IL-22-induced antimicrobial peptides are key determinants of mucosal vaccine-induced protection against H. pylori in mice.

Despite the recent description of the mucosal vaccine-induced reduction of Helicobacter pylori natural infection in a phase 3 clinical trial, the absence of immune correlates of protection slows the final development of the vaccine. In this study, we evaluated the role of interleukin (IL)-22 in mucosal vaccine-induced protection. Gastric IL-22 levels were increased in mice intranasally immunized with urease+cholera toxin and challenged with H. felis, as compared with controls. Flow cytometry analysis showed that a peak of CD4+IL-22+IL-17+ T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice. The inhibition of the IL-22 biological activity prevented the vaccine-induced reduction of H. pylori infection. Remarkably, anti-microbial peptides (AMPs) extracted from the stomachs of vaccinated mice, but not from the stomachs of non-immunized or immunized mice, injected with anti-IL-22 antibodies efficiently killed H. pylori in vitro. Finally, H. pylori infection in vaccinated RegIIIß-deficient mice was not reduced as efficiently as in wild-type mice. These results demonstrate that IL-22 has a critical role in vaccine-induced protection, by promoting the expression of AMPs, such as RegIIIß, capable of killing Helicobacter. Therefore, it can be concluded that urease-specific memory Th17/Th22 cells could constitute immune correlates of vaccine protection in humans.

Tollip, an early regulator of the acute inflammatory response in the substantia nigra.

BACKGROUND: Tollip is a ubiquitously expressed protein, originally described as a modulator of the IL-1R/TLR-NF-κB signaling pathways. Although this property has been well characterized in peripheral cells, and despite some evidence of its expression in the central nervous system, the role of Tollip in neuroinflammation remains poorly understood. The present study sought to explore the implication of Tollip in inflammation in the substantia nigra pars compacta, the structure affected in Parkinson's disease. METHODS: We first investigated Tollip distribution in the midbrain by immunohistochemistry. Then, we addressed TLR4-mediated response by intra-nigral injections of lipopolysaccharide (LPS), a TLR4 agonist, on inflammatory markers in Tollip knockout (KO) and wild-type (WT) mice. RESULTS: We report an unexpectedly high Tollip immunostaining in dopaminergic neurons of the mice brain. Second, intra-nigral injection of LPS led to increased susceptibility to neuroinflammation in Tollip KO compared to Tollip WT mice. This was demonstrated by a significant increase of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), and interferon gamma (IFN-γ) messenger RNA (mRNA) in the midbrain of Tollip KO mice upon LPS injection. Consistently, brain rAAV viral vector transduction with a nuclear factor kappa B (NF-κB)-inducible reporter gene confirmed increased NF-κB activation in Tollip KO mice. Lastly, Tollip KO mice displayed higher inducible NO synthase (iNOS) production, both at the messenger and protein level when compared to LPS-injected WT mice. Tollip deletion also aggravated LPS-induced oxidative and nitrosative damages, as indicated by an increase of 8-oxo-2'-deoxyguanosine and nitrotyrosine immunostaining, respectively. CONCLUSIONS: Altogether, these findings highlight a critical role of Tollip in the early phase of TLR4-mediated neuroinflammation. As brain inflammation is known to contribute to Parkinson's disease, Tollip may be a potential target for neuroprotection.

[An update on celiac disease]. / Maladie coeliaque: état des lieux.

Celiac disease is a well-known entity in pediatrics and pediatric gastroenterology that is now also frequently encountered in the adult population. Apart from typical symptoms, celiac disease can present with a wide range of manifestations that are sometimes atypical, scarce or purely extraintestinal. Serologic and genetic testing are useful tools in case of low clinical probability in the early diagnostic algorithm. Upper gastrointestinal endoscopy remains the mainstay to confirm the diagnosis especially in atypical clinical presentations. Complications are rare but can be severe. Although gluten-free diet often leads to complete recovery, compliance is not universal and alternative treatment strategies are under investigation.

Macrophages promote epithelial repair through hepatocyte growth factor secretion.

