Immunization with attenuated Salmonella typhimurium producing catalase in protection against gastric Helicobacter pylori infection in mice

Aim. To evaluate the protective effect of live attenuated Salmonella typhimurium expressing catalase against gastric Helicobacter pylori infection in mice, and to explore the underlying mechanisms of the protective immune reaction. Materials and Methods The H. pylori catalase gene was introduced into attenuated S. typhimurium strain SL3261. C₅₇BL/6 mice were orally immunized with the SL3261 vaccine strain expressing catalase or with SL3261 alone or phosphate‐buffered saline (PBS). Mice were sacrificed 4 weeks after immunization and 5 weeks after H. pylori challenge, respectively. Results. All PBS control mice were infected. Eight of 13 (61.5%) mice immunized with the SL3261 vaccine strain and three of 14 (21%) mice immunized with SL3261 alone showed protection against H. pylori infection. Serum anti‐H. pylori IgG2a levels of S. typhimurium‐immunized mice were higher than those of PBS controls, both before and after H. pylori challenge, while there were no differences for IgG1 and IgA. Similarly, mRNA expression of interleukin (IL)‐2, IL‐12 and interferon‐γ in the gastric mucosa of S. typhimurium‐immunized mice was significantly higher than that of PBS controls both before and after challenge. Moreover, S. typhimurium‐immunized mice were characterized by marked infiltration of lymphocyte and mononuclear cells in the gastric mucosa after challenge. IL‐4 and IL‐10 were not detected in any of the three groups. IL‐6 expression was increased in the PBS group compared with the S. typhimurium‐immunized groups after challenge. Conclusions. This study demonstrates that oral immunization of mice with catalase delivered by an attenuated S. typhimurium strain offers protection against H. pylori infection. This protective immunity was mediated through a predominantly Th1‐type response and was associated with post‐immunization gastritis.     

IL‐17 is Involved in Helicobacter pylori‐Induced Gastric Inflammatory Responses in a Mouse Model

Background: Helicobacter pylori (H. pylori) is the major cause of chronic active gastritis and peptic ulcer disease. Recent studies have shown that H. pylori produces various cytokines that are related to neutrophil or mononuclear cell accumulation. Interleukin‐17 (IL‐17) is the founding member of an emerging family of inflammatory cytokines whose biological activities remain incompletely defined. In this study, the contributions of IL‐17 to the induction of gastric inflammation and to the protection from H. pylori infection were investigated using IL‐17 gene‐knockout (IL‐17^?/–) mice. Materials and Methods: IL‐17^?/–and wild‐type C57BL/6 mice were challenged with H. pylori CPY2052 (2 × 10⁸ CFU/mL) and the histological and microbiological evaluation were carried out at specified times. IL‐17 and myeloperoxidase (MPO) protein levels in tissues were assayed in duplicate using ELISA kits. Results: In wild‐type mice, IL‐17 was undetected at baseline; however, the protein expression of IL‐17 was induced after infection with H. pylori. A severe infiltration of neutrophils appeared in the submucosa and the lamina propria in wild‐type mice. In contrast, the degree of neutrophil infiltration in IL‐17^?/– mice was significantly lower than that in wild‐type mice. Although wild‐type mice infected with H. pylori showed drastically higher MPO activity compared with uninfected wild‐type mice, any significant increase in the enzyme activity was not revealed in infected IL‐17^?/– mice. The number of H. pylori colonized in the stomach of IL‐17^?/– mice was significantly lower than that of wild‐type mice from 1 to 6 months after infection. Conclusions: These results suggest that IL‐17 may play an important role in the inflammatory response to the H. pylori infection and ultimately influence the outcome of the H. pylori‐associated disease.     

Helicobacter pylori modulation of gastric and duodenal mucosal T cell cytokine secretions in children compared with adults

