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A cohort of 321 children was followed from birth up to 2 years of age to determine the incidence of enterotoxigenic Escherichia coli (ETEC) in Bangladesh. The average number of diarrheal days and incidence rates were 6.6 and 2.3/child/year, respectively. ETEC was the most common pathogen and was isolated in 19.5% cases, with an incidence of 0.5 episode/child/year. The prevalence of rotavirus diarrhea was lower (10%). ETEC expressing the heat-stable enterotoxin (ST) was predominant. Strains isolated from diarrheal cases were positive for colonization factors (CFs) in higher frequency (66%) than from healthy children (33%) (P < 0.001). The heat-labile toxin (LT)-positive strains from healthy children were more often CF negative (92%) than those isolated from children with diarrhea (73%) (P < 0.001). In children with symptomatic or asymptomatic infections by CFA/I, CS1 plus CS3, CS2 plus CS3, or CS5 plus CS6 strains, a repeat episode of diarrhea or infection by the homologous CF type was uncommon. Repeat symptomatic infections were noted mostly for LT- and ST-expressing ETEC. ETEC diarrhea was more prevalent in children in the A and AB groups than in those in the O blood group (P = 0.032 to 0.023). Children with ETEC diarrhea were underweight and growth stunted at the 2-year follow-up period, showing the importance of strategies to prevent and decrease ETEC diarrheal morbidity in children.

Based on the very high prevalence of diseases caused by Helicobacter pylori, particularly in the developing world, and the rapid emergence of antibiotic resistance among clinical isolates, there is a strong rationale for an effective vaccine against H. pylori. In this review we describe recent promising candidate vaccines and prophylactic or therapeutic immunization strategies for use against H. pylori, as well as studies to identify immune responses that are related to protection in experimental animals. We also describe identification of different types of immune responses that may be related to protection against symptoms based on comparisons of H. pylori-infected patients with duodenal ulcers or gastric cancer and asymptomatic carriers. We conclude that there is still a strong need to clarify the main protective immune mechanisms against H. pylori as well as to identify a cocktail of strong protective antigens, or recombinant bacterial strains that express such antigens, that could be administered by a regimen that gives rise to effective immune responses in humans.

We tested the efficacy of a killed oral vaccine for enterotoxigenic Escherichia coli (ETEC) diarrhoea to determine if two doses of vaccine with colonization factor antigens (CF) and cholera B subunit would protect against ETEC diarrhoea of travellers. Six hundred seventy-two healthy travellers going to Mexico or Guatemala were studied in a prospective, randomised, placebo-controlled trial. The primary outcome was a vaccine preventable outcome (VPO), defined as an episode of ETEC diarrhoea with an ETEC organism producing heat labile toxin (LT) or CF homologous with the vaccine, without other known causes. The vaccine was safe and stimulated anti-heat labile toxin antibodies. There was a significant decrease in more severe VPO episodes (PE=77%, p=0.039) as defined by symptoms that interfered with daily activities or more than five loose stools in a day, although the total number of VPO events did not differ significantly in the vaccine and placebo groups. We conclude that the new oral ETEC vaccine reduces the rate of more severe episodes of traveller's diarrhoea (TD) due to VPO-ETEC, but it did not reduce the overall rate of ETEC diarrhoea or of travellers' diarrhoea due to other causes.

Colonization factor CS6 expressed by enterotoxigenic Escherichia coli (ETEC) is a nonfimbrial polymeric protein. A substantial proportion of ETEC strains isolated from patients in endemic settings and in people who travel to regions where ETEC is endemic are ETEC strains expressing CS6, either alone or in combination with fimbrial colonization factor CS5 or CS4. However, relatively little is known about the natural immune responses elicited against CS6 expressed by ETEC strains causing disease. We studied patients who were hospitalized with diarrhea (n = 46) caused by CS6-expressing ETEC (ETEC expressing CS6 or CS5 plus CS6) and had a disease spectrum ranging from severe dehydration (27%) to moderate or mild dehydration (73%). Using recombinant CS6 antigen, we found that more than 90% of the patients had mucosal immune responses to CS6 expressed as immunoglobulin (IgA) antibody-secreting cells (ASC) or antibody in lymphocyte supernatant (ALS) and that about 57% responded with CS6-specific IgA antibodies in feces. More than 80% of the patients showed IgA seroconversion to CS6. Significant increases in the levels of anti-CS6 antibodies of the IgG isotype were also observed in assays for ASC (75%), ALS (100%), and serum (70%). These studies demonstrated that patients hospitalized with the noninvasive enteric pathogen CS6-expressing ETEC responded with both mucosal and systemic antibodies against CS6. Studies are needed to determine if the anti-CS6 responses protect against reinfection and if protective levels of CS6 immunity are induced by vaccination.

In the present study, we evaluated the capacity of Helicobacter pylori adhesin A (HpaA), a H. pylori specific colonization factor, to induce therapeutic protection against H. pylori infection in mice. We found that oral immunization of H. pylori infected mice with HpaA induced protection, i.e. significant reduction in bacterial load in the stomach. This was even more pronounced when a combination of HpaA and urease was used. The protection was strongly related to specific mucosal CD4+ T cell responses with a Th1 profile as well as to mucosal IgA responses locally in the stomach. These findings suggest that HpaA is a promising vaccine candidate antigen for use in a therapeutic vaccine against H. pylori.

A total of 99 isolates out of 370 colonization factor (CF)-positive, well-characterized enterotoxigenic Escherichia coli (ETEC) strains belonging to 13 different CF types isolated from diarrhoeal patients admitted to the hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, were tested. The isolates were selected at random based on expression of the major CFs prevailing in Dhaka, Bangladesh, from 1996 to 1998. These isolates were characterized by O-antigenic serotyping, randomly amplified polymorphic DNA (RAPD) analysis and biochemical fingerprinting using the PhenePlate (PhP) system. The 99 ETEC isolates belonged to 10 O serogroups, the predominant ones being O6 (n=28), O115 (n=20) and O128 (n=20). Most isolates of serogroup O6 (CS1+CS3, 11/14; CS2+CS3, 5/8) belonged to the same PhP/RAPD type (H/f), whereas other isolates of serogroup O6 (n=12) belonged to different PhP/RAPD types (Si/f and F/c). Eleven serogroup O128 (CFA/I) isolates belonged to the same PhP/RAPD type (E/b), whereas the other O128 isolates formed different PhP/RAPD types. Fifteen (75%) serogroup O115 isolates (together with fourteen isolates from serogroups O25, O114, O142 and O159) demonstrated two closely related common groups by PhP typing (A and A1) and belonged to the same PhP/RAPD type (A/a). Three major clonal groups were identified among the ETEC strains in this study, largely based on O-antigenic type, CF expression pattern and toxin profile.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children. Although the prevalence of ETEC is high in Bangladesh and infections can be spread through food and contaminated water, limited information is available about ETEC in the surface water. We carried out studies to isolate ETEC from surface water samples from ponds, rivers, and a lake from a site close to field areas known to have a high incidence of diarrhea in Dhaka, Bangladesh, and Matlab, Bangladesh. ETEC strains isolated from the water sources were compared with ETEC strains isolated from patients with diarrhea at two hospitals in these areas. ETEC were isolated from 30% (45 of 150) of the samples from the surface water sources and 19% (518 of 2700) of the clinical specimens. One hundred ETEC strains isolated from patients with similar phenotypes as the environmental strains were compared for phenotypic and genotypic properties. The most common O serogroups on ETEC were O6, O25, O78, O115, and O126 in both types of strains. Pulsed-field gel electrophoresis analyses of the ETEC strains showed that multiple clones of ETEC were present within each colonization factor type and that some clones detected in the environment were also isolated from the stools of patients. The strains showed multiple and similar antibiotic resistance patterns. This study shows that ETEC is prevalent in surface water sources in Bangladesh suggesting a possible reason for the endemicity of this pathogen in Bangladesh.

