with acute and convalescent serum from microbiologically confirmed Shigella infections. RESULTS: Several Shigella antigens displayed IgG and IgA seroconversion, with no difference in sero-reactivity across by sex or age. IgG sero-reactivity to key Shigella antigens was observed at birth, indicating transplacental antibody transfer. Six antigens (FepA, EmrK, FhuA, MdtA, NlpB, and CjrA) were identified in in vivo testing as capable of producing binding IgG and complement-mediated bactericidal antibody. CONCLUSIONS: These findings provide six novel immunogenic Shigella proteins that could serve as candidate vaccine antigens, species-specific carrier proteins, or targeted adjuvants.

Monoclonal immunoglobulin A antibody directed against serotype-specific epitope of Shigella flexneri lipopolysaccharide protects against murine experimental shigellosis.

To determine the role of humoral mucosal immune response in protection against shigellosis, we have obtained a monoclonal dimeric immunoglobulin A (IgA) antibody specific for Shigella flexneri serotype 5a lipopolysaccharide (mIgA) and used a murine pulmonary infection model that mimics the lesions occurring in natural intestinal infection. Adult BALB/c mice challenged with 10(7) S. flexneri organisms developed a rapid inflammatory response characterized by polymorphonuclear cell infiltration around and within the bronchi and strong systemic interleukin 6 response. Implantation of hybridoma cells in the back of mice, resulting in the development of a myeloma tumor producing mIgA in the serum and subsequently secretory mIgA in local secretions, or direct intranasal administration of these antibodies, protected the animals against subsequent intranasal challenge with S. flexneri serotype 5a. Absence of histopathological lesion and significant decrease in bacterial load of the lungs and of systemic interleukin 6 response were the three major criteria of protection. This protection was shown to be serotype-specific and dependent on local concentration of mIgA. These data demonstrate that mucosal antibodies directed against a single polysaccharidic surface epitope of Shigella can protect against the disease.

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.

[Proinflammatory cytokines in children with acute enteric infections caused by enterobacteria].

The levels of the proinflammatory cytokines including interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) was investigated in 73 children in the age range from 3 to 17 years with slight and medium-heavy diarrheal illness caused by pathogenic and conditionally pathogenic entrobacteria. Strong positive correlation was found between: the levels of IL-1beta, TNF-alpha and intoxication rate (r=0.65 and r=0.49); height of temperature and duration of fever (r=0.86 and r=0.66); dyspepsia (r=0.48 and r=0.41); diarrhea (r=0.37 and r=0.54) and changes in blood including number of leucocytes (r=0.40 and r=0.52) and level of erythrocyte sedimentation rate (ESR) (r=0.65 and r=0.52). Medium positive relationship between the level of IL-6 and intoxication rate (r=0.40 and r=0.52), height of temperature and duration of fever (r=0.45), changes in blood including ESR (r=0.42) and number of leucocytes (r=0.46) was shown. There was a strong positive correlation between IL-1beta and TNF-alpha (r=0.74), between IL-6 and TNF-alpha (r=0.71) and a medium positive correlation between IL-1beta and IL-6 (r=0.61). Considerable decrease in concentration of all cytokines during early period of convalescent at disease with no complications was revealed. Change in concentration of IL-1beta and TNF-alpha determines both intoxication rate and fever. High level of IL-6 is related with complications and lingering course of disease.

Immunoproteome analysis of soluble and membrane proteins of Shigella flexneri 2457T.

AIM: To profile the immunogenic proteins of Shigella flexneri (S. flexneri) expressed during human infection using a proteomic approach. METHODS: Soluble and membrane protein extractions of S. flexneri 2457T were separated by two-dimensional gel electrophoresis (2-DE). Proteins were transferred to PVDF membrane and immunoblotted with sera from shigellosis patients. Reactive protein spots were matched to Coomassie stained gels run in parallel, cut out and trypsin digested. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) was used to determine the peptide mass fingerprints, which were searched in the MASCOT database to identify the protein. RESULTS: A total of 8 immunoreactive proteins were successfully identified from the Coomassie stained gels in three repeats. Six of these proteins have not previously been reported as immunogenic in S. flexneri. These proteins could be potential candidates for vaccine or attenuation studies. CONCLUSION: Soluble and membrane proteins of S. flexneri 2457T have been screened by 2-DE and immunoblotting with sera from shigellosis patients. Eight proteins are identified as immunogenic.