Background. In contrast to adults, ulcers are un‐common in Helicobacter pylori‐infected children. Since immunological determinants influence the outcome of H. pylori infection, we have investigated mucosal T cell responses in H. pylori‐infected children and compared them with those of adults and negative controls. Material and Methods. Mucosal biopsies were obtained from 43 patients undergoing an upper GI endoscopy for dyspeptic symptoms. The concentrations of released cytokines and the density of CD3⁺, CD25⁺ and CD69⁺cells were evaluated by flow cytometry, and the numbers of cytokine‐secreting cells were measured by ELISPOT. Results. The numbers of isolated antral CD3⁺ lymphocytes were only significantly raised in infected adults compared with noninfected controls (p < 0.05), whereas the proportion of CD3⁺ cells expressing activation markers (CD25 or CD69) remained low. In the stomach, IFN‐γ concentrations increased in infected children and infected adults compared with controls (p < 0.05), but IFN‐γ concentrations were tenfold lower in children than in adults (p < 0.01). IL‐2, IL‐4, IL‐10 and TNF‐α concentrations were similar in infected and in uninfected children and adults. In contrast, in the duodenum, IFN‐γ, as well as IL‐4 and IL‐10 concentrations were only increased in infected children compared with controls (p < 0.05). The concentrations of these cytokines were similar in both groups of adults who, however, like children, displayed a higher number of duodenal IL‐4‐secreting cells compared to controls (p < 0.05). Conclusion. These results suggest that IFN‐γ secretion in the stomach of H. pylori‐infected patients is lower in children than in adults. This could protect children from development of severe gastro‐duodenal diseases such as ulcer disease. In addition, infected patients are characterised by a dysregulation of the mucosal cytokine secretion at distance from the infection site.     

Helicobacter pylori-pulsed dendritic cells induce H. pylori-specific immunity in mice

Background: The growing concern over the emergence of antibiotic‐resistant Helicobacter pylori infection is propelling the development of an efficacious vaccine to control this highly adaptive organism. Aim: We studied the use of a dendritic cell (DC)‐based vaccine against H. pylori infection in mice. Methods: The cellular immune responses to murine bone marrow‐derived DCs pulsed with phosphate‐buffered saline (PBS‐DC) or live H. pylori SS1 (HP‐DC) were assessed in vitro and in vivo. The protective immunity against H. pylori SS1 oral challenge was compared between HP‐DC or PBS‐DC immunized mice. The effect of regulatory T‐cell (Treg) depletion by anti‐CD25 antibody on HP‐DC vaccine efficacy was also evaluated. Results: HP‐DC induced a Th1‐dominant response in vitro. In vivo, HP‐DC immunized mice were characterized by a mixed Th1/Th2 peripheral immune response. However, in the stomach, HP‐DC immunized mice expressed a higher level of IFN‐γ compared to PBS‐DC immunized mice; no difference was found for interleukin‐5 expressions in the stomach. A lower bacterial colonization post‐H. pylori challenge was observed in HP‐DC immunized mice compared to PBS‐DC immunized mice with no significant difference in gastritis severity. H. pylori‐specific Th1 response and protective immunity were further enhanced in vivo by depletion of Treg with anti‐CD25 antibody. Conclusion: DC‐based anti‐H. pylori vaccine induced H. pylori‐specific helper T‐cell responses capable of limiting bacterial colonization. Our data support the critical role of effector cellular immune response in the development of H. pylori vaccine.     

Activities of muscadine grape skin and quercetin against Helicobacter pylori infection in mice

Aims: To explore the preventative potential of muscadine grape skin (MGS) and the single flavonoid, quercetin, as an alternative means for ameliorating Helicobacter pylori infection and/or the H. pylori‐induced inflammatory response in mice. Methods and Results: The antimicrobial and anti‐inflammatory properties of MGS and quercetin, a major phenolic constituent, were evaluated against H. pylori in vitro and in vivo. The antimicrobial activity of quercetin was evaluated against 11 H. pylori strains in vitro with inhibition of all strains at 128–64 μg ml^?1. In vivo studies showed a moderate reduction in H. pylori counts following treatment with 5 and 10% MGS or quercetin (25 mg kg^?1 body weight) in addition to significantly reduced inflammatory cytokines (TNF‐α, IL‐1β and IFN‐γ) when compared with untreated mice. Conclusions: MGS and quercetin did not significantly reduce H. pylori growth in a mouse model. However, these products were effective in regulating the inflammatory response to H. pylori infection. Significance and Impact of the Study: Our results suggest that H. pylori infection may be reduced or prevented via the consumption of fruits rich in certain phenolic compounds (e.g. quercetin) such as muscadine grapes.     