Background: A protective effect of breastfeeding against cholera has been demonstrated in areas endemic of cholera. To assess the protection offered by breast milk from mothers living in an area that had been free from cholera for 7 years, we investigated mothers with cholera and their children during an epidemic with Vibrio cholerae El Tor in the capital of Guinea-Bissau., Methods: Eighty mothers with clinical cholera and their children were identified, and interviewed. Blood samples for vibriocidal and antitoxin antibodies were collected from mother-and-child pairs. Breast milk samples were collected from lactating mothers. Cholera was defined as acute watery diarrhea during the epidemic and a vibriocidal reciprocal titer of 20 or above., Results: Three (7%) of 42 breastfed children had cholera as defined above compared with 9 (24%) of 38 nonbreastfed children (RR for breastfed children, 0.19; 95% CI, 0.04-0.91, adjusted for age). The 3 breastfed children who developed cholera received milk containing lower concentrations of anticholera toxin IgA/total IgA (median, 2.0 units/mL) than 14 children who had serologic signs of colonization but did not develop the disease (median, 17.4 units/mL)., Conclusions: The protective effect of breast milk against cholera is not confined to endemic areas. Lactating mothers with cholera should receive supportive care to continue breastfeeding.

Because of the important role of membrane proteins in adhesion, invasion, and intracellular survival of pathogens in the host, membrane proteins are of potential interest in the search for drug targets or biomarkers. We have established a mass spectrometry-based method that allows characterization of the outer membrane protein (OMP) profile of clinical isolates from of the human gastric pathogen Helicobacter pylori. Subcellular fractionation and one-dimensional gel electrophoresis (1D-GE) analysis was combined with nano-liquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FT-ICR MS) and tandem mass spectrometry (MS/MS) analysis of fifteen H. pylori strains associated either with duodenal ulcers, gastric cancer, or isolated from asymptomatic H. pylori infected carriers. Over 60 unique membrane or membrane-associated proteins, including 30 of the 33 theoretically predicted OMPs, were identified from the strains. Several membrane proteins, including Omp11 and BabA, were found to be expressed by all strains. In the search for clinical markers we found that Omp26 was expressed by all disease-related strains but was only present in one out of five strains from asymptomatic carriers, which makes Omp26 a potential target for further investigation in the search for proteins unique to disease-related H. pylori strains. In addition, presence of Omp30 and absence of Omp6 seemed to be associated with H. pylori strains causing duodenal ulcer.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea among children in developing countries and in travelers to areas of ETEC endemicity. ETEC strains isolated from humans may produce a heat-labile enterotoxin (LT) and two types of the heat-stable enterotoxin STa, called STh and STp, encoded by the estA gene. Two commonly used assay methods for the detection of STa, the infant mouse assay or different enzyme-linked immunosorbent assays, are unable to distinguish between the two subtypes of ST. Different genotypic methods, such as DNA probes or PCR assays, may, however, allow such discrimination. Using gene probes, it has recently been reported that ETEC strains producing STp as the only enterotoxin are not associated with diarrhea. In this study, we have used highly specific PCR methods, including newly designed primers for STh together with previously described STp primers, to compare the relative distribution of STh and STp in ETEC isolated from children with diarrhea in three different geographically distinct areas, i.e., Bangladesh, Egypt, and Guatemala, and from travelers to Mexico and Guatemala. It was found that ETEC strains producing STp were as commonly isolated from cases of diarrhea as strains producing STh both in Egypt and Guatemala, whereas STp strains were considerably less common in Bangladesh. No difference was found in the relative distribution of STh and STp in ETEC strains isolated from travelers with diarrhea and from asymptomatic carriers. Irrespective of ST genotype, the disease symptoms were also similar in both children and travelers.

We determined the types of cholera toxin (CT) produced by a collection of 185 Vibrio cholerae O1 strains isolated in Bangladesh over the past 45 years. All of the El Tor strains of V. cholerae O1 isolated since 2001 produced CT of the classical biotype, while those isolated before 2001 produced CT of the El Tor biotype.

Enterotoxigenic Escherichia coli (ETEC) colonize the intestine and adhere to the epithelium by means of different host specific colonization factors (CFs). Colonizing ETEC produce one or both of two enterotoxins; the heat stable (ST) and heat labile (LT) toxins which are both able to cause diarrhoea. The regulation of virulence genes in ETEC during infection of the human intestine is mainly unknown. In this study we analysed the level of mRNA expression of estA, coding for ST, and eltB, coding for the B subunit of LT, during human infection. The expressions of the toxins in ETEC strains expressing both ST and LT were investigated in bacteria isolated directly from patient stool without sub-culturing, (in vivo) and compared to the expression pattern of the corresponding ST/LT strains grown in liquid broth (in vitro) by quantitative competitive RT-PCR using fluorescent primers. We found that estA and eltB are expressed in the in vivo samples but no significant up-or down regulation of the expression levels of either estA or eltB could be determined in vivo as compared to in vitro.

We have studied homologous (HoM) and cross-reacting (CR) immunoglobulin A (IgA) antibody responses to colonization factors (CFs) in Bangladeshi children with diarrhea due to enterotoxigenic E. coli (ETEC) strains of the CF antigen I (CFA/I) group (CFA/I, n = 25; coli surface antigen 4 [CS4], n = 8; CS14, n = 11) and the CS5 group (CS5, n = 15; CS7, n = 8), respectively. The responses to the HoM, CR, and heterologous (HeT) CF antigens in each group of patient were studied and compared to that seen in healthy children (n = 20). In the CFA/I group (CFA/I and CS14), patients responded with antibody-secreting cell (ASC) responses to HoM CFs (geometric mean, 156 to 329 ASCs/10(6) peripheral blood mononuclear cells [PBMCs]) and to CR CFs ( approximately 15 to 38 ASCs/10(6) PBMCs) but least of all to the HeT CS5 antigen (2 to 4 ASCs/10(6) PBMCs). For the CS5 group of patients with ETEC (CS5 and CS7), likewise, responses to HoM CFs (230 to 372 ASCs/10(6) PBMCs) and CR CFs (27 to 676 ASCs/10(6) PBMCs) were seen, along with lower responses to the HeT CFA/I antigen (9 to 38 ASCs/10(6) PBMCs). Both groups of patients responded with CF-specific IgA antibodies to HoM and CR antigens in plasma but responded less to the HeT CFs. The responses in patients were seen very soon after the onset of diarrhea and peaked around 1 week after onset. Vaccinees who had received two doses of the oral, killed whole-cell ETEC vaccine (CF-BS-ETEC) responded with plasma IgA antibodies to CFA/I, a component of the vaccine, but also to the CR CS14 antigen, which was not included in the vaccine, showing that antibody responses can be stimulated by a CFA/I-containing ETEC vaccine to a CR-reacting antigen in individuals in countries where ETEC is endemic.

