幽门螺杆菌在胃内定植的相关影响因素

胃内定植是引起幽门螺杆菌（Helicobacter pylori,H. pylori）感染的先决条件。H. pylori可穿过胃黏液层并与胃上皮细胞相互作用。这个定植过程主要受到H. pylori动力和尿素酶的影响。同时H. pylori形态、胃内pH、外膜蛋白及益生菌等也在其中扮演重要角色。该研究主要对H. pylori胃内定植过程中的相关影响因素进行综述。     

Helicobacter pylori dampens gut epithelial self-renewal by inhibiting apoptosis, a bacterial strategy to enhance colonization of the stomach

Colonization of the gastric pits in the stomach by Helicobacter pylori (Hp) is a major risk factor for gastritis, gastric ulcers, and cancer. Normally, rapid self-renewal of gut epithelia, which occurs by a balance of progenitor proliferation and pit cell apoptosis, serves as a host defense mechanism to limit bacterial colonization. To investigate how Hp overcomes this host defense, we use the Mongolian gerbil model of Hp infection. Apoptotic loss of pit cells induced by a proapoptotic agent is suppressed by Hp. The ability of Hp to suppress apoptosis contributed to pit hyperplasia and persistent bacterial colonization of the stomach. Infection with WT Hp but not with a mutant in the virulence effector cagA increased levels of the prosurvival factor phospho-ERK and antiapoptotic protein MCL1 in the gastric pits. Thus, CagA activates host cell survival and antiapoptotic pathways to overcome self-renewal of the gastric epithelium and help sustain Hp infection.     

Importance of the host genetic background on immune responses to Helicobacter pylori infection and therapeutic vaccine efficacy

In order to investigate the role of host factors in Helicobacter pylori infection and immunity, two different strains of inbred mice, C57BL/6 and BALB/c, were infected with a standard H. pylori strain, SS1. A month later, infected mice were immunized orally with whole-cell lysates of H. pylori SS1 and cholera toxin on days 1, 3, 6, 30, and 54. Ten days after the last immunization, mice were sacrificed and the stomach was collected to assess H. pylori colonization density by quantitative culture. H. pylori SS1 colonization was significantly greater in C57BL/6 than in BALB/c (P<0.02 and P<0.003 at 2 and 13 weeks post-inoculation, respectively). Colonization in C57BL/6 persisted at equivalent levels for 13 weeks but the colonization density in BALB/c decreased significantly during this period. In contrast to the pattern of bacterial colonization, antibody responses following H. pylori SS1 infection were greater in BALB/c than in C57BL/6, suggesting that host factors may modulate the immune responses to H. pylori infection. Following therapeutic immunization, H. pylori colonization in BALB/c mice was also significantly reduced (P<0.03), while no significant differences in bacterial density were observed in C57BL/6. These observations collectively demonstrate the great importance of host factors in H. pylori infection and the development of effective immune responses.     

Bacterial factors that mediate colonization of the stomach and virulence of Helicobacter pylori

Helicobacter pylori is a Gram-negative microaerophilic organism that colonizes the gastric mucosa of humans. Helicobacter pylori is one of the most common infections in humans and results in the development of gastritis in all infected individuals, although the majority of people are asymptomatic. A subset of infected people develop serious disease including duodenal ulceration and gastric cancer. Helicobacter pylori exhibits many striking characteristics. It lives in the hostile environment of the stomach and displays a very strict host and tissue tropism. Despite a vigorous immune response, infection persists for the lifetime of the host unless eradicated with antimicrobials. Why H. pylori is so pathogenic in some individuals and not in others is unknown but is thought to be due to a variety of host, environmental and bacterial factors. In this review, some of the bacterial factors that mediate colonization of the gastric mucosa and play a role in the pathogenesis of this organism have been considered.     

Intraperitoneal immunization led to T cell hyporesponsiveness to Helicobacter pylori infection in mice

During Helicobacter pylori infection, T cell response is critical in the development of active gastritis and in protective immunity against infection. We studied gastric inflammation and T cell response in H. pylori-challenged mice following an intraperitoneal immunization, using whole H. pylori lysate (HpAg) in the absence of adjuvants. H. pylori-challenged mice without immunization developed moderate to severe gastric inflammation, and splenocytes from these mice produced Th1 polarizing cytokines in response to HpAg and Con A during the acute infection. On the other hand, immunized-challenged mice (those inoculated with H. pylori following immunization) had little or no gastric inflammation despite persistent H. pylori colonization. Our immunization primed splenocytes to produce IL-2, IFN-gamma, and IL-4 in response to HpAg and Con A before infection. However, these cells became hyporesponsive to both stimulants immediately after live bacterial challenge in terms of the production of these cytokines, especially IL-2 and IFN-gamma. CTLA-4 has been documented to be a negative regulator of IL-2 production and lymphoproliferation that induces peripheral tolerance and functions 24-72 hr after the initiation of T cell activation. Compared with challenged mice, T cells from immunized-challenged mice showed higher levels of CTLA-4 expression at 72 hr after oral challenge. These data suggested that our immunization inhibited the development of H. pylori-associated gastritis and induced T cell hyporesponsiveness to H. pylori infection, which might be mediated by the early induction of CTLA-4 following challenge.     