Macrophages play a critical role in intestinal wound repair. However, the mechanisms of macrophage-assisted wound repair remain poorly understood. We aimed to characterize more clearly the repair activities of murine and human macrophages. Murine macrophages were differentiated from bone marrow cells and human macrophages from monocytes isolated from peripheral blood mononuclear cells of healthy donors (HD) or Crohn's disease (CD) patients or isolated from the intestinal mucosa of HD. In-vitro models were used to study the repair activities of macrophages. We found that murine and human macrophages were both able to promote epithelial repair in vitro. This function was mainly cell contact-independent and relied upon the production of soluble factors such as the hepatocyte growth factor (HGF). Indeed, HGF-silenced macrophages were less capable of promoting epithelial repair than control macrophages. Remarkably, macrophages from CD patients produced less HGF than their HD counterparts (HGF level: 84 ± 27 pg/mg of protein and 45 ± 34 pg/mg of protein, respectively, for HD and CD macrophages, P < 0·009) and were deficient in promoting epithelial repair (repairing activity: 90·1 ± 4·6 and 75·8 ± 8·3, respectively, for HD and CD macrophages, P < 0·0005). In conclusion, we provide evidence that macrophages act on wounded epithelial cells to promote epithelial repair through the secretion of HGF. The deficiency of CD macrophages to secrete HGF and to promote epithelial repair might contribute to the impaired intestinal mucosal healing in CD patients.

Low dose endoluminal photodynamic therapy improves murine T cell-mediated colitis.

BACKGROUND AND STUDY AIMS: Low dose photodynamic therapy (LDPDT) may modify the mucosal immune response and may thus provide a therapy for Crohn's disease. We evaluated the efficacy and safety of this technique in a murine T cell-mediated colitis model. METHODS: The safety of LDPDT was first tested in BALB/c mice. Naïve T cells were used to induce colitis in mice with severe combined immunodeficiency, which were followed up endoscopically, and a murine endoscopic index of colitis (MEIC) was developed. The efficacy of LDPDT (10 J/cm (2); delta-aminolevulinic acid, 15 mg/kg bodyweight) was then tested on mice with moderate colitis, while a disease control group received no treatment. The MEIC, weight, length, and histology of the colon, cytokine expression indices, number of mucosal CD4 (+) T cells, percentage of apoptotic CD4 (+) T cells, body weight, and systemic side effects were evaluated. RESULTS: LDPDT improved the MEIC ( P = 0.011) and the histological score ( P = 0.025), diminished the expression indices of the proinflammatory cytokines, interleukin-6 ( P = 0.042), interleukin-17 ( P = 0.029), and interferon-gamma ( P = 0.014), decreased the number of mucosal CD4 (+) T cells, and increased the percentage of apoptotic CD4 (+) T cells compared with the disease control group. No local or systemic side effects occurred. CONCLUSION: LDPDT improves murine T cell-mediated colitis, decreases the proinflammatory cytokines interleukin-6, interleukin-17, and interferon-gamma, and decreases the number of CD4 (+) T cells. No adverse events were observed. Therefore, this technique is now being evaluated in patients with inflammatory bowel disease.

Synthesis, refolding and protective immune responses of a potential antigen for human Respiratory Syncytial Virus vaccine.

The design of new antigens with both high immunogenic and safety properties is of particular interest to vaccine against infectious diseases. In the present study, we describe the synthesis and the refolding of peptide G20 derived from the Human Respiratory Syncytial Virus (hRSV) G-protein. G20 (MEF G140-190 G144-158) is a peptide of 69 amino acids with two disulfide bridges, which comprises multiple protective B-cell epitopes. It was deleted of the T helper cell epitope 184-198 of the RSV G-protein, which was found to induce pulmonary pathology after RSV challenge in mice. Interestingly, we showed in the present study that G20 generated a highly protective antibody response against RSV challenge in Balb/c mice. Therefore, G20 represents a new potential antigen for an RSV vaccine.

Lipophilic quaternary ammonium salt acts as a mucosal adjuvant when co-administered by the nasal route with vaccine antigens.

Nasal administration of vaccines is an attractive approach which offers several significant advantages over traditional intramuscular vaccine delivery. These advantages include easier administration and induction of immune responses in the mucosal secretions of the body. In this study we describe a new potent nasal adjuvant, dimethyldioctadecylammonium bromide (DDA), that induces both mucosal and systemic immune responses when co-administered with diphtheria toxoid (DT), tetanus toxoid (TT) and BBG2Na antigens. In particular, we show that the nasal delivery of recombinant fragment (BBG2Na) of the G protein of respiratory syncytial virus (RSV) mixed with DDA induces both local and systemic anti-RSV immune responses and protects against viral challenge. Furthermore, we provide evidence that the DDA+BBG2Na vaccine does not induce lung immunopathology upon subsequent RSV challenge.

BBG2Na an RSV subunit vaccine candidate intramuscularly injected to human confers protection against viral challenge after nasal immunization in mice.