Bacterial adherence to mucosal surfaces is an important virulence trait of pathogenic bacteria. Adhesion of enterotoxigenic Escherichia coli (ETEC) to the intestine is mediated by a number of antigenically distinct colonization factors (CFs). One of the most common CFs is CFA/I. This has a fimbrial structure composed of a major repeating subunit, CfaB, and a single tip subunit, CfaE. The potential carbohydrate recognition by CFA/I was investigated by binding CFA/I-fimbriated bacteria and purified CFA/I fimbriae to a large number of variant glycosphingolipids separated on thin-layer chromatograms. For both fimbriated bacteria and purified fimbriae, specific interactions could be identified with a number of nonacid glycosphingolipids. These included glucosylceramide, lactosylceramide with phytosphingosine and/or hydroxy fatty acids, neolactotetraosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, the H5 type 2 pentaglycosylceramide, the Lea-5 glycosphingolipid, the Lex-5 glycosphingolipid, and the Ley-6 glycosphingolipid. These glycosphingolipids were also recognized by recombinant E. coli expressing CFA/I in the absence of tip protein CfaE, as well as by purified fimbriae from the same strain. This demonstrates that the glycosphingolipid-binding capacity of CFA/I resides in the major CfaB subunit.

Operation Bright Star (OBS) is a biennial, multinational exercise in Egypt involving 15000 US troops. Consistent with past observations in deployed troops, diarrhea is the most significant cause of morbidity. Focused efforts are ongoing to develop vaccines against the most common pathogens affecting our troops. As part of these efforts, diarrhea surveillance was conducted during OBS to monitor pathogens associated with illness and to identify new vaccine targets. A retrospective review was conducted of prior studies with similar methods. Soldiers with diarrhea presenting to the OBS clinic provided a stool sample that was inoculated into Carey-Blair transport media. Within 3 days, the Cary-Blair tubes were transported to the Naval Medical Research Unit no. 3 in Cairo where bacterial culture was performed. As part of the evaluation, 5 Escherichia coli-like colonies were collected and tested for toxin production using the GM1-ELISA. Toxin-positive isolates were further tested for colonization factors (CF) by a dot-blot assay using a standardized panel of monoclonal antibodies against CFA/I, CS1-CS7, CS17, CS8 (CFA/III), CS12 (PCFO159), and CS14 (PCFO166). Enterotoxigenic E. coli (ETEC) was the most frequently isolated pathogen during each OBS from which data were collected. The rate of ETEC-associated diarrhea ranged from 22% to 58%. Over time, there were dramatic shifts in the frequency and distribution of CFs. Over the 5 years of study, an increasing number of ETEC isolates had no known CF identified, and in 2001, only 40% of ETEC was associated with known CFs. The most commonly identified CF was CS6. Diarrheal disease, particularly ETEC, continues to be a common malady among US military personnel deployed to Egypt. We have identified ETEC CF types, especially CS6, which should be considered potential vaccine candidates. However, despite intensive testing, CFs could not be identified in most of the ETEC isolated, highlighting the need for further studies to identify novel CFs and alternative vaccine targets.

The oral-formalin inactivated whole cell enterotoxigenic Escherichia coli (ETEC) vaccine needs to be further tested in developing countries in order to determine the dose at which it will be safe and immunogenic for infants who are the target population for the vaccine. To determine the immunogenicity of reduced doses, studies were first carried out in children, 2-12 years of age (n = 60). The full, half or a quarter doses of the vaccine were comparable in immunogenicity with similar frequency of responses seen to the different antigens (P = NS). Following this result, a pilot study carried out in infants, 6-17 months of age (n = 50), showed that the frequency of episodes of vomiting was lowest when a quarter of the full dose was used. The infants however showed comparable immune responses to the half and quarter dose of vaccine that was tested (P = NS). Based on these results in the infants, a randomized double blind placebo-controlled Phase II study was carried out in 158 children, 6-17 months of age, where a quarter dose of the ETEC vaccine was tested. Adverse events of mild vomiting were seen in only 4% of vaccinees and in 2.5% of placebo recipients. The IgA-antibody secreting cell (ASC) responses to CFA/I (GM: 28.1 ASC/10(7) PBMC) and BS (GM: 55.7 ASC/10(7) PBMC) were elevated compared to placebo recipients (CFA/I-2.0; BS-4.8 ASC/10(7) PBMC) (P = 0.01 to < 0.001). The plasma-IgA antibody titers in vaccinees were also significantly elevated to CFA/I (GM-93.00), CS1 (GM-62.0), CS2 (GM-55.0), CS4 (GM-66.0) and BS (1057.0) compared to preimmune levels or responses or levels in placebo recipients (P < or = 0.05-0.001). This study thus demonstrates that reduced doses of the ETEC vaccine is immunogenic in children and infants as well as safe in infants down to 6 months of age.

Helicobacter pylori infection induces chronic inflammation in the gastric mucosa with a marked increase in the number of lymphoid follicles consisting of infiltrating B and T cells, neutrophils, dendritic cells (DC) and macrophages. It has been suggested that an accumulation of mature DC in the tissue, resulting from a failure of DC to migrate to lymph nodes, may contribute to this chronic inflammation. Migration of DC to lymph nodes is regulated by chemokine receptor CCR7, expressed on mature DC, and the CCR7 ligands CCL19 and CCL21. In this study we analysed the maturation, in vitro migration and cytokine production of human DC after stimulation with live H. pylori. For comparison, DC responses to non-pathogenic Escherichia coli bacteria were also evaluated. Stimulation with H. pylori induced maturation of DC, i.e. up-regulation of the chemokine receptors CCR7 and CXCR4 and the maturation markers HLA-DR, CD80 and CD86. The H. pylori-stimulated DC also induced CD4(+) T-cell proliferation. DC stimulated with H. pylori secreted significantly more interleukin (IL)-12 compared to DC stimulated with E. coli, while E. coli-stimulated DC secreted more IL-10. Despite low surface expression of CCR7 protein following stimulation with H. pylori compared to E. coli, the DC migrated equally well towards CCL19 after stimulation with both bacteria. Thus, we could not detect any failure in the migration of H. pylori stimulated DC in vitro that may contribute to chronic gastritis in vivo, and our results suggest that H. pylori induces maturation and migration of DC to lymph nodes where they promote T cell responses.

