Streptococcus thermophilus NCIMB 41856 ameliorates signs of colitis in an animal model of inflammatory bowel disease.

Treatment of inflammatory bowel disease (IBD) is mainly based on suppression of symptoms, often with numerous side effects. Trials of probiotics in IBD have frequently produced disappointing results. The majority of probiotics are unusual, since they do not require iron for growth, unlike many bacteria resident in the intestine. The IBD intestine is iron-rich due to bleeding and use of oral iron supplements; conventional probiotics would be rapidly outcompeted. We have evaluated an iron-responsive Streptococcus thermophilus strain for its potential to reduce signs of colitis. Efficacy of S. thermophilus was evaluated in the dextran sodium sulphate mouse model of colitis. Treated animals were given 1×108 cfu S. thermophilus per day and clinical observations were taken daily. At termination, gross and histopathological signs of disease, cellular infiltration, location of bacteria, and cytokine expression in the intestine were determined. S. thermophilus delayed onset of colitis and reduced clinical signs of disease, including bodyweight loss and gastrointestinal bleeding. It reduced bacterial translocation into the colonic tissue. Increased numbers of CD8+ intraepithelial lymphocytes were seen in control animals treated with S. thermophilus. S. thermophilus had no effect on gross pathology, histopathology or cytokine production in either colitic or control animals. We propose that S. thermophilus promotes maintenance of mucosal barrier function which reduces bacterial translocation, thereby reducing immune stimulation and associated inflammation. This allows mucosal healing, reducing gastrointestinal bleeding and weight loss. This could be studied as a locally-acting adjunct or alternative to current IBD treatments.

Body esteem, peer difficulties and perceptions of physical health in overweight and obese urban children aged 5 to 7 years.

OBJECTIVE: To determine whether there is an association between body mass index (BMI) and body esteem in young overweight and obese urban children, and to test peer relationship difficulties and perceived physical health as mediators of this relationship. METHODS: Child self-reported body esteem, and parent-reported child peer relationship difficulties (being bullied by peers and peer rejection) and physical health perceptions were obtained from 218 overweight and obese children aged 5-7 years (81% racial/ethnic minority, M BMI = 25.3) and their primary caregivers. RESULTS: Higher BMI was associated with lower body esteem for both girls and boys. This relation was mediated by poor physical health for boys but not for girls. Peer relationship difficulties did not mediate the observed association between BMI and body esteem in either group; however, girls with higher BMI experienced more bullying and being bullied by peers was associated with lower body esteem in girls. CONCLUSIONS: Intervening with perceptions of physical health may buffer overweight and obese boys from developing low body esteem in early childhood.

Polysaccharide-protein conjugate vaccination induces antibody production but not sustained B-cell memory in the human nasopharyngeal mucosa.

Colonization of the nasopharyngeal mucosa by meningococcus and other polysaccharide (PS)-encapsulated bacteria precedes invasion. PS-conjugate vaccines induce PS-specific B-cell memory (B(MEM)) and also prevent colonization, thus blocking person-to-person transmission, generating herd protection. However, in isolation the B(MEM) are unable to sustain immunity. Furthermore, the duration of herd protection the vaccines induce appears limited. We demonstrate that, despite the persistence of PS-specific B(MEM), the population is not maintained within the nasopharynx. Although booster immunization results in the transient appearance of PS-specific B(MEM) within the mucosa, this reflects the re-circulation of systemic B(MEM) through the site rather than the generation of resident mucosal B(MEM). The induction of sustained PS-specific B(MEM) in the nasopharynx would allow the population to be activated by colonization, thus inhibiting subsequent invasion. It would also be expected to boost local mucosal immunity, thus extending herd protection. Strategies to generate PS-specific B(MEM) in the mucosa warrant further investigation.

The Escherichia coli heat-labile enterotoxin B subunit protects from allergic airway disease development by inducing CD4+ regulatory T cells.

The B subunit of E. coli heat-labile enterotoxin (EtxB) protects against the development of T helper type 1 (Th1)-mediated autoimmune pathologies in mice. Protection was transferable with splenic CD4(+) T cells and was less effective following CD25 depletion; implying a T regulatory cell (Treg)-mediated process. We hypothesized that if this were the case, then EtxB would also control a Th2-mediated disorder. We tested the effect of EtxB treatment on asthma development in ovalbumin (OVA)-sensitized mice. EtxB treatment diminished eosinophilia in bronchoalveolar lavage samples, reduced OVA-specific immunoglobulin E and interleukin 4 production locally and systemically, and reduced airway hyper-reactivity. EtxB induced a dose-dependent increase in Foxp3(+)CD4(+) T cells, and adoptive transfer of splenic CD4(+) T cells partially suppressed lung pathology. Importantly, EtxB treatment increased OVA-specific CD4(+)Foxp3(+) T cells in the lung and systemically. These data demonstrate that EtxB modulates the differentiation of allergen-specific T cells causing inducible Treg induction and preventing disease.