Respiratory syncytial virus (RSV) is a major respiratory pathogen responsible for severe pulmonary disease. We have developed a parenterally administered vaccine, BBG2Na, which is currently in a phase III clinical trial. BBG2Na comprises residues 130--230 of RSV-A G protein (G2Na) fused to the BB carrier protein. In this study, we show that BBG2Na can be delivered by the nasal route and generates both mucosal and systemic antibody responses when co-administered with cholera toxin B or a newly described delivery system, zwittergent 3--14. We found that nasal BBG2Na administration protects against RSV challenge and does not induce lung immunopathology upon subsequent RSV challenge.

Delivery systems and adjuvants for vaccination against HIV.

Epidemiological studies have revealed that HIV-1 infections occur through contact with contaminated blood or during unprotected vaginal or anal intercourse. Hence, to protect against HIV infection, vaccines should ideally induce both mucosal and systemic immune responses. We present a brief review of the different delivery systems and adjuvants which can be used to elicit mucosal immune responses. Oral or nasal administration of recombinant attenuated bacteria or viruses can induce both mucosal and systemic immune responses against the carried antigen. The oral delivery of mucosal adjuvants (such as cholera toxin) in association with antigens has been shown to enhance mucosal and systemic immune responses against them. Recently developed vaccination strategies using naked DNA or other antigen delivery systems are also discussed.

Systemic antibodies can inhibit mouse mammary tumor virus-driven superantigen response in mucosa-associated lymphoid tissues.

Many mucosal pathogens invade the host by initially infecting the organized mucosa-associated lymphoid tissue (o-MALT) such as Peyer's patches or nasal cavity-associated lymphoid tissue (NALT) before spreading systemically. There is no clear demonstration that serum antibodies can prevent infections in o-MALT. We have tested this possibility by using the mouse mammary tumor virus (MMTV) as a model system. In peripheral lymph nodes or in Peyer's patches or NALT, MMTV initially infects B lymphocytes, which as a consequence express a superantigen (SAg) activity. The SAg molecule induces the local activation of a subset of T cells within 6 days after MMTV infection. We report that similar levels of anti-SAg antibody (immunoglobulin G) in serum were potent inhibitors of the SAg-induced T-cell response both in peripheral lymph nodes and in Peyer's patches or NALT. This result clearly demonstrates that systemic antibodies can gain access to Peyer's patches or NALT.

Delivery systems and adjuvants for vaccination against HIV.

Epidemiologic studies have revealed that HIV-1 infections occur through contact with contaminated blood or during unprotected vaginal or anal intercourse. Hence, to protect against HIV infection, vaccines should induce both mucosal and systemic immune responses. We present a brief review of the different delivery systems and adjuvants which can be used to elicit mucosal immune responses. Oral or nasal administration of recombinant Salmonella vaccines can induce both mucosal and systemic immune responses against the carried antigen. The oral delivery of mucosal adjuvants (such as cholera toxin) in association with antigens has been shown to enhance mucosal and systemic immune responses against them. Recently developed vaccination strategies using naked DNA or recombinant adenovirus are also discussed.

The nasal-associated lymphoid tissue of adult mice acts as an entry site for the mouse mammary tumor retrovirus.

Mouse mammary tumor virus (MMTV) is a B type retrovirus transmitted to the suckling offspring through milk. MMTV crosses the intestinal barrier of neonates, initially infects the lymphoid cells of the Peyer's patches, and later spreads to all lymphoid organs and to the mammary gland. Adult mice can be infected systemically, but not by oral MMTV administration. In this study, we show that nasal administration of infected milk induces the infection of adult mice. Nasal MMTV infection shared the main features of systemic and neonatal intestinal MMTV infections: deletion of the superantigen (SAg)-reactive T cell subset from the peripheral T cell population, presence of viral DNA in lymphoid cells, and transmission of MMTV from mother to offspring. Viral DNA was restricted to the lungs and nasal-associated lymphoid tissue (NALT) 6 d after nasal infection. Furthermore, SAg-induced T cell proliferation was only detected in NALT. These results demonstrate that MMTV crosses the intact epithelium of the upper respiratory tract of adult mice and infects the lymphoid follicles associated with these structures.

The neonatal Fc receptor is not required for mucosal infection by mouse mammary tumor virus.