The aim of this study was to investigate the capacity of oral and parenteral therapeutic immunization to reduce the bacterial colonization in the stomach after experimental Helicobacter pylori infection, and to evaluate whether any specific immune responses are related to such reduction. C57BL/6 mice were infected with H. pylori and thereafter immunized with H. pylori lysate either orally together with cholera toxin or intraperitoneally (i.p.) together with alum using immunization protocols that previously have provided prophylactic protection. The effect of the immunizations on H. pylori infection was determined by quantitative culture of H. pylori from the mouse stomach. Mucosal and systemic antibody responses were analyzed by ELISA in saponin extracted gastric tissue and serum, respectively, and mucosal CD4+ T cell responses by an antigen specific proliferation assay. Supernatants from the proliferating CD4+ T cells were analyzed for Th1 and Th2 cytokines. The oral, but not the parenteral therapeutic immunization induced significant decrease in H. pylori colonization compared to control infected mice. The oral immunization resulted in markedly elevated levels of serum IgG+M as well as gastric IgA antibodies against H. pylori antigen and also increased H. pylori specific mucosal CD4+ T cell proliferation with a Th1 cytokine profile. Although the parenteral immunization induced dramatic increases in H. pylori specific serum antibody titers, no increases in mucosal antibody or cellular immune responses were observed after the i.p. immunization compared to control infected mice. These findings suggest that H. pylori specific mucosal immune responses with a Th1 profile may provide therapeutic protection against H. pylori.

Infection with the human gastric pathogen Helicobacter pylori can give rise to chronic gastritis, peptic ulcer, and gastric cancer. All H. pylori strains express the surface-localized protein HpaA, a promising candidate for a vaccine against H. pylori infection. To study the physiological importance of HpaA, a mutation of the hpaA gene was introduced into a mouse-adapted H. pylori strain. To justify that the interruption of the hpaA gene did not cause any polar effects of downstream genes or was associated with a second site mutation, the protein expression patterns of the mutant and wild-type strains were characterized by two different proteomic approaches. Two-dimensional differential in-gel electrophoresis analysis of whole-cell extracts and subcellular fractionation combined with nano-liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for outer membrane protein profiling revealed only minor differences in the protein profile between the mutant and the wild-type strains. Therefore, the mutant strain was tested for its colonizing ability in a well-established mouse model. While inoculation with the wild-type strain resulted in heavily H. pylori-infected mice, the HpaA mutant strain was not able to establish colonization. Thus, by combining proteomic analysis and in vivo studies, we conclude that HpaA is essential for the colonization of H. pylori in mice.

Helicobacter pylori infection causes chronic gastritis that may progress to peptic ulcers or gastric adenocarcinoma and thereby cause major world-wide health problems. Previous studies have shown that CD4+ T cells are important in the immune response to H. pylori in humans, but the role of CD8+ T cells is less clear. In order to study the CD8+ T cell response to H. pylori in greater detail, we have evaluated efficient conditions for activation of CD8+ T cells in vitro. We show that H. pylori-reactive CD8+ T cells can be activated most efficiently by B cells or dendritic cells pulsed with H. pylori antigens. We further show that the majority of CD8+ T cells in H. pylori-infected gastric mucosa are memory cells, and that memory CD8+ T cells sorted from peripheral blood of H. pylori-infected individuals respond 15-fold more to H. pylori urease compared to memory cells from uninfected subjects. We conclude that CD8+ T cells do participate in the immune response to H. pylori, and this may have implications for the development of more severe disease outcomes in H. pylori-infected subjects.

Worldwide, gastric adenocarcinoma (GC) is the second most common cause of death from malignant disease. The reason why immune responses are unable to clear the tumour is not fully understood, although aberrant lymphocyte recruitment to the tumour site might be one factor. Therefore, we investigated the homing phenotype of mucosal T lymphocytes in GC, compared to tumour-free mucosa. We could detect significantly decreased frequencies of mucosal homing alpha4beta7+ T cells in the tumour tissues and increased frequencies of L-selectin+ T cells. This was probably due to the correlated decrease in MAdCAM-1 positive and increase in PNAd positive blood vessels in the tumour mucosa. There were also fewer CXCR3+ T lymphocytes in the tumour tissue. These findings provide evidence that endothelial cells within tumours arising at mucosal sites do not support extravasation of typical mucosa-infiltrating T cells. This may be of major relevance for future immunotherapeutic strategies for treatment of GC.

Vibrio cholerae O139 emerged in 1992 as a major cause of epidemic cholera. However, the incidence of disease due to this new serogroup subsequently decreased for almost a decade. In April 2002, there was a dramatic resurgence of V. cholerae O139 in Bangladesh. We compared the phenotypic properties of the bacterial isolates and the immunological responses in patients with disease due to V. cholerae O139 during the 2002 epidemic with those dating to the emergence of this disease in 1993 to 1995. Strains isolated from patients in the two time periods were compared with respect to capsular polysaccharide, their resistance to the bactericidal effect of serum, and their capacity to be used as target strains in complement-mediated vibriocidal assays. Phase-contrast microscopy showed that strains isolated in 2002 had less capsular material than those isolated from 1993 to 1995 (P = <0.001), a finding confirmed by electron microscopic studies. Strains isolated in 2002 were more susceptible to the bactericidal activity of serum compared to strains from 1993 to 1995 (P = 0.013). Compared to results using a standard O139 strain, a modified vibriocidal assay utilizing a 2002 strain, CIRS 134, as the target organism detected higher vibriocidal responses in both O139-infected cholera patients as well as O139 vaccine recipients. The vibriocidal assay utilizing the less encapsulated 2002 strain, CIRS 134, is a more sensitive indicator of adaptive immune responses to recent infection with V. cholerae O139. Consequently, this assay may be useful in studies of both O139-infected patients and recipients of O139 vaccines.

Helicobacter pylori infects the stomach and duodenal mucosa. T cells are important components of the H. pylori-induced immune response, but little is currently known about how these cells are recruited to the infected mucosa. Here, we have characterized stomach and duodenal T cells isolated from H. pylori-infected and noninfected subjects with regard to subtype, expression of homing and chemokine receptors, and in vitro reactivity to H. pylori antigens. Higher numbers of CD4(+) but similar numbers of CD8(+) lamina propria T cells were isolated from stomach biopsies from H. pylori-positive compared to H. pylori-negative individuals. CD4(+) T cells from infected stomach expressed increased levels of the homing receptor L-selectin and the chemokine receptor CCR4 compared to CD4(+) T cells from uninfected stomach. Infected stomach mucosa also contained increased levels of the CCR4 chemokine ligand MDC/CCL22. In contrast, comparable numbers of CD4(+) T cells with similar receptor expression were isolated from the duodenum of H. pylori-positive and H. pylori-negative individuals. In vitro proliferation of mucosal T cells was strongly enhanced by the addition of interleukin-2 (IL-2) and IL-7 to the cell cultures. Using this approach, H. pylori-specific T-cell responses were detected in stomach CD4(+) T cells from H. pylori-positive but not H. pylori-negative individuals. Duodenal T cells from only a few individuals responded to H. pylori stimulation, and the responsiveness was not restricted to H. pylori-positive individuals, suggesting limited H. pylori specificity in the duodenum and possible cross-reactivity with antigens from other bacteria in this compartment. In conclusion, these results suggest that H. pylori-specific CD4(+) T cells preferentially home to and accumulate in the infected stomach and that L-selectin and CCR4/MDC are important for this recruitment.