幽门螺杆菌感染引起的免疫应答及其对疫苗研制的启示

幽门螺杆菌是一种全世界范围的人类感染病原菌,人感染该菌后可以被诱导产生较强的体液免疫应答和一定程度的细胞免疫应答反应;但是,自然感染往往不能使机体产生有效的免疫保护,相反可引起对机体的免疫病理损伤,有效的疫苗应该被设计为能够诱导机体产生非病理损伤的特异的免疫保护反应,以此来预防和治疗幽门螺杆菌相关性疾病。本综述了这些方面的研究进展。     

Micronuclei in mucose cells and colonization of human stomach epithelium with coccoid forms of Helicobacter pylori

International Agency for Research on Cancer recognized as sufficient the evidence of Helicobacter pylori (HP) infection carcinogenicity and placed it into the 1 st group of carcinogens. Micronucleus level in gastric epithelial cells of antral stomach region of patients with chronic non-atrophy gastritis (n = 62) was studied. 40 patients of 62 had HP-associated gastritis. The HP-bacterium exists in a spiral and coccoid form. Both morphological forms were examined using immunocytochemistry. Significantly increased micronucleus number was observed in the cells of HP-infected patients compared with non-infected person (P < 0.05). The frequency of stomach epithelium cells with micronuclei was enhanced considerably in the patients infected with the coccoid HP form. Therefore the patients with HP-associated chronic gastritis caused by the coccoid form with high degree of colonization must be considered as a group of enhanced risk of gastric carcinogenesis.     

Docosahexaenoic acid inhibits Helicobacter pylori growth in vitro and mice gastric mucosa colonization

H. pylori drug-resistant strains and non-compliance to therapy are the major causes of H. pylori eradication failure. For some bacterial species it has been demonstrated that fatty acids have a growth inhibitory effect. Our main aim was to assess the ability of docosahexaenoic acid (DHA) to inhibit H. pylori growth both in vitro and in a mouse model. The effectiveness of standard therapy (ST) in combination with DHA on H. pylori eradication and recurrence prevention success was also investigated. The effects of DHA on H. pylori growth were analyzed in an in vitro dose-response study and n in vivo model. We analized the ability of H. pylori to colonize mice gastric mucosa following DHA, ST or a combination of both treatments. Our data demonstrate that DHA decreases H. pylori growth in vitro in a dose-dependent manner. Furthermore, DHA inhibits H. pylori gastric colonization in vivo as well as decreases mouse gastric mucosa inflammation. Addition of DHA to ST was also associated with lower H. pylori infection recurrence in the mouse model. In conclusion, DHA is an inhibitor of H. pylori growth and its ability to colonize mouse stomach. DHA treatment is also associated with a lower recurrence of H. pylori infection in combination with ST. These observations pave the way to consider DHA as an adjunct agent in H. pylori eradication treatment.     

Essential role of Helicobacter pylori gamma-glutamyltranspeptidase for the colonization of the gastric mucosa of mice

Constitutive expression of gamma-glutamyltranspeptidase (GGT) activity is common to all Helicobacter pylori strains, and is used as a marker for identifying H. pylori isolates. Helicobacter pylori GGT was purified from sonicated extracts of H. pylori strain 85P by anion exchange chromatography. The N-terminal amino acid sequences of two of the generated endo-proteolysed peptides were determined, allowing the cloning and sequencing of the corresponding gene from a genomic H. pylori library. The H. pylori ggt gene consists of a 1681 basepair (bp) open reading frame encoding a protein with a signal sequence and a calculated molecular mass of 61 kDa. Escherichia coli clones harbouring the H. pylori ggt gene exhibited GGT activity at 37 degrees C, in contrast to E. coli host cells (MC1061, HB101), which were GGT negative at 37 degrees C. GGT activity was found to be constitutively expressed by similar genes in Helicobacter felis, Helicobacter canis, Helicobacter bilis, Helicobacter hepaticus and Helicobacter mustelae. Western immunoblots using rabbit antibodies raised against a His-tagged-GGT recombinant protein demonstrated that H. pylori GGT is synthesized in both H. pylori and E. coli as a pro-GGT that is processed into a large and a small subunit. Deletion of a 700 bp fragment within the GGT-encoding gene of a mouse-adapted H. pylori strain (SS1) resulted in mutants that were GGT negative yet grew normally in vitro. These mutants, however, were unable to colonize the gastric mucosa of mice when orally administered alone or together (co-infection) with the parental strain. These results demonstrate that H. pylori GGT activity has an essential role for the establishment of the infection in the mouse model, demonstrating for the first time a physiological role for a bacterial GGT enzyme.     