Mucosal administration of the B subunit of E. coli heat-labile enterotoxin promotes the development of Foxp3-expressing regulatory T cells.

Understanding the processes by which certain mucosal pathogens and their products induce regulatory T cells (Tregs) is important in determining mechanisms of pathogenicity and may point toward their use in treating immunological disorders. Accordingly, we have studied the events that follow mucosal administration of the B subunit of E. coli heat-labile enterotoxin (EtxB). EtxB modulates the response to co-administered antigens and can prevent autoimmune disease. Our data show that EtxB translocates across the nasal epithelium, modulating the expression of interleukin-10 (IL-10) and transforming growth factor-ß(1) (TGF-ß(1)). The modulated microenvironment drives an increase in Forkhead box P3 (Foxp3)-positive T cells, predominantly in the CD4(+)CD25(-) subset. Adoptive transfer experiments showed that enhanced Foxp3 expression was particularly evident in recently activated T cells by concomitant unrelated antigen challenge, and was both TGF-ß(1) and IL-10 dependent. This ability to alter T-cell differentiation pathways following mucosal delivery explains how EtxB may modify mucosal immune environments and prevent unwanted pathologies.

IgA1 antibodies specific for outer membrane protein PorA modulate the interaction between Neisseria meningitidis and the epithelium.

Despite high carriage rates of Neisseria meningitidis, incidence of meningococcal disease remains low, partially due to development of natural immunity. We have previously demonstrated an inverse relationship between salivary anti-meningococcal IgA and disease incidence, but little is known about the contribution of IgA to immunity at mucosal surfaces. Here we show strong immunoreactivity by human salivary IgA against the meningococcal outer membrane porin, PorA. Monomeric anti-PorA IgA1 (humanized chimeric antibodies) but not IgG increased the association of unencapsulated serogroup B N. meningitidis (H44/76) with Chang (conjunctival) but not with either Detroit (pharyngeal) cells or with A549 (alveolar) epithelial cells. Association of encapsulated N. meningitidis was not increased. Epithelial binding of IgA was Fc fragment dependent and not inhibited by IgM. Together these data suggest the presence of a specific epithelial IgA receptor that could influence the effect of both naturally acquired and vaccine induced IgA antibodies at the epithelial surface.

Influence of age and carriage status on salivary IgA to Neisseria meningitidis.

Asymptomatic carriage of Neisseria meningitidis is common (5-35% of individuals) while the incidence of invasive meningococcal disease is fairly low (<1-5 per 100,000 per annum in Europe). Naturally acquired protective immunity may account for this difference. In this study, we investigated the relationship between anti-meningococcal salivary IgA and age and carriage. We showed that salivary IgA to a range of meningococcal antigens increased successively with age with some specificity for commonly circulating serosubtypes. In a group of 258 students 37 (14%) of whom were carriers of N. meningitidis serogroup B, higher levels of specific IgA were associated with carriage. Stratified analysis revealed a positive relationship between smoking and specific anti- N. meningitidis IgA independent of current carriage, weighted odds ratio (OR) 4.1 (95% CI 1.1-18) and OR 3.8 (95% CI 0.96-16) for reference strains B:1:P1.14 and B:4:P1.5,4 respectively. These data implicate IgA as a factor in host defence from meningococcal invasion, although the precise mechanisms remain uncertain.

Increased NF-kappaB DNA binding but not transcriptional activity during apoptosis induced by the COX-2-selective inhibitor NS-398 in colorectal carcinoma cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit colorectal neoplasia, an effect that is associated with their ability to induce apoptosis. Although NSAIDs have been reported to inhibit NF-kappaB, more recent studies show activation of NF-kappaB by NSAIDs. NF-kappaB commonly shows antiapoptotic activity and is implicated in the therapeutic resistance of cancer cells. The effects of highly COX-2-selective NSAIDs such as NS-398 on NF-kappaB in colorectal tumour cells have not been reported. Therefore, we addressed whether NF-kappaB has a role in NS-398-induced apoptosis of colorectal cancer cells. Treatment of HT-29 colorectal carcinoma cells with doses of NS-398 (50-75 microM) known to induce apoptosis had no effect on NF-kappaB for up to 48 h. However after 72 and 96 h NF-kappaB DNA-binding activity was increased by NS-398, in parallel with apoptosis induction. NS-398-treated HT-29 cells showed increased p50 homodimer binding and an induction of p50/p65 heterodimers, as demonstrated by supershift assay. However, although NS-398 increased NF-kappaB DNA binding it did not increase NF-kappaB-dependent reporter activity and inhibition of NF-kappaB DNA binding did not enhance NS-398-induced apoptosis. This indicates that NF-kappaB activated by NS-398 is transcriptionally inactive and is an encouraging result for the use of COX-2-selective NSAIDs not only in chemoprevention but also as novel therapies for colon cancer.