The milk-borne mouse mammary tumor virus (MMTV) infects newborn mice via the intestine. Infection is initially restricted to Peyer's patches and later spreads to the epithelial cells of the mammary gland. The receptor that mediates uptake and transport of MMTV across the intestinal barrier has not yet been identified, The neonatal Fc receptor (nFcR), which is expressed by enterocytes during the first two weeks of life, is downregulated at weaning, and its disappearance correlates with the onset of intestinal resistance to MMTV. To test whether the nFcR mediates transport and allows infection, we foster nursed on infected MMTV mothers beta2 microglobulin-deficient (beta2m-deficient) newborn mice that are unable to express the nFcR at the surface of their enterocytes. Exposure of beta2m-deficient mice to milk-borne virus resulted in the deletion of peripheral blood T cells reactive to the superantigen encoded by MMTV. Since beta2m-deficient newborn mice are susceptible to MMTV infection despite the lack of the nFcR, we conclude that the nFcR is not required for MMTV transport.

Serological abnormalities induced by engraftment of viable motheaten hematopoietic cells in nude beige recipients.

C57BL/6J (B6) mice homozygous for the viable motheaten (mev) mutation are short-lived and display severe immunodeficiency, autoimmunity and inflammatory disease. B6 mice doubly homozygous for the nude (nu) and beige (bg) mutations (nubg mice) are also short-lived and immunodeficient. Nevertheless, grafts of mev lympho-hematopoietic cells increased life expectancy of nubg recipients. Such [mev --> nubg] chimeras did not develop any mev-like inflammatory pathology but showed autoimmunity features, particularly hyperglobulinemia which, unlike the mev one, was due to IgG rather than IgM. Serological studies of [mev IgHb --> nubg Igha] chimeras surprisingly revealed the exclusive host B-cell origin of the IgG2a overproduced by these chimeras. Yet, about half of such chimera serum IgM being IgMb, mev B cells had actually engrafted the nubg hosts. Together with the lack of transfer of the inflammatory pathology, this suggests that a non-mev environment might succeed acting as a regulator of some mev-induced dysfunctions.

gld and lpr hematopoietic cell transfers: common and different serological features of the C57BL/6 chimeras.

Homozygosity for either the lpr (lymphoproliferation) or the gld (generalized lymphoproliferative disease) mutation in mice causes the development of strikingly similar hyperglobulinemia and lymphoproliferative syndromes. Nevertheless, previous studies of various C57BL/6 chimeras obtained by reconstitution of irradiated recipients with hematopoietic cells (HC), differing at the bg, gld, lpr, and/or nu loci, showed that the lpr and gld syndromes had distinct etiologies. The [lpr-->lpr], [gld-->gld], and [gld-->wild] chimeras developed lymphoid hyperplasia, while the [lpr-->wild, bg, or gld] and [nulpr-->wild or bg] chimeras developed a severe persistent lymphoid aplasia. We now show that the serological status (immunoglobulin (Ig) levels and Ig isotype distribution) of the [lpr-->lpr], [gld-->gld], and [gld-->wild] chimeras were roughly equivalent to those of genetic lpr and gld mice. Despite their lymphoid aplasia, all the [lpr-->non-lpr] chimeras displayed surprisingly normal serum Ig levels, similar to [wild-->wild] control chimeras, although always with some abnormal isotype profile. In fact, an early but transient increase of serum IgG1 levels was found in all [lpr-->wild, bg, or gld], [lpr-->lpr], [nulpr-->wild or bg], [wild-->lpr], and [gld-->wild or gld] types of chimeras. Despite a common early behavior, the host type and/or the gld or lpr HC origin may cause later divergences of the gld or lpr HC grafted chimeras.

Different nature of the proliferation defects of GLD, LPR and MEV C57BL/6 mouse lymphoid cells.

The three non-allelic gld, lpr and mev mutations in the mouse all lead to profound immunodeficiency besides a splenomegaly and a generalized autoimmunity. Spleen cells from young B6 gld, B6 lpr and B6 mev mice all display a decreased proliferative response to the T-cell mitogen concanavalin A (ConA), but the nature of the deficiency seems very different. No restoration of proliferation could be obtained by adding exogenous recombinant rIL2 to ConA-treated mev spleen cells, this lack of IL2-responsiveness suggesting a lack of (functional) IL2-receptors. In young mice of both gld and lpr strains, a B6 wild-type level of proliferation could be reached by rIl2 addition to ConA-treated spleen cells, this normal responsiveness to exogenous IL2 suggesting a normal expression of IL2-receptors. The endogenous IL2 production by ConA-treated spleen cells decreased very much with ageing in both B6 gld and B6 lpr mice. Yet, IL2 production in young mice revealed an earlier deficiency of the B6 lpr mice: the young B6 gld IL2 levels reached about 60% of age-matched B6 wild cell levels, but the B6 lpr levels reached 14% only. Finally the addition of exogenous rIL2 to ConA-pretreated cells from old B6 gld and B6 lpr mice, while enhancing the proliferative responses, could not restore the B6 wild-type levels. This suggests that, with ageing, the expression of functional IL2-receptors may become as abnormal in these gld and lpr mutants as it is from birth in the mev mutant mice.