Continuous recruitment of neutrophils into the inflamed gastric mucosal tissue is a hallmark of Helicobacter pylori infection in humans. In this study, we examined the ability of H. pylori to induce transendothelial migration of neutrophils using a transwell system consisting of a cultured monolayer of human endothelial cells as barrier between two chambers. We showed for the first time that live H. pylori, but not formalin-killed bacteria, induced a significantly increased transendothelial migration of neutrophils. H. pylori conditioned culture medium also induced significantly increased transendothelial migration, whereas heat-inactivated culture filtrates had no effect, suggesting that the chemotactic factor was proteinaceous. Depletion of H. pylori-neutrophil activating protein (HP-NAP) from the culture filtrates resulted in significant reduction of the transmigration. Culture filtrates from isogenic HP-NAP deficient mutant bacteria also induced significantly less neutrophil migration than culture filtrates obtained from wild-type bacteria. HP-NAP did not induce endothelial cell activation, suggesting that HP-NAP acts directly on the neutrophils. In conclusion, our results demonstrate that secreted HP-NAP is one of the factors resulting in H. pylori induced neutrophil transendothelial migration. We propose that HP-NAP contributes to the continuous recruitment of neutrophils to the gastric mucosa of H. pylori infected individuals.

Flooding in Dhaka in July 2004 caused epidemics of diarrhea. Enterotoxigenic Escherichia coli (ETEC) was almost as prevalent as Vibrio cholerae O1 in diarrheal stools. ETEC that produced heat-stable enterotoxin alone was most prevalent, and 78% of strains had colonization factors. Like V. cholerae O1, ETEC can cause epidemic diarrhea.

ETEC is an underrecognized but extremely important cause of diarrhea in the developing world where there is inadequate clean water and poor sanitation. It is the most frequent bacterial cause of diarrhea in children and adults living in these areas and also the most common cause of traveler's diarrhea. ETEC diarrhea is most frequently seen in children, suggesting that a protective immune response occurs with age. The pathogenesis of ETEC-induced diarrhea is similar to that of cholera and includes the production of enterotoxins and colonization factors. The clinical symptoms of ETEC infection can range from mild diarrhea to a severe cholera-like syndrome. The effective treatment of ETEC diarrhea by rehydration is similar to treatment for cholera, but antibiotics are not used routinely for treatment except in traveler's diarrhea. The frequency and characterization of ETEC on a worldwide scale are inadequate because of the difficulty in recognizing the organisms; no simple diagnostic tests are presently available. Protection strategies, as for other enteric infections, include improvements in hygiene and development of effective vaccines. Increases in antimicrobial resistance will dictate the drugs used for the treatment of traveler's diarrhea. Efforts need to be made to improve our understanding of the worldwide importance of ETEC.

Helicobacter pylori infection is one of the most common gastrointestinal infections worldwide. Although the majority of the infected individuals remain asymptomatic carriers of the bacteria, approximately 15% develop peptic ulcers, which are most prevalent in the duodenum. H. pylori induce a vigorous immune response which, however, fails to clear the infection. Instead, the chronic inflammation that arises in the infected gastroduodenal mucosa may be involved in the development of H. pylori-associated peptic ulcers. We have previously shown that duodenal ulcer (DU) patients have a significantly lower epithelial cytokine, e.g. IL-8, response in the duodenum than asymptomatic (AS) carriers. In this study we have further investigated the mechanisms behind this finding, i.e. whether it can be explained by bacterial factors, down-regulation of epithelial cytokine production by regulatory T cells, or an impaired ability of the duodenal epithelium in DU patients to produce cytokines. Gastric AGS, and intestinal T84 epithelial cell lines were stimulated with H. pylori strains isolated from DU patients and AS carriers, respectively. All strains were found to induce comparable cytokine and cytokine receptor expression in epithelial cells. Regulatory T cells (CD4+ CD25(high)), isolated from human peripheral blood and cocultured with H. pylori stimulated AGS cells, were found to slightly suppress H. pylori-induced epithelial cytokine production. Furthermore, primary cultures of duodenal epithelial cells from DU patients were found to produce markedly lower amounts of cytokines than epithelial cells isolated from AS carriers. These results suggest that the lower epithelial cytokine responses in the duodenum of DU patients, which may be of importance for the pathogenesis of H. pylori-induced duodenal ulcers, most likely can be explained by host factors, i.e. mainly a decreased ability of the duodenal epithelium to produce cytokines, but possibly partly also down-regulation by regulatory T cells.

Mucosal immunisation may be used both to protect the mucosal surfaces against infections and as a means for immunological treatment of peripheral immunopathological disorders through the induction of systemic antigen-specific tolerance ('oral tolerance'). The development of mucosal vaccines, whether for prevention of infectious diseases or for oral tolerance immunotherapy, requires efficient antigen delivery and adjuvant systems that can help to present the appropriate vaccine or immunotherapy antigens to the mucosal immune system. The most potent (but also toxic) mucosal adjuvants are cholera toxin (CT) and the closely related Escherichia coli heat-labile enterotoxin (LT), and much effort and significant progress have been made recently to generate toxicologically acceptable derivatives of these toxins with retained adjuvant activity. Among these are the non-toxic, recombinantly produced cholera toxin B-subunit (CTB). CTB is a specific protective antigen component of a widely registered oral cholera vaccine as well as a promising vector for either giving rise to mucosal anti-infective immunity or for inducing peripheral anti-inflammatory tolerance to chemically or genetically linked foreign antigens administered mucosally. CT and CTB have also recently been used as combined vectors and adjuvants for markedly promoting ex vivo dendritic cell (DC) vaccination with different antigens and also steering the immune response to the in vivo-reinfused DCs towards either broad Th1 + Th2 + CTL immunity (CT) or Th2 or tolerance (CTB). Another type of mucosal adjuvants is represented by bacterial DNA or synthetic oligodeoxynucleotides containing CpG-motifs, which especially when linked to CTB have been found to effectively stimulate both innate and adaptive mucosal immune responses. The properties and clinical potential of these different classes of adjuvants are being discussed.