Protection against recurrent ocular herpes simplex virus type 1 disease after therapeutic vaccination of latently infected mice.

The potential of therapeutic vaccination of animals latently infected with herpes simplex virus type 1 (HSV-1) to enhance protective immunity to the virus and thereby reduce the incidence and severity of recurrent ocular disease was assessed in a mouse model. Mice latently infected with HSV-1 were vaccinated intranasally with a mixture of HSV-1 glycoproteins and recombinant Escherichia coli heat-labile enterotoxin B subunit (rEtxB) as an adjuvant. The systemic immune response induced was characterized by high levels of virus-specific immunoglobulin G1 (IgG1) in serum and very low levels of IgG2a. Mucosal immunity was demonstrated by high levels of IgA in eye and vaginal secretions. Proliferating T cells from lymph nodes of vaccinated animals produced higher levels of interleukin-10 (IL-10) than were produced by such cells from mock-vaccinated animals. This profile suggests that vaccination of latently infected mice modulates the Th1-dominated proinflammatory response usually induced upon infection. After reactivation of latent virus by UV irradiation, vaccinated mice showed reduced viral shedding in tears as well as a reduction in the incidence of recurrent herpetic corneal epithelial disease and stromal disease compared with mock-vaccinated mice. Moreover, vaccinated mice developing recurrent ocular disease showed less severe signs and a quicker recovery rate. Spread of virus to other areas close to the eye, such as the eyelid, was also significantly reduced. Encephalitis occurred in a small percentage (11%) of mock-vaccinated mice, but vaccinated animals were completely protected from such disease. The possible immune mechanisms involved in protection against recurrent ocular herpetic disease in therapeutically vaccinated animals are discussed.

MHC matching and mechanisms of alloactivation in corneal transplantation.

BACKGROUND: In human corneal transplantation the value of matching, particularly for MHC class II, is unclear and controversial. The contribution of the direct pathway to T cell activation is also uncertain. We have determined the relative contribution of class I, II and non-MHC antigens to graft rejection and of the direct and indirect pathways to T cell activation in a rat model mimicking human incompatibilities. METHODS: DA (RT1a) strain recipients received fully mismatched PVG (RT1c) strain grafts or grafts from one of three recombinant strains bearing DA MHC genes on a PVG background. Graft survival was assessed and the specificity of T cells generated in the draining lymph nodes was determined in mixed lymphocyte (MLR) proliferation assays. To assess the contribution of the direct pathway, fully mismatched graft were performed and allospecific proliferation was measured after depletion of recipient APC from the MLR reaction. RESULTS: There was no significant difference in survival of grafts between the four grades of mismatch, which ranged from a full mismatch to non-MHC mismatches alone (median survival 12.5, 11, 13 and 12.5 days respectively). In conformity with clinical results, strong secondary responses were generated against targets matched for MHC with the recipient. Depletion of recipient APC from a fully allogeneic secondary MLR did not fully abrogate donor-specific proliferation. CONCLUSIONS: Class II matching is of no benefit in this model. Strong indirect responses to non-MHC mismatches are sufficient to induce the rapid rejection, but the small numbers of class II+ cells in the donor appear sufficient to generate a direct response.

The genetic and immunopathological processes underlying collagen-induced arthritis.

Animal models of rheumatoid arthritis (RA) have provided substantial insights into basic pathogenic mechanisms of chronic inflammatory arthritis and autoimmune disease in general. Of the variety of models reported, collagen-induced arthritis (CIA) has been the most characterized in terms of both its pathogenesis and its underlying immunological basis. Collagen-induced arthritis has also been the model of choice in terms of testing potential new therapeutic agents for the treatment of human RA. Nevertheless, the complex nature of the balance between T-cell cytokines and the chronic inflammatory processes is only recently becoming clear. This review focuses on these developments, highlighting their implications for our understanding of RA and for the use of CIA as a suitable animal model.

Escherichia coli enterotoxin B subunit triggers apoptosis of CD8(+) T cells by activating transcription factor c-myc.