NOD1 is required for Helicobacter pylori induction of IL‐33 responses in gastric epithelial cells

Helicobacter pylori (H. pylori) causes chronic inflammation which is a key precursor to gastric carcinogenesis. It has been suggested that H. pylori may limit this immunopathology by inducing the production of interleukin 33 (IL‐33) in gastric epithelial cells, thus promoting T helper 2 immune responses. The molecular mechanism underlying IL‐33 production in response to H. pylori infection, however, remains unknown. In this study, we demonstrate that H. pylori activates signalling via the pathogen recognition molecule Nucleotide‐Binding Oligomerisation Domain‐Containing Protein 1 (NOD1) and its adaptor protein receptor‐interacting serine–threonine Kinase 2, to promote production of both full‐length and processed IL‐33 in gastric epithelial cells. Furthermore, IL‐33 responses were dependent on the actions of the H. pylori Type IV secretion system, required for activation of the NOD1 pathway, as well as on the Type IV secretion system effector protein, CagA. Importantly, Nod1^+/+ mice with chronic H. pylori infection exhibited significantly increased gastric IL‐33 and splenic IL‐13 responses, but decreased IFN‐γ responses, when compared with Nod1^?/? animals. Collectively, our data identify NOD1 as an important regulator of mucosal IL‐33 responses in H. pylori infection. We suggest that NOD1 may play a role in protection against excessive inflammation.     

Toll-like receptor 9 signaling has anti-inflammatory effects on the early phase of Helicobacter pylori-induced gastritis

Helicobacter pylori (H. pylori)-induced immune responses in the gastric mucosa are skewed toward T helper (Th) 1 phenotype, which is characterized by predominant production of tumor necrosis factor (TNF)-α and interferon (IFN)-γ by helper T cells. Toll-like receptors (TLRs) play an essential role in mucosal defense against microbes through the recognition of bacterial molecules. Among the members of the TLR family, TLR9 recognizes bacterial unmethylated CpG DNA sites, and signal transduction of TLR9 induces production of a variety of cytokines, including type-I IFN (IFN-α/β). We investigated the expression and role of TLR9 in H. pylori-induced gastritis in mice. Expression of TLR9 mRNA in the gastric tissue increased after infection with H. pylori. TLR9 was mainly expressed in the macrophages, dendritic cells, and CD3⁺ cells in the gastric mucosa. Neutrophil infiltration and the expression levels of TNF-α and IFN-γ mRNA were higher in TLR9 knockout (KO) mice than in wild-type mice at 2 and 4 months after H. pylori inoculation. These differences in inflammatory parameters between H. pylori-infected wild-type and TLR9 KO mice disappeared 6 months after H. pylori inoculation. Expression of interleukin-4 mRNA, typical Th2 cytokine, in the gastric tissue did not differ between H. pylori-infected wild-type and TLR9 KO mice. Expression level of IFN-α/β mRNA in the TLR9 KO mice was lower than that in wild-type mice by 4 months after inoculation. Administration of IFN-α reduced H. pylori infection-induced increase in neutrophil infiltration and the expression levels of TNF-α and IFN-γ mRNA in TLR9 KO mice. Our findings suggest that TLR9 signaling plays important roles in the suppression of H. pylori-induced gastritis in the early phase via downregulation of Th1-type cytokines modulated by IFN-α.     

Discordant susceptibility of inbred C57BL/6 versus outbred CD1 mice to experimental fungal sepsis

Individual susceptibility differences to fungal infection following invasive and/or immunosuppressive medical interventions are an important clinical issue. In order to explore immune response‐related factors that may be linked to fungal infection susceptibility, we have compared the response of inbred C57BL/6J and outbred CD1 mouse strains to different experimental models of fungal sepsis. The challenge of animals with the zymosan‐induced generalised inflammation model revealed poorer survival rates in C57BL/6J, consistent with lower Th1 cytokine interferon (IFN)‐γ serum levels, compared with CD1 mice. Likewise, ex vivo exposure of C57BL/6J splenocytes to zymosan but also bacterial lipopolisaccharide or lipoteichoic acid, resulted in lower IFN‐γ secretion compared with CD1 mice. C57BL/6J susceptibility could be reverted by rescue infusion of relative low IFN‐γ doses (0.2 μg/kg) either alone or in combination with the ß‐glucan‐binding CD5 protein (0.7 mg/kg) leading to improved post zymosan‐induced generalised inflammation survival. Similarly, low survival rates to systemic Candida albicans infection (2.86 × 10⁴ CFU/gr) were ameliorated by low‐dose IFN‐γ infusion in C57BL/6J but not CD1 mice. Our results highlight the importance of strain choice in experimental fungal infection models and provide a susceptibility rationale for more specific antifungal immunotherapy designs.     