Predictors of serum retinol in children with shigellosis.

BACKGROUND: Low serum retinol can be useful as an indicator of depleted liver vitamin A stores, particularly in population-based studies. However, serum retinol concentrations decrease transiently during infection, independent of any changes in liver stores. The magnitude of the decrease in serum retinol is often proportional to indicators of disease severity. OBJECTIVE: We examined the relation of serum retinol in children with culture-positive shigellosis with severity of illness, anthropometric indicators of nutritional status, urinary retinol excretion, and serum concentrations of C-reactive protein, alpha1-acid glycoprotein, retinol binding protein, and transthyretin. DESIGN: This was a prospective study assessing the clinical and laboratory measurements at admission and recovery of 90 children with dysentery (66 with shigellosis) hospitalized in Bangladesh. RESULTS: Serum retinol concentrations were low at admission but were significantly greater at discharge even though no vitamin A supplements were given during the illness (0.36 +/- 0.22 compared with 1.15 +/- 0.50 micromol/L, P < 0.001). Serum retinol concentrations were lower in children with Shigella dysenteriae type 1 infection than in children with shigellosis due to less virulent strains of Shigella. Low serum retinol was independently associated with S. dysenteriae type 1, high serum C-reactive protein concentrations, and low weight-forage in multiple regression analysis. CONCLUSIONS: This study showed that shigellosis was associated with a significant, transient decrease in serum retinol concentrations of approximately 0.8 micromol/L, and that this change was significantly associated with severity of disease and poor underlying nutritional status, particularly low weight-for-age.

Urinary retinol excretion and kidney function in children with shigellosis.

BACKGROUND: Acute infections, including diarrhea, are associated with an increased risk of vitamin A deficiency. Urinary retinol excretion during such infections may contribute to this risk. The mechanism accounting for urinary retinol loss has not been clearly defined. OBJECTIVE: This study attempted to determine whether urinary retinol loss in children with acute infection is associated with impaired kidney function, particularly impaired tubular protein reabsorption. DESIGN: Urinary retinol excretion and kidney function were examined in 66 hospitalized children 5 mo to 5 y of age with acute Shigella dysentery. RESULTS: Urinary retinol loss occurred in 59% of children and was substantial (>0.1 micromol/d) in 8% of them. Children with more severe disease excreted higher concentrations of urinary retinol; those with a body temperature > or =40 degrees C excreted a mean of 0.10 +/- 0.18 micromol/d compared with 0.005 +/- 0.008 micromol/d for other children (P < 0.0001). Children with more severe disease also had impaired tubular reabsorption of low-molecular-weight proteins beta2-microglobulin and retinol binding protein (RBP)], although other measures of tubular and glomerular function were not similarly impaired. In multiple regression analysis, severity of disease indicators were the best predictors of tubular reabsorption of beta2-microglobulin (R2 = 0.53) whereas tubular reabsorption of beta2-microglobulin and RBP were found to be the best predictors of urinary retinol loss (R2 = 0.69). CONCLUSIONS: A significant amount of retinol was excreted in the urine in children with shigellosis: 8% excreted >0.10 micromol/d (15% of the daily metabolic requirement). Impaired tubular reabsorption of low-molecular-weight proteins, such as RBP transporting retinol, appeared to be the cause of this urinary retinol loss.

HLA-B27M1M2 and high immune responsiveness to Shigella flexneri in post-dysenteric arthritis.