Helicobacter pylori is known to induce a local immune response, which is characterized by activation of lymphocytes and the production of IFN-gamma in the stomach mucosa. Since not only T cells, but also natural killer (NK) cells, are potent producers of gamma interferon (IFN-gamma), we investigated whether NK cells play a role in the immune response to H. pylori infection. Our results showed that NK cells were present in both the gastric and duodenal mucosae but that H. pylori infection did not affect the infiltration of NK cells into the gastrointestinal area. Furthermore, we could show that NK cells could be activated directly by H. pylori antigens, as H. pylori bacteria, as well as lysate from H. pylori, induced the secretion of IFN-gamma by NK cells. NK cells were also activated without direct contact when separated from the bacteria by an epithelial cell layer, indicating that the activation of NK cells by H. pylori can also occur in vivo, in the infected stomach mucosa. Moreover, the production of IFN-gamma by NK cells was greatly enhanced when a small amount of interleukin-12 (IL-12) was added, and this synergistic effect was associated with increased expression of the IL-12 receptor beta2. It was further evident that bacterial lysate alone was sufficient to induce the activation of cytotoxicity-related molecules. In conclusion, we demonstrated that NK cells are present in the gastroduodenal mucosa of humans and that NK cells produce high levels of IFN-gamma when stimulated with a combination of H. pylori antigen and IL-12. We propose that NK cells play an active role in the local immune response to H. pylori infection.

Background: We conducted a nested case-control study in 397 rural Egyptian children <36 months of age to assess the correlation between serum levels of antibodies against toxin and colonization factors (CFs) and the risk of homologous enterotoxigenic Escherichia coli (ETEC) diarrhea., Methods: Active case detection was performed via semiweekly home visits, and blood was obtained at 3-month intervals. After each serosurvey, case subjects were selected from children experiencing a CF antigen (CFA)/I-, CFA/II-, CFA/IV-, or heat-labile enterotoxin (LT)-ETEC diarrheal episode during the subsequent 3 months. Up to 5 control subjects per case subject were selected from children who did not experience an ETEC diarrheal episode during the corresponding interval. Serum titers of immunoglobulin G antibodies against CFA/I, coli surface antigen (CS) 3, CS6, and LT were measured by enzyme-linked immunosorbant assay., Results: The distribution of serum titers of LT, CS3, and CS6 antibodies did not differ between the case and control subjects. For children <18 months of age, serum titers of CFA/I antibody were inversely related to the risk of CFA/I-ETEC diarrhea; reciprocal serum titers of CFA/I antibody > or =76 were associated with a 77% reduction in the odds of CFA/I-ETEC diarrhea., Conclusion: Induction of reciprocal serum titers of antibodies against CFA/I within or above the 76-186 range should be further evaluated as a predictor for assessment of the ability of candidate vaccines to protect against CFA/I-ETEC diarrhea.

Helicobacter pylori chronically colonizes the stomach and duodenum and causes peptic ulcers or gastric adenocarcinoma in 10 to 20% of infected individuals. We hypothesize that the inability of patients to clear H. pylori infections is a consequence of active suppression of the immune response. Here we show that H. pylori-infected individuals have increased frequencies of CD4(+) CD25(high) T cells in both the stomach and duodenal mucosa compared to uninfected controls. These cells have the phenotype of regulatory T cells, as they express FOXP3, a key gene for the development and function of regulatory T cells, as well as high levels of the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) protein. In contrast, mucosal CD4(+) CD25(low) and CD4(+) CD25(-) cells express little FOXP3 mRNA and low levels of the CTLA-4 protein. Mucosal CD4(+) CD25(high) T cells are present in individuals with asymptomatic H. pylori infections as well as in duodenal ulcer patients. The frequencies of CD4(+) CD25(high) cells are also increased in the stomachs of H. pylori-infected patients with gastric adenocarcinoma, particularly in cancer-affected tissues. These findings suggest that regulatory T cells may suppress mucosal immune responses and thereby contribute to the persistence of H. pylori infections.

Enterotoxigenic Escherichia coli (ETEC) causes substantial diarrheal morbidity and mortality in young children in countries with limited resources. We determined the phenotypic profiles of 915 ETEC diarrheal isolates derived from Egyptian children under 3 years of age who participated in a 3-year population-based study. For each strain, we ascertained enterotoxin and colonization factor (CF) expression, the O:H serotype, and antimicrobial susceptibility. Sixty-one percent of the strains expressed heat-stable enterotoxin (ST) only, 26% expressed heat-labile enterotoxin (LT) alone, and 12% expressed both toxins. The most common CF phenotypes were colonization factor antigen I (CFA/I) (10%), coli surface antigen 6 (CS6) (9%), CS14 (6%), and CS1 plus CS3 (4%). Fifty-nine percent of the strains did not express any of the 12 CFs included in our test panel. Resistance of ETEC strains to ampicillin (63%), trimethoprim-sulfamethoxazole (52%), and tetracycline (43%) was common, while resistance to quinolone antibiotics was rarely detected. As for the distribution of observed serotypes, there was an unusually wide diversity of O antigens and H types represented among the 915 ETEC strains. The most commonly recognized composite ETEC phenotypes were ST CS14 O78:H18 (4%), ST (or LTST) CFA/I O128:H12 (3%), ST CS1+CS3 O6:H16 (2%), and ST CFA/I O153:H45 (1.5%). Temporal plots of diarrheal episodes associated with ETEC strains bearing common composite phenotypes were consistent with discrete community outbreaks either within a single or over successive warm seasons. These data suggest that a proportion of the disease that is endemic to young children in rural Egypt represents the confluence of small epidemics by clonally related ETEC strains that are transiently introduced or that persist in a community reservoir.

Background: To design an efficacy trial of a killed oral vaccine against enterotoxigenic Escherichia coli (ETEC) diarrhea in Egyptian children, we derived for ETEC diarrhea an empirical definition that increased the probability that diarrhea associated with excretion of ETEC was caused by the detected ETEC., Methods: We conducted a cohort study of 397 Egyptian children <24 months old and monitored them until they were 3 years old. Vaccine-preventable (VP) ETEC was defined as ETEC expressing >/=1 of the toxin- (heat-labile [LT] toxin) and colonization-factor antigens (CFA I, II, and IV) in the vaccine., Results: Although fecal excretion of VP-ETEC was highly associated with diarrhea, excretion of LT-ETEC per se was not related to diarrhea (adjusted odds ratio [OR(A)], 1.16 [95% confidence interval [CI], 0.90-1.49]). The fecal excretion of antigenic types of VP-ETEC other than LT-ETEC (non-LT VP-ETEC) was highly associated with diarrheal symptoms (OR(A), 3.91 [95% CI, 2.78-5.49]; P<.001), and this association was greater for nonbloody than for bloody diarrhea., Conclusions: Because the vaccine had been anticipated to protect primarily against symptomatic ETEC diarrhea, these results indicate that the primary-outcome definition of ETEC diarrhea for the trial should be restricted to nonbloody diarrheal episodes associated with fecal excretion of non-LT VP-ETEC.

Enteric infections resulting in diarrhoea are among the most important causes of morbidity and mortality, particularly in children in developing countries. They are also a common cause of disease among travellers to Africa, Asia and Latin America. Recently, effective, live and inactivated oral and parenteral vaccines against some of the most severe enteric infections-cholera and typhoid fever-have been licensed in several countries. Different candidate vaccines against rotavirus, Shigella and ETEC diarrhoea have also been developed and tested for safety and immunogenicity in developed as well as in developing countries. The protective efficacy of several of these vaccines has also been tested, either in human volunteer challenge studies or in field trials. In this chapter we describe the properties and availability of the recently licensed vaccines and present an update on the diverse efforts being made to achieve new or improved vaccines against the most prevalent enteropathogens.