Heat-labile enterotoxin from enterotoxinogenic Escherichia coli is not only an important cause of diarrhea in humans and domestic animals but also possesses potent immunomodulatory properties. Recently, the nontoxic, receptor-binding B subunit of heat-labile enterotoxin (EtxB) was found to induce the selective death of CD8(+) T cells, suggesting that EtxB may trigger activation of proapoptotic signaling pathways. Here we show that EtxB treatment of CD8(+) T cells but not of CD4(+) T cells triggers the specific up-regulation of the transcription factor c-myc, implicated in the control of cell proliferation, differentiation, and death. A concomitant elevation in Myc protein levels was also evident, with peak expression occurring 4 h posttreatment. Preincubation with c-myc antisense oligodeoxynucleotides demonstrated that Myc expression was necessary for EtxB-mediated apoptosis. Myc activation was also associated with an increase of IkappaBalpha turnover, suggesting that elevated Myc expression may be dependent on NF-kappaB. When CD8(+) T cells were pretreated with inhibitors of IkappaBalpha turnover and NF-kappaB translocation, this resulted in a marked reduction in both EtxB-induced apoptosis and Myc expression. Further, a non-receptor-binding mutant of EtxB, EtxB(G33D), was shown to lack the capacity to activate Myc transcription. These findings provide further evidence that EtxB is a signaling molecule that triggers activation of transcription factors involved in cell survival.

A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity.

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Antigen-receptor cross-linking and lipopolysaccharide trigger distinct phosphoinositide 3-kinase-dependent pathways to NF-kappa B activation in primary B cells.

The NF-kappaB/Rel transcription factors play an important role in the expression of genes involved in B cell development, differentiation and function. Nuclear NF-kappaB is induced in B cells by engagement of either the BCR or CD40 or by stimulation with lipopolysaccharide (LPS). Despite the importance of NF-kappaB to B cell function, little is known about the signaling pathways leading to NF-kappaB activation. In this report we address the role of phosphoinositide 3'-kinase (PI 3-kinase) in BCR- and LPS-induced NF-kappaB activation using populations of primary murine resting B cells. Using the specific pharmacological inhibitors of PI 3-kinase, Wortmannin and LY294002, we demonstrate that PI 3-kinase activity is vital for BCR-induced NF-kappaB DNA-binding activity. Furthermore, we show that this is achieved via protein kinase C-dependent degradation of IkappaBalpha. Similar analyses reveal that PI 3-kinase is also critical in triggering NF-kappaB DNA-binding activity and IkappaBalpha degradation following LPS stimulation. Interestingly, a PKC inhibitor which blocked the BCR-induced IkappaBalpha degradation had no effect on the degradation of IkappaBalpha after LPS stimulation. Taken together, our results indicate the involvement of PI 3-kinase in at least two distinct signaling pathways leading to activation of NF-kappaB in B cells.

Immunomodulation using bacterial enterotoxins.

Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination by a mucosal route can effectively induce immune suppression. However, some bacterial-derived proteins, e.g. cholera toxin and the heat labile toxin of Escherichia coli, are immunogenic and immunomodulatory at mucosal surfaces and can effectively adjuvant immune responses to codelivered bystander antigens. This review summarizes some of the structural and biological characteristics of these toxins and provides examples of how these properties have been exploited for tolerance induction and mucosal vaccine development.

Protective mucosal immunity to ocular herpes simplex virus type 1 infection in mice by using Escherichia coli heat-labile enterotoxin B subunit as an adjuvant.

The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 microg of rEtxB or above with 10 microg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 microg) with a trace (0.5 microg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 microg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.

Immune modulation by the cholera-like enterotoxin B-subunits: from adjuvant to immunotherapeutic.

Cholera toxin (Ctx) and its close relative, Escherichia coli heat-labile enterotoxin (Etx) have long been established as potent mucosal and systemic adjuvants. Problems arising from their inherent toxicity have, however, precluded human use. Here we describe findings which demonstrate that contrary to the established dogma the non-toxic B-subunit of Etx (EtxB) is a highly potent mucosal adjuvant capable of potentiating protective immunity to viral infection. The mechanisms which underlie this activity arise from an ability to trigger specific signaling processes in lymphocyte populations which modulate differentially their activation, differentiation and survival. The elucidation of these properties has led to the further use of EtxB as an agent capable of preventing the establishment of autoimmune diseases. The basis for these activities and their potential applicability to human therapies are discussed.

An amphioxus Emx homeobox gene reveals duplication during vertebrate evolution.

Members of the Emx homeobox gene class are expressed during embryogenesis in the brain and/or other head structures of phylogenetically diverse phyla. Here, we describe sequence, genomic structure, and molecular phylogenetic analysis of a cephalochordate (amphioxus) Emx class gene termed AmphiEmxA. The genomic structure of AmphiEmxA is very similar to that of vertebrate Emx genes, with two conserved intron sites. The Drosophila homolog empty spiracles (ems) has just one intron, which may be shared with chordates; the other has been secondarily lost in this Drosophila gene and in a cnidarian Emx-related gene. We identify a highly conserved peptide motif close to the amino terminus of Emx proteins, demonstrate its similarity to a sequence found in a variety of transcription factors, and argue that it arose through convergent evolution in homeobox and forkhead genes. Finally, our molecular phylogenetic analysis strongly supports the presence of a single Emx gene in the ancestor of chordates and gene duplication along the vertebrate lineage.