The heterogeneous HLA-B27 antigen is closely associated with post-infectious or reactive arthritis (ReA) and is comprised of two serologically defined variants: B27M1+M2+ and B27M1+M2-. An outbreak of dysentery (n = 120) caused by a Shigella flexneri 2a strain, which possessed cell envelope antigens with epitopes resembling B27M2, resulted in five B27M1+M2+ patients with ReA. The remaining seven B27M1+M2+, one B27M1+M2- and all but three B27-negative patients remained free of joint symptoms; the latter three displayed arthralgia. IgM, IgG and IgA serum titers were statistically raised in all patient groups, but were exceptionally and persistently high in the B27M1+M2+ patients with ReA, especially IgA, as determined in acute-phase sera and sera sampled 1 year after dysentery. B27M1+M2+ thus appears to be a marker for a subset of disease, characterized by a high immune response. It is concluded that the B27M2 epitope is not unequivocally disease-related to Shigella ReA, that B27M1+M2+ is not likely to be the only immune-response-regulating gene involved in this form of ReA and that cross-reactivity between bacterial antigenic epitopes and B27 can only be part of a multifactorial process leading to ReA and in itself not sufficient to produce ReA. The intensity of the immune response appears to be another important factor.

Disease-dependent changes in T-cell populations in patients with shigellosis.

In shigellosis, bacterial infection is associated with an extensive inflammation of the rectal mucosa, resulting in bloody dysentery. The role of T-cell-mediated pro-inflammatory mechanisms has been implicated in this process, but the specific role of T-cell subsets is still not well understood. In this study we attempted to identify the changes in T-cell populations in patients with shigellosis during the disease course. The T-cell subset distribution was analyzed by immunohistochemistry in the rectal mucosa and by immuno-flow cytometry in the peripheral blood. Blood and rectal biopsies were studied from patients with Shigella dysenteriae 1 (n= 11) and S. flexneri (n= 11) infection and 20 healthy age-matched controls. We found an expansion of gammadelta+T cells in the rectal mucosa, but a decrease in the percentage of gammadelta+T cells in the blood in acute shigellosis. There was also a preferential increase in CD8+ T cells in the surface epithelium of rectal tissue in patients infected with S. dysenteriae 1, but not in patients infected with S. flexneri. Our findings suggest that the rectal mucosal inflammation in shigellosis is associated with an expansion of T cells, in particular CD8+ and gammadelta+T-cell subsets in the gut mucosa, which may be of importance for the pathogenesis of shigellosis.

Sociodemographic factors associated with serum anti-Shigella lipopolysaccharide antibodies and shigellosis.

The association between various sociodemographic variables and the presence of anti-Shigella sonnei lipopolysaccharide (LPS) antibodies was examined in a random sample (N = 383) of male Israeli conscripts. Of the male conscripts, 190 (49.6%) had pre-existing antibodies against S. sonnei LPS (defined as HA titres of greater than or equal to 1:10 after treatment of sera with 2-mercaptoethanol). Univariate analysis revealed a significant positive association between the presence of humoral anti-S. sonnei LPS antibodies and sociodemographic variables including Eastern origin (p = 0.007), low socioeconomic status (p = 0.0016), and the number of siblings (p = 0.023). When multiple logistic regression was used to control simultaneously for the effects of the other variables, ethnic origin emerged as the strongest correlate of anti-S. sonnei LPS antibodies. On the other hand, the association of the sociodemographic variables in subjects suffering from S. sonnei infection during their military service, was in the opposite direction (p less than 0.001 for both socioeconomic status and ethnicity). These findings suggest differences between subpopulations in acquired immunity to S. sonnei due to differences in exposure to the homologous organism prior to military service.

Significance of the secreted form of IpaC, a 45 kDa protein of Shigella dysenteriae 1, in the invasive process as determined by monoclonal antibodies.

Invasion plasmid antigen C (IpaC), a 45 kDa plasmid encoded protein, is associated with the virulence of virulent Shigella spp. In S. dysenteriae type 1 the 45 kDa IpaC protein is secreted to a greater extent into the surrounding medium in comparison to other Shigella spp. Monoclonal antibodies (mAbs) to the secreted form of IpaC protein were raised in this study. Of the four secretory hybrid cells, one (3G4) was found to have a very high antibody titre as determined by ELISA. The specificity of 3G4 was confirmed by immunoblotting of whole cell extract of Escherichia coli strain MC1061 carrying the plasmid pHW756 which synthesizes both the IpaB and C proteins. The effect of the mAbs on plaque formation by virulent Shigella dysenteriae 1 was determined and it was found that the clone 3G4 substantially (55%) reduced plaque formation on HeLa cell monolayer. The epitope specificity of the mAb 3G4 was competitively inhibited by the convalescent phase sera from human, suggesting that the epitope recognized by clone 3G4 was expressed during the natural course of infection and also indicating that the 45 kDa (IpaC) protein in secreted form has a definite role in the invasive process.

Antibodies to Shiga toxin in the serum of children with Shigella-associated haemolytic uraemic syndrome.

Antibodies to Shiga toxin (Stx) were measured in the sera of 49 children with Shigella dysenteriae serotype 1 infection, of whom 17 had haemolytic uraemic syndrome (HUS) and 32 had no complications (uncomplicated shigellosis, UCS). Children with HUS had lower levels of total IgG and IgM and lower IgM titres to Stx than those with UCS. The number of children with neutralising antibodies was similar in the two groups. Of the children with HUS, 11 had HUS on enrolment and six developed HUS subsequent to enrolment. Antibody titres in children who subsequently developed HUS were compared with those in children with UCS to assess whether differences in antibody titres occurred before the development of HUS. IgA titres to Stx were found to be higher in children who subsequently developed HUS than in those with UCS. However, logistic regression analysis revealed that titres of Stx antibodies in the serum were not significant risk factors for the development of HUS. Thus, although the levels of Stx antibodies were different in children with HUS, and higher IgA titres to Stx were identifiable in children who subsequently developed HUS compared with those with UCS, the relevance of these findings in the development of HUS remains to be elucidated.

Sub-populations of peripheral lymphocytes from patients with shigella bacillary dysentery with associated leucocytosis.

In this study we ascertained the proportion and absolute number of T, B and nul cell peripheral lymphocytes from Shigella dysenteriae type I patients and compared these results with those from age matched controls. Single blood samples were collected from 13 Bangladeshi children who complained of symptoms for one or more weeks. Patients were divided into two categories, with and without a leukaemoid response. The leukaemoid response was primarily granulocytic in nature, but there was a substantial increase in the mean number of lymphocytes. Over one third of the lymphocytes were nul cells. These cells rose proportionally to the decrease of T-cells, as the proportion of B-cells remained within normal range. One leukaemoid patient died. The absolute number of lymphocytes was normal and the B-cells were normal in respect of both proportions and absolute numbers. T-cell percentage and numbers were well below normal values.

Salmonella and shigella bacteraemia in Zimbabwe.

In patients with HIV infection, non-typhoidal salmonellae are a recognised cause of bacteraemia. This association was initially demonstrated in the United States, but has more recently been found in Kenyan patients. This prompted us to review the cases of patients with enterobacteriaceae bacteraemia admitted to Parirenyatwa Hospital, Harare. Non-typhoidal salmonella bacteraemia as compared with typhoid fever was significantly more common in HIV infected patients than in non-HIV infected patients (p < 0.01). It was also a cause of bacteraemia in patients with other immuno-suppressive conditions and in some patients without identifiable risk factors.

PIP: The case notes of patients with blood cultures positive for enterobacteriaceae were examined retrospectively over a 6-month period in Parirenyatwa Hospital, Harare, Zimbabwe. Speciation was possible for Salmonella typhi and shigellae only. Nontyphoidal salmonellae were serotyped. Salmonella or shigella bacteremia was identified in 51 patients. There were 14 isolates of S. typhi, 32 isolates of nontyphoidal salmonellae, and 5 isolates of shigellae species. The case notes of 38 patients could be identified for review, and of these HIV serology was available for 15 seropositive and 15 seronegative patients. The male to female ratio was approximately 3:1 for both groups and the mean age was 29.7 +or- 21. Nontyphoidal bacteremias as compared with typhoid fever were strongly associated with HIV seropositivity [p 0.01]. 3 out of 8 HIV-negative patients with nontyphoidal bacteremia had another underlying immunosuppressive disease [2 had myeloma and 1 patient had cirrhosis with complicating hepatoma]. 2 patients with nontyphoidal bacteremia whose HIV status was unknown also had another immunosuppressing disease [acute myeloid leukemia and idiopathic pancytopenia]. 13 out of 15 HIV-positive patients showed other signs of HIV infection [oral candida, herpes zoster, persistent generalized lymphadenopathy]. 3 out of 11 patients [27%] with typhoid died, while 11 out of 27 patients [40.7%] with nontyphi bacteremia died. Most strains of S. typhimurium were included in serogroup B, which accounted for 37% of nontyphoidal isolates. Earlier studies identified invasive salmonellosis in patients with other AIDS defining diseases. In Nairobi clinical features of HIV infection were found in 64% of bacteremic HIV-positive patients, but only 28% of patients fulfilled the CDC clinical case definition for AIDS. A more recent study from Nairobi demonstrated that S. typhimurium bacteremia is a common cause of intercurrent infection in HIV-positive tuberculous patients.