Adoptive transfer of viable motheaten (mev) humoral autoimmunity: IgM to IgG switch of the hyperglobulinaemia in nude, beige recipient mice.

Homozygous C57BL/6 nude, beige mice (B6 nu,bg) were used as recipients for the transfer of lymphoid cells from autoimmune homozygous B6 'viable motheaten' mice (B6 mev) and from either normal B6 mice (B6 wild) or B6 bg mice as controls. Surprisingly, the mev cell grafts prolonged survival of these short-living doubly immunodeficient recipients. Although the [mev----nu,bg] chimeras did not develop the mev external necrosis phenotype, they showed a hyperglobulinaemia and a significant increase of their anti-single-stranded DNA (ssDNA) antibody titres, compared to control chimeras ([bg----nu,bg] and [wild----nu,bg]). However, this hyperglobulinaemia was quite different from the mev-type hyperglobulinaemia, with poor contribution of the IgM isotype. Moreover, the anti-ssDNA antibodies were more distributed among the various Ig classes than the anti-ssDNA antibodies of the mev homozygous mice. Though the adoptive transfer of some mev-type humoral autoimmunity symptoms were achieved in this chimera model, the recipient mice did not suffer from the several other features of the mev syndrom, such as the severe pathology and the extremely high IgM serological levels.

Adoptive transfer of viable motheaten humoral autoimmunity in cyclophosphamide-immunodepressed beige recipient mice.

Cyclophosphamide-pretreated homozygous C57BL/6 beige mice (B6 bg) were used as recipients for the transfer of lymphoid cells either of short-living autoimmune homozygous B6 'viable motheaten' mice (B6 mev) or of normal B6 mice (B6+) or B6 bg mice as controls. The grafts had no incidence on the survival of the recipients, whatever protocol used. The [mev----bg] chimeras did not develop the mev external phenotype, but there was a transfer of humoral autoimmunity. Compared to control Compared to control chimeras ([bg----bg] and [+----bg]), recipients of mev cells always showed an increase in anti-single-stranded DNA (ssDNA) antibody titres, reaching 2/3 of the mev ones 40 weeks after the cell transfers. Moreover, the anti-ssDNA were mainly of IgM class, correlating with the higher total IgM level found in [mev----bg] chimeras, thus reflecting the serological phenotype of the mev homozygous mice. Though the adoptive transfer of some mev-type humoral autoimmunity symptoms was clearly achieved in this chimera model, the recipient mice did not suffer from the several other features of the mev syndrome, such as the hyperglobulinemia and the severe pathology. This indicates that microenvironmental influences act in concert with B cells to produce pathology in mev mice.

Enterotoxin production by Yersinia enterocolitica in food samples.

Milk and cold meat samples were contaminated with 7 various serogroups of Yersinia enterocolitica strains. The infected food samples were incubated under different conditions of growth, at different temperatures and for different periods of time, then the number of colony forming units was determined and enterotoxin production was assayed by the suckling mice test. The Y. enterocolitica strains multiplied well under varying conditions of growth, but enterotoxin production could be detected only in the meat samples when incubated under shaking at 25 degrees C for 48 h. It may be assumed that performed yersinia enterotoxin is absent from food stored under normal conditions.

The stability of enterotoxin production in Yersinia enterocolitica and the methanol solubility of heat-stable enterotoxin.

Twenty five strains of Yersinia enterocolitica serogroup O3, were isolated from human enteritis and studied for heat-stable enterotoxin production. Enterotoxin production was found even in the crude supernatant fluid of cultures that had been stored in stock agar for a year. According to the suckling mice and rabbit gut loop tests, after 1 to 5 years storage the filtrate showed heat-stable enterotoxin activity only in a purified and concentrated form. Following more than 5 years storage positive results could be obtained only in rabbit gut loop test. After 9 years the freeze dried strains still showed a full capacity of heat-stable enterotoxin production. Studies with concentrated substances showed that even after more than 9 years, there was no spontaneous loss of heat-stable enterotoxin production, only quantitative changes occurred. The methanol solubility of the heat-stable enterotoxin of Y. enterocolitica is--as distinct from the heat-stable enterotoxin of Escherichia coli--homogeneous and only the methanol soluble fractions showed any activity. The activity of methanol soluble enterotoxin from several years old subcultures could be demonstrated in an isolated rabbit gut loop model even when it failed to show any activity in suckling mice.