Mucosal production of antigastric autoantibodies in Helicobacter pylori gastritis

Background. Apart form bacterial virulence factors of Helicobacter pylori , certain host factors influence the pathogenesis of H. pylori gastritis. In particular, antigastric autoantibodies that are detectable in the sera of a substantial proportion of H. pylori were shown to correlate with the development of gastric atrophy. The aim of this study was to analyze the possible antigastric autoimmune response in H. pylori gastritis at the site where the action is, i.e., in the gastric mucosa.
Material and Methods. Gastric biopsy specimens from antrum and corpus mucosa of 24 H. pylori –infected and of 33 noninfected patients were cultured for 3 days, and tissue culture supernatants were analyzed for the amount of locally produced IgA and IgG. Antigastric autoantibodies were screened in the sera and in the supernatants by means of immunohistochemistry.
Results. The infected patients had significantly higher concentrations of locally produced IgA, whereas the IgG concentrations were virtually the same in infected and noninfected patients. IgG or IgA antigastric autoantibodies, or both, were detectable only in the sera (38%) and supernatants (17%) of infected patients. Interestingly, the patient with the strongest local autoimmune response showed body-predominant H. pylori gastritis, with destruction of gastric glands and atrophy of the body mucosa.
Conclusions. These results demonstrate that antigastric autoimmune reactions are detectable at the site of the disease and might be relevant for the pathogenesis of gastric mucosa atrophy in H. pylori gastritis.     

FecA1, a bacterial iron transporter, determines the survival of Helicobacter pylori in the stomach

Helicobacter pylori encodes a single iron-cofactored superoxide dismutase (SodB), which is regulated by the ferric uptake regulator (Fur). Ferrous ion (Fe(2+)) is necessary for the activation of SodB. The activity of SodB is an important determinant of the capability of H. pylori for long-term colonization of the stomach and of the development of metronidazole (Mtz) resistance of the bacterium. This study is conducted to characterize the Fe(2+)-supply mechanisms for the activation of SodB in H. pylori, which, as mentioned above, is associated with the host-colonization ability and Mtz resistance of H. pylori. In this study, we demonstrate that fecA1, a Fe(3+)-dicitrate transporter homolog, is an essential gene for SodB activation, but not for the biogenic activity of H. pylori. H. pylori with SodB inactivation by fecA1 deletion showed reduced resistance to H(2)O(2), reduced gastric mucosal-colonization ability in Mongolian gerbils, and also reduced resistance to Mtz. Our experiment demonstrated that FecA1 is an important determinant of the host-colonization ability and Mtz resistance of H. pylori through Fe(2+) supply to SodB, suggesting that FecA1 may be a possible target for the development of a novel bactericidal drug.     

Acute inflammatory response in the stomach of BALB/c mice challenged with coccoidal Helicobacter pylori

An experimental murine model was used to verify the viability and pathogenicity of coccoid Helicobacter pylori. For this purpose, 27 BALB/c mice were inoculated intragastrically with 1 ml broth culture (10(8)organisms/ml) of a coccoid H. pylori clinical isolate. The animals were divided into two groups. Nine were infected on a one-time basis (GA1) and 18 were infected on two consecutive days (GA2). Other 27 mice were inoculated with Brucella broth and divided in the same way; they composed the control group. Mice were killed at 2, 3, 7, 14 and 21 days post inoculation (pi). Fragments of stomach and duodenum were collected, fixed with 12% formalin and stained by hematoxilin-eosin and Giemsa for histopathological examination. Until the 14th()day, only reinfected mice had mild-to-moderate inflammatory infiltrate in the stomach. The infiltration was predominantly lymphomonocytic, although plasma cells and eosinophils could be seen. However, at 21st day, severe eosinophilic infiltration was present in the lamina propria and submucosa of gastric corpus. In subgroup GA1, animals presented lymphomonocytic infiltration in the stomach from 14th()day pi. Our results showed that coccoid H. pylori was able to induce an acute inflammatory response in stomach of reinfected mice since the initial periods of infection.     

Helicobacter pylori virulence factors and the host immune response: implications for therapeutic vaccination

Helicobacter pylori colonizes the human gastric mucosa and is associated with specific gastric disease. Virulence factors, such as urease, the vacuolating toxin (VacA), the cytotoxin-associated antigen CagA or blood-group-antigen-binding adhesin (BabA), an adherence factor, might account for the development of different diseases. Vaccination trials exploiting the antigenic properties of some of these proteins have not been successful in preventing infection in humans. A more in-depth understanding of the immune response to H. pylori infection as well as additional information on suitable epitopes and adjuvants will be required before a successful vaccine can be developed.     

Mucosal and systemic immune responses of mice to tetanus toxoid coadministered nasally with AFCo1

Mucosal immune responses are an early and important line of defense against pathogens. The current understanding of the mucosal immune system allows us to consider the use of nasal immunization for induction of antigen-specific immune responses at the mucosal surface and the systemic compartment. Mucosal adjuvants are key for developing novel mucosal vaccines and represent 1 approach to improving mucosal and systemic immunity. However, few mucosal vaccine