Monoclonal antibodies (MAbs) were prepared against toxin-coregulated pili (TCP) isolated from Vibrio cholerae O1 El Tor. Despite their limited bactericidal potential, two MAbs were able to mediate biotype-specific protection against experimental cholera in infant mice. These MAbs were used in immunoblotting studies to assess seroconversion to El Tor TCP following cholera. Clear anti-pilus responses were observed in five of nine patients.

Determination of optimal routes to induce mucosal immune responses locally in the stomach and duodenum are important steps in the development of vaccines against Helicobacter pylori infection. In this study, we immunized H. pylori-infected individuals either nasally or rectally with a model antigen, i.e. cholera toxin B subunit, and compared the immune responses after these routes with the responses after oral or intrajejunal vaccination. Specific antibody levels in serum as well as specific antibody levels and antibody-secreting cells in biopsies from antrum and duodenum were determined by ELISA and ELISPOT methods. In contrast to oral vaccination, nasal and rectal vaccination did not induce significant increases in specific antibody-secreting cells either in the antrum or duodenum. Furthermore, when analyzing the antibody levels in saponin extracted biopsies, intrajejunal vaccination was superior to both nasal and rectal vaccination in inducing antigen-specific IgA levels in the stomach. We conclude that oral vaccination is the optimal route for induction of antigen-specific IgA antibody responses in the stomach and duodenum of humans, while nasal or rectal vaccination is less suitable for this purpose.

Background and Aims: The general concept is that as Vibrio cholerae is not invasive, it mediates a non-inflammatory type of infection. This is being re-evaluated based on available data that natural cholera infection or cholera toxin induces a Th2-type of immune profile and stimulates the humoral immune response, innate cells, and mediators in the host., Methods: To perform a comprehensive analyses of the inflammatory components, we studied mucosal biopsies from patients, both adults and children with acute watery diarrhoea caused by V cholerae O1 and O139. Patients with cholera, adults (n = 30) and children (n = 18), as well as healthy controls (n = 24) were studied. Histochemical, immunohistochemical, and ultrastructural studies were carried out to elucidate the contribution of the different factors using paraffin and frozen duodenal and/or rectal sections as appropriate. Samples were collected during the acute stage and during early and/or late convalescence., Results: Following natural cholera infection, patients responded with increases in neutrophil polymorphs during the acute stage (p<0.001) compared with healthy controls whereas mucosal mast cells (MMC) (p = 0.008) and eosinophils (p = 0.034) increased in the gut during convalescence. Electron microscopic analyses of duodenal biopsies from adult patients showed increased piecemeal degranulation in both MMC and eosinophils and accumulation of lipid bodies in MMC. Duodenal biopsies from V cholerae O1 infected patients showed upregulation of myeloperoxidase, lactoferrin, PGHS-1, SCF, tryptase, tumour necrosis factor alpha, alpha-defensin, and eotaxin during the acute stage and chymase, interleukin 3 and major basic protein during convalescence., Conclusion: We have shown that innate cells and their mediators are upregulated in acute watery diarrhoea. These cells and factors of the innate arm may be important in the host's defence against cholera. Such effects may need to be simulated in a vaccine to achieve long lasting protection from cholera.

The incidence of enterotoxigenic Escherichia coli diarrhea among Egyptian children was 1.5 episodes per child per year and accounted for 66% of all first episodes of diarrhea after birth. The incidence increased from 1.7 episodes per child per year in the first 6 months of life to 2.3 in the second 6 months and declined thereafter.

This review deals with the induction and expression of immune responses in the human stomach following mucosal immunisation. As prerequisites for developing a Helicobacter pylori vaccine, the role of gastric inflammation, in particular inflammation induced by H. pylori infection, as well as lymphocyte homing within the common mucosal immune system, and the importance of immunisation route are discussed.

A phase II safety and immunogenicity study of an oral-formalin inactivated enterotoxigenic Escherichia coli (ETEC) vaccine containing six colonization factors (CFA/I, CS1, CS2, CS3, CS4, CS5) and 1mg of recombinant cholera toxin B subunit (the CF-BS-ETEC vaccine) was carried out in an urban slum of Dhaka city in Bangladesh. The study was carried out in a double blinded, placebo controlled design in 158 children, 18-36 months of age. Children were given two doses of the CF-BS-ETEC vaccine or the placebo which consisted of E. coli K12. The vaccine was well tolerated. The immune response was studied in 60 children (30 each in the placebo and vaccine group). Significant vaccine specific IgA antibody-secreting cell (ASC) responses were seen 7 days after ingestion of the first and second dose of the vaccine. The responses to CFA/I (P

Helicobacter pylori induces symptomatic chronic gastritis in a subpopulation of infected individuals. The mechanism(s) determining the development and severity of pathology leading to symptoms are not fully understood. In a mouse model of H. pylori infection we analysed the influence of immunoregulatory CD4+CD25+ T cells on H. pylori colonization and gastritis. Athymic C57BL/6 nu/nu mice were reconstituted with (a) lymph node (LN) cells (b) LN cells depleted of CD25+ T cells (CD25(-) LN) or (c) not reconstituted at all. Mice were then infected orally with 3 x 10(8)H. pylori SS1 bacteria. At 2 and 6 weeks after the inoculation there was a significant (P < 0.001) reduction in H. pylori colonization in athymic mice transferred with CD25(-) LN cells compared to mice transferred with LN cells. Colonization was still reduced at 12 weeks after inoculation. Mice transferred with CD25(-) LN cells showed an earlier onset and increased severity of gastritis as compared to mice receiving LN cells. Splenic cells isolated from mice receiving CD25(-) LN cells produced the highest level of IFN-gamma on stimulation with H. pylori antigens in vitro, had a higher H. pylori-specific DTH response and increased infiltration of CD4+ T cells and macrophages in the gastric mucosa. Athymic mice not transferred with T cells had persistent high H. pylori colonization and displayed a normal gastric epithelium without inflammatory cells. In conclusion, CD4+CD25+ cells reduce immunopathology in H. pylori infection, possibly by reducing the activation of IFN-gamma producing CD4+ T cells, even at the expense of a higher H. pylori load in the gastric mucosa.