Blood group and shigellosis.

A study on the distribution of ABO blood groups was carried out on 85 patients with clinically and bacteriologically proven shigellosis. A significant association (P less than 0.01) of blood group B was observed with shigellosis cases in comparison to controls from whom no Shigella species or other enteropathogen could be isolated. Patients with isoagglutinin B or those who possess blood group B antigen may be at a relatively increased risk of shigellosis.

[Species and serovar-distribution, and drug-resistance of Shigella strains isolated from imported and domestic cases from 1990 through 1994 in Tokyo].

A total of 446 Shigella strains consisting of 336 imported and 110 domestic strains isolated in Tokyo from 1990 to 1994 were examined for their species and serovar-distribution, and their drug-resistance. In both imported and domestic strains, S. sonnei was found to be the most prevalent species, followed by S. flexneri. In imported strains, however, the isolation frequency of S. flexneri, S. boydii and S. dysenteriae were higher than that of domestic strains, and the serovar of each species was distributed in a wider range than that of the domestic strains. Provisional new serovar Shigella strains were isolated from 8 imported cases and 2 domestic cases. The drug-resistance-test using 9 drugs (CP, TC, SM, KM, ABPC, ST, NA, FOM, and NFLX) showed that 82.7% of the imported strains and 89.1% of the domestic strains were resistant to any drugs examined. Drugs of a high resistant rate were SM, TC, ST for the both groups. Drug-resistance-patterns of the resistant isolates varied to 21 types. Among those, a triple drug-resistance-type with TC SM ST was found the most frequently in both groups. None of the strains were resistant to FOM or NFLX.

[Effect of infectious factors on human and animal cytogenetic structures]. / Vliianie infektsionnykh faktorov na tsitogeneticheskie struktury cheloveka i zhivotnykh

The analysis of blood leucocyte chromosomes has been carried out on 60 patients with different infectious diseases (influenza, measles, scarlet fever, and disentery), and on 47 patients immunized against measles, tick-born encephalitis, typhoid fever and brucellosis. The mutagenic influence of viruses on the genital cells of mice and on the human somatic cells in vitro was studied. Both viruses and bacteria appeared to be able to bring about different breaks in human and animal cells.

Serological identification of Shigella flexneri strains by the coagglutination reaction.

Several variants of reagents were prepared by coupling type (I, II III, IV, V and VI) and group (3, 4, 6 and 7, 8, 9) anti-Shigella flexneri sera with protein A--containing staphylococcal suspension. For most serum lots coagglutination led to a 1/10 minimum dilution. However, different efficiencies were reported between serum lots and even within the same type or group. No improvement by the coagglutination reaction could be obtained for one lot from group 3, 4 and 7, 8, 9 sera and for both type IV serum lots. The coagglutination reactions were specific both with collection strains and with recently isolated strains and the two staphylococcal suspension lots showed an identical behaviour. Coagglutination may be used for obtaining a better efficiency of type and group. Shigella flexneri sera but this varies in terms of the serum lot and the serum/Staphylococcus combination used.

[Clinico-immunological characteristics of dysentery in persons with different blood groups]. / Kliniko-immunologicheskaia kharakteristika dizenterii u lits s raznymi gruppami krovi.

The authors studied the frequency of distribution of the ABO system blood groups in 392 adults and 322 children suffering from acute dysentery. X2 criterion and double-factor dispersion analysis were used for statistical analysis. Deficiency of persons with B III and AB IV blood group. Phagocytosis indices were decreased in them with the elevated neutrophil damage index.