Role of 13C-urea breath test in experimental model of Helicobacter pylori infection in mice

Background: Animal models have been widely used to study Helicobacter pylori infection. Evaluation of H. pylori infection status following experimental inoculation of mice usually requires euthanasia. The ¹³C‐urea breath test (¹³C‐UBT) is both sensitive and specific for detection of H. pylori in humans. Thus, it would be very useful to have such a test with the same accuracy for the follow‐up of this infection in animal models of gastric infection. Accordingly, the purpose of this study was to develop and evaluate a ¹³C‐UBT method for following the course of H. pylori infection in a mouse model. Material and Methods: A total of 50 female C57BL/6 mice were gavaged three times with either 10⁸ colony‐forming units of H. pylori (n = 29) or saline solution only (n = 21). After 2 months of infection, mice were fasted for 14 hours and ¹³C‐UBT was performed using 300 μg of ¹³C‐urea. The mice were killed, and the stomach was removed and processed for immunohistochemistry and PCR. Results: The optimal time for breath sample collection in mice was found to be 15 minutes. The ¹³C‐UBT cutoff was set at 3.0‰δPDB. Using PCR as the gold standard, the sensitivity of ¹³C‐UBT and immunohistochemistry was 96.6 and 72.4%, respectively, while the specificity was 85.7 and 95.2%, respectively. Conclusions: ¹³C‐UBT was shown to be a reliable method for the detection of H. pylori infection in C57BL/6 mice and was even more accurate than immunohistochemistry. The use of ¹³C‐UBT in the mouse model of H. pylori infection can be very useful to detect the bacterium without the need to kill the animals in long‐term time course studies.     

Genome-wide DNA methylation profiles in noncancerous gastric mucosae with regard to Helicobacter pylori infection and the presence of gastric cancer

Background and Aims: To determine genome‐wide DNA methylation profiles induced by Helicobacter pylori (H. pylori) infection and to identify methylation markers in H. pylori‐induced gastric carcinogenesis. Methods: Gastric mucosae obtained from controls (n = 20) and patients with gastric cancer (n = 28) were included. A wide panel of CpG sites in cancer‐related genes (1505 CpG sites in 807 genes) was analyzed using Illumina bead array technology. Validation of the results of Illumina bead array technique was performed using methylation‐specific PCR method for four genes (MOS, DCC, CRK, and PTPN6). Results: The Illumina bead array showed that a total of 359 CpG sites (269 genes) were identified as differentially methylated by H. pylori infection (p < .0001). The correlation between methylation‐specific PCR and bead array analysis was significant (p < .0001, Spearman coefficient = 0.5054). Methylation profiles in noncancerous gastric mucosae of the patients with gastric cancer showed quite distinct patterns according to the presence or absence of the current H. pylori infection; however, 10 CpG sites were identified to be hypermethylated and three hypomethylated in association with the presence of gastric cancer regardless of H. pylori infection (p < .01). Conclusions: Genome‐wide methylation profiles showed a number of genes differentially methylated by H. pylori infection. Methylation profiles in noncancerous gastric mucosae from the patients with gastric cancer can be affected by H. pylori‐induced gastritis. Differentially methylated CpG sites in this study needs to be validated in a larger population using quantitative methylation‐specific PCR method.     

A coordinated cytotoxic effect of IFN-gamma and cross-reactive antibodies in the pathogenesis of Helicobacter pylori gastritis

Background. Helicobacter pylori (H. pylori) infection is associated with chronic infiltration into the stomach by T cells and plasma cells producing IFN‐γ and antibodies of various specificities, respectively. It is unknown whether these lymphocyte‐products may play coordinated roles in the gastric pathology of this infection. Aims. To know how IFN‐γ may relate to anti‐H. pylori antibodies in their roles in pathogenesis, we determined the isotype subclass of those antibodies as well as their cross‐reactivity and cytotoxicity to gastric epithelium. Methods and Results. We infected BALB/c mice with H. pylori (SS1, Sydney Strain 1) and generated monoclonal antibodies, which were comprised of 240 independent clones secreting immunoglobulin and included 80 clones reactive to SS1. Ninety percent of the SS1‐reactive clones had IgG2a isotype. Two clones, 2B10 and 1A9, were cross reactive to cell surface antigens in H. pylori and to antigens of 28 KDa and 42 KDa, respectively, which were present on the cell surface of and shared by both mouse and human gastric epithelial cells. The antigens recognized by these monoclonal antibodies localized a distinctive area in the gastric glands. In the presence of complement, 2B10 showed cytotoxicity to gastric epithelial cells. The effect was dose dependant and augmented by IFN‐γ. Finally, administration of 2B10 to mice with SS1 infection aggravated gastritis by increasing cellular infiltration. Conclusion. IFN‐γ by gastric T cells may participate in pathogenesis of the H. pylori infected stomach by directing an isotype‐switch of anti‐H. pylori antibodies to complement‐binding subclass and by augmenting cytotoxic activity of a certain autoantibody. This may explain a host‐dependent diversity in gastric pathology of the patients with H. pylori infection.     

Crystal Structure of the TNF-α-Inducing Protein (Tipα) from Helicobacter pylori: Insights into Its DNA-Binding Activity

Helicobacter pylori infection is one of the highest risk factors for gastroduodenal diseases including gastric cancer. Tumor necrosis factor-alpha (TNF-α) is one of the essential cytokines for tumor promotion, and thus, an H. pylori protein that induces TNF-α is believed to play a significant role in gastric cancer development in humans. The HP0596 gene product of H. pylori strain 26695 was identified as the TNF-α-inducing protein (Tipα). Tipα is secreted from H. pylori as dimers and enters the gastric cells. It was shown to have a DNA-binding activity. Here, we have determined the crystal structure of Tipα from H. pylori. Its monomer consists of two structural domains (“mixed domain” and “helical domain”). Tipα exists as a dimer in the crystal, and the dimeric structure represents a novel scaffold for DNA binding. A positively charged surface patch formed across the two monomers of the Tipα dimer by the loop between helices α1 and α2 may be important in DNA binding.     

Production of chemokines and reactive oxygen species by human neutrophils stimulated by Helicobacter pylori

Background. Bacteria have different characteristics in stimulation of human neutrophils to produce reactive oxygen species (ROS) and chemokines. This study examined the ability of Helicobacter pylori to induce production of ROS and chemokines by human neutrophils. Methods. H. pylori strains (1.5 × 10⁸ CFU/ml) were cocultured with 5 × 10⁴ neutrophils isolated from healthy subjects. Samples were incubated with human serum with or without IgG antibodies to H. pylori. ROS production was measured using luminol‐dependent chemiluminescence (LmCL), and the concentrations of chemokines (IL‐8, RANTES, MIP‐1α and MCP‐1) were measured by ELISA. Results. The mean of the highest LmCL (peak height; PH) value stimulated by H. pylori was 3318 in the absence of serum. PH increased to 4687 when incubated with anti‐H. pylori antibody‐positive sera (p < .001) but antibody‐negative sera did not affect LmCL response. The mean final concentration of IL‐8 produced in the absence of serum was 142.6 pg/ml. Increased IL‐8 production was seen by addition of antibody positive serum (p < .01). IL‐8 production was not significantly correlated with production of ROS. On the other hand, H. pylori stimulation did not induce neutrophil production of RANTES, MIP‐1α or MCP‐1. Conclusions. H. pylori was capable of inducing IL‐8 production by human neutrophils, but not C‐C chemokines. Production of C‐X‐C dominant chemokine by neutrophils is consistent with the pathological characteristics of H. pylori‐induced gastritis, where persistent neutrophil infiltration is present.     

Mechanism of action of low recurrence of gastritis caused by Helicobacter pylori with the type II urease B gene

Background. Low recurrence of gastritis is seen in patients infected with Helicobacter pylori carrying the type II urease B gene, compared with H. pylori carrying types I and III. The underlying mechanism has been studied in terms of the urease activity and interleukin (IL)‐8 production capacity of different strains of H. pylori. Materials and Methods. Forty‐five patients infected with different strains of H. pylori (type I; 15, type II; 15 and type III; 15) were enrolled in the study. H. pylori was isolated from gastric mucosa and cultured in the presence of urea at pH 5.5 to evaluate urease activity. The capacity of different strains of H. pylori to induce IL‐8 mRNA and IL‐8 from a human gastric cancer cell line and human peripheral blood mononuclear cells was evaluated. Results. The urease activity of type II H. pylori[523 ± 228 µg of ammonia/dl/10⁸ colony‐forming units (CFU)/ml] was significantly lower than that of type I (1355 ± 1369 µg of ammonia/dl/10⁸ CFU/ml) and type III (1442 ± 2229 µg of ammonia/dl/10⁸ CFU/ml) (p < .05). Gastric cancer cells cocultured with type II H. pylori produced lower levels of IL‐8 mRNA compared with type I and type III H. pylori. The levels of IL‐8 were also significantly lower in cultures induced by type II H. pylori compared with those induced by type I and type III H. pylori. Peripheral blood mononuclear cells also produced lower levels of IL‐8 when cocultured with type II compared with type I H. pylori. Conclusions. These results indicate that both the lower level of urease activity and the low IL‐8‐inducing capacity of type II H. pylori might underlie the lower recurrence rate of gastritis caused by type II H. pylori.     

Attenuated accumulation of regulatory T cells and reduced production of interleukin 10 lead to the exacerbation of tissue injury in a mouse model of acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a pathological condition that involves diffuse lung injury and severe hypoxemia caused by pulmonary and systemic diseases. We have established a mouse model of severe ARDS, developed by intratracheal injection of α‐galactosylceramide (α‐GalCer), an activator of natural killer T (NKT) cells, followed by LPS. In the present study, we used this model to investigate the regulatory mechanism in the early inflammatory response during acute lung injury. In α‐GalCer/LPS‐treated mice, the number of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and the expression of a Treg cell‐tropic chemokine, secondary lymphoid‐tissue chemokine (SLC), in the lungs was significantly lower than in mice treated with LPS alone. Giving recombinant (r)SLC increased the number of Treg cells in α‐GalCer/LPS‐treated mice. Treatment with anti‐IFN‐γ mAb enhanced the expression of SLC and the accumulation of Treg cells in the lungs of α‐GalCer/LPS‐treated mice, whereas giving recombinant (r)IFN‐γ reduced the number of Treg cells in mice treated with LPS alone. IL‐10 production was significantly lower in α‐GalCer/LPS‐treated mice than in mice treated with LPS alone. Giving rIL‐10 prolonged survival and attenuated lung injury as a result of reduced production of inflammatory cytokines (such as IL‐1β, IL‐6, TNF‐α, and IFN‐γ) and chemokines (including MCP‐1, RANTES, IP‐10, Mig, MIP‐2, and KC) in α‐GalCer/LPS‐treated mice. Treatment with anti‐IFN‐γ mAb enhanced IL‐10 production in α‐GalCer/LPS‐treated mice. These results suggest that the attenuated accumulation of Treg cells may be involved in the development of severe ARDS through a reduction in the synthesis of IL‐10.

     

The role of interleukin-17 in the Helicobacter pylori induced infection and immunity

Background: Helicobacter pylori infection is regarded as the major cause of various gastric diseases and induces the production of several cytokines including interleukin‐17 (IL‐17) recently recognized as an important player in the mammalian immune system. Objective: This review deals with the role of IL‐17 on the H. pylori‐induced infection and immunity in humans and experimental animals. Results: H. pylori infection increases IL‐17 in the gastric mucosa of humans and experimental animals. In humans, IL‐17 induces the secretion of IL‐8 by activating the ERK 1/2 MAP kinase pathway and the released IL‐8 attracts neutrophils promoting inflammation. IL‐23 is increased in patients with H. pylori‐related gastritis and regulates IL‐17 secretion via STAT3 pathway. Studies in H. pylori‐infected mice indicate that IL‐17 is primarily associated with gastric inflammation. The early events in the immune response of immunized and challenged mice include the recruitment of T cells and the production of IL‐17. Neutrophil attracting chemokines are released, and the bacterial load is considerably reduced. IL‐17 plays a dual role in infection and vaccination. In infection, T regulatory cells (Tregs) suppress the inflammatory reaction driven by IL‐17 thereby favoring bacterial persistence. Immunization produces Helicobacter‐specific memory T‐helper cells that can possibly alter the ratio between T‐helper 17 and Treg responses so that the IL‐17‐driven inflammatory reaction can overcome the Treg response leading to bacterial clearance. Conclusion: IL‐17 plays an important role in H. pylori‐related gastritis and in the reduction of Helicobacter infection in mice following immunization.     

Vaccine-induced immunity against Helicobacter pylori in the absence of IL-17A

Background: Helicobacter pylori (H. pylori) is a gram negative bacterium that can cause diseases such as peptic ulcers and gastric cancer. IL‐17A, a proinflammatory cytokine that can induce the production of CXC chemokines for neutrophil recruitment, has recently been shown to be elevated in both H. pylori‐infected patients and mice. Furthermore, studies in mouse models of vaccination have reported levels significantly increased over infected, unimmunized mice and blocking of IL‐17A during the challenge phase in immunized mice reduces protective immunity. Because many aspects of immunity had redundant or compensatory mechanisms, we investigated whether mice could be protectively immunized when IL‐17A function is absent during the entire immune response using IL‐17A and IL‐17A receptor knockout (KO) mice immunized against H. pylori. Materials and Methods: Gastric biopsies were harvested from naïve, unimmunized/challenged, and immunized/challenged wild type (WT) and KO mice and analyzed for inflammation, neutrophil, and bacterial levels. Groups of IL‐17A KO mice were also treated with anti‐IFNγ or control antibodies. Results: Surprisingly, all groups of immunized KO mice reduced their bacterial loads comparably to WT mice. The gastric neutrophil counts did not vary significantly between IL‐17A KO and WT mice, whereas IL‐17RA KO mice had on average a four‐fold decrease compared to WT. Additionally, we performed an immunization study with CXCR2 KO mice and observed significant gastric neutrophils and reduction in bacterial load. Conclusion: These data suggest that there are compensatory mechanisms for protection against H. pylori and for neutrophil recruitment in the absence of an IL‐17A‐CXC chemokine pathway.     

减毒沙门氏菌介导的小鼠肝炎病毒S1基因DNA疫苗的免疫特性分析

根据GenBank公开序列自行设计一对引物,通过RT-PCR扩增出小鼠肝炎病毒的全长S1基因,并将其插入真核表达质粒pVAX1中,构建出重组真核表达质粒pVAX1-S1。将重组质粒转染COS-7细胞,采用间接免疫荧光检测出S1蛋白的体外表达。将重组质粒转入减毒鼠伤寒沙门氏菌SL7207中,构建出运送DNA疫苗的重组沙门氏菌SL7207(pVAX1-S1)。分别以5×108CFU、1×109CFU、2×109CFU剂量的重组菌口服接种6周龄BALB/c小鼠,试验结果表明,重组菌对小鼠具有良好的安全性。以1×109CFU剂量的重组菌口服免疫小鼠,抗体检测结果显示,在二免后两周和三免后两周,重组菌免疫组的血清抗体水平与SL7207(pVAX1)空载体免疫组间分别存在显著性差异(P<0.05)和极显著性差异(P<0.01)。在三免后两周重组菌免疫组出现了较高水平的肠黏膜抗体。     

Anti‐infectious activity of synbiotics in a novel mouse model of methicillin‐resistant Staphylococcus aureus infection

The anti‐infectious activity of synbiotics against methicillin‐resistant Staphylococcus aureus (MRSA) infection was evaluated using a novel lethal mouse model. Groups of 12 mice treated with multiple antibiotics were infected orally with a clinical isolate of MRSA at an inoculum of 10⁸ CFU on day 7 after starting the antibiotics. A dose of 400 mg/kg 5‐fluorouracil (5‐FU) was injected intraperitoneally on day 7 after the infection. A dose of 10⁸ CFU Bifidobacterium breve strain Yakult and 10 mg of galactooligosaccharides (GOS) were given orally to mice daily with the antibiotic treatment until day 28. The intestinal population levels of MRSA in the mice on multiple antibiotics were maintained stably at 10⁸ CFU/g of intestinal contents after oral MRSA infection and the subsequent 5‐FU treatment killed all the mice in the group within 14 days. B. breve administration saved most of the mice, but the synbiotic treatment saved all of the mice from lethal MRSA infection. The synbiotic treatment was effective for the treatment of intestinal infection caused by four MRSA strains with different toxin productions. There was a large difference among the six Bifidobacteria strains that were naturally resistant to the antibacterial drugs used. B. breve in combination with GOS is demonstrated to have valuable preventive and curative effects against even fatal MRSA infections.