Helicobacter pylori colonize the human stomach and duodenum. The infection has been shown to induce a strong T-cell response in the stomach, whereas the response within the duodenum has been poorly characterized. Furthermore, it remains to be elucidated whether the T-cell response may contribute to ulcer formation in the host. In this study, the frequency of different T-cell subsets, their degree of activation and expression of co-stimulatory receptors in biopsies from the duodenum as well as the antrum were studied by immunohistochemistry and flow cytometry. It was also evaluated whether there are differences in the T-cell responses between duodenal ulcer patients and asymptomatic carriers that might explain why only 10-15% of the infected subjects develop duodenal ulcers. The frequencies of CD4+, CD8+ and CD45RO+, i.e. memory T-cells, were significantly increased in the antrum, and the number of CD25+ cells was considerably higher in both the antrum and duodenum of duodenal ulcer patients and asymptomatic carriers as compared to uninfected individuals. Interestingly, the levels of immunosuppressive CTLA-4+ cells were significantly higher in the duodenum of duodenal ulcer patients, as compared to the asymptomatic carriers. H. pylori cause activation of T-cells in the duodenum as well as in the stomach. Our observation of higher levels of CTLA-4+ cells in the duodenum of duodenal ulcer patients than in the asymptomatic carriers suggests that a suppressive T-cell response may be related to the development of duodenal ulcers.

The capacity of an oral live attenuated Salmonella enterica serovar Typhi Ty21a vaccine to induce immune responses in patients who had undergone colectomies because of ulcerative colitis was evaluated, and these responses were compared with those of healthy volunteers. Purified CD4(+) and CD8(+) T cells from peripheral blood were stimulated in vitro by using the heat-killed Ty21a vaccine strain, and the proliferation and gamma interferon (IFN-gamma) production were measured before and 7 or 8 days after vaccination. Salmonella-specific immunoglobulin A (IgA) and IgG antibody responses in serum along with IgA antibody responses in ileostomy fluids from the patients who had undergone colectomies were also evaluated. Three doses of vaccine given 2 days apart failed to induce proliferative T-cell responses in all the six patients who had undergone colectomies, and increases in IFN-gamma production were found only among the CD8(+) cells from three of the patients. In contrast, both proliferative responses and increased IFN-gamma production were observed among CD4(+) and CD8(+) T cells from 3 and 6 of 10 healthy volunteers, respectively. Salmonella-specific IgA and/or IgG antibody responses in serum were observed for five (56%) of nine patients who had undergone colectomies and in 15 (88%) of 17 healthy volunteers. In ileostomy fluids, significant anti-Salmonella IgA antibody titer increases were detected in six (67%) of nine patients who had undergone colectomies. The impaired T- and B-cell immune responses found after vaccination in the circulation of patients who have undergone colectomies may be explained by a diminished colonization of the Ty21a vaccine strain due to the lack of a terminal ileum and colon.

Helicobacter pylori colonizes the gastric and duodenal mucosa. The infection normally persists for life and causes peptic ulcers and gastric cancer in a subset of infected individuals. We hypothesized that the inability to clear the infection may be a consequence of H. pylori-specific regulatory T cells that actively suppress T-cell responses. Therefore, we characterized the T-cell responses to H. pylori in H. pylori-infected individuals without any subjective symptoms and in uninfected control subjects and investigated the role of regulatory CD4+ CD25(high) T cells during infection. The stimulation of CD4+ peripheral blood T cells with monocyte-derived dendritic cells pulsed with a membrane preparation of H. pylori resulted in proliferation and gamma interferon production in both infected and uninfected individuals. Sorted memory cells from infected individuals responded less than cells from uninfected subjects, and the unresponsiveness could be abolished by depletion of CD4+ CD25(high) regulatory T cells or the addition of interleukin 2. Furthermore, CD4+ CD25(high) T cells suppressed H. pylori-induced responses in cocultures with CD25(low/-) cells. Tetanus toxoid induced comparable responses in memory cells from infected and uninfected individuals in both the presence and the absence of regulatory T cells, suggesting that the suppression was H. pylori specific. In conclusion, we have shown that H. pylori-infected individuals have impaired memory CD4+ T-cell responses to H. pylori that are linked to the presence of H. pylori-specific regulatory T cells that actively suppress the responses.

The mechanisms by which Helicobacter pylori escape the host immune response remains an important topic. Regulatory T cells appear to play a role in the persistence of the infection and the control of tissue damage. In the thought that the host genetic background influences the cross-talk between pathogens and hosts, the impact of cytokine polymorphisms on the outcome of H. pylori has been further delineated in the review period. On the other hand, several additional genes of H. pylori have been shown to participate in the pro-inflammatory cytokine and chemokine response to the infection. Finally, progress has been achieved in vaccine development, with new vaccine delivery systems and routes of immunization tested in animal models and human volunteers.

Helicobacter pylori colonizes the human stomach and areas of gastric metaplasia in the duodenum, but only a minority of those that are infected develop symptoms, e.g., peptic ulcers. Although most ulcers occur in the duodenum, almost all studies of mucosal immune responses against the infection have been limited to responses in the stomach. In the present study we evaluated whether there are differences in the levels of proinflammatory cytokines as well as immunoregulatory cytokines in the duodenal mucosa of duodenal ulcer (DU) patients and asymptomatic (AS) carriers which may be related to the development of duodenal ulcers. Duodenal biopsy specimens collected from normal mucosa as well as metaplastic mucosa of DU patients, AS carriers, and uninfected controls were analyzed for a number of cytokines by immunohistochemistry. Interestingly, the level of epithelial staining for several cytokines, e.g., interleukin-8 (IL-8), transforming growth factor beta (TGF-beta), and gamma interferon (IFN-gamma), was found to be significantly lower in DU patients than in AS carriers and uninfected individuals. No differences were observed when cytokine staining in normal and metaplastic biopsy specimens was compared. However, larger numbers of IL-8-, IL-6-, TGF-beta-, and IFN-gamma-positive mononuclear cells were observed in the duodenal lamina propria of both DU patients and AS carriers than in that of the uninfected controls. Our finding that a number of cytokines that may be important for the mucosal host defense against H. pylori are strongly decreased in the duodenal epithelium of ulcer patients suggests that a down-regulated immune response plays a role in the development of duodenal ulcers.

The protective effect of therapeutic oral immunization with homologous and heterologous formalin-inactivated Helicobacter pylori cells given together with cholera toxin as an adjuvant was evaluated with C57BL/6 mice infected with H. pylori Sydney strain 1 (SS1). The bacteria used for immunization were strains that were either homologous or heterologous with regard to the O antigen (i.e., the Lewis antigen [Le antigen]) expressed by the lipopolysaccharide of the infecting H. pylori SS1 strain. We found that repeated oral immunization with inactivated H. pylori SS1 cells can significantly inhibit an existing infection (P < 0.001) and that the protection induced by such therapeutic immunization extends to protection against reinfection (P < 0.001). A similar level of protection was also achieved by immunization with another inactivated H. pylori strain having the same O antigen (Le antigen) as the infecting H. pylori SS1 strain. In contrast, immunization with inactivated strains expressing a heterologous O antigen, Le(x), provided less protection or no protection. Immunization with H. pylori lysate preparations, on the other hand, resulted in significant comparable protection whether the lysates were prepared from an Le(x) strain or an Le(y) strain. Postimmunization gastritis was seen in mice that were protected after vaccination but not in unimmunized or unprotected mice. In conclusion, therapeutic immunization with inactivated H. pylori whole-cell vaccines may provide strong protection both against experimental H. pylori infection and against later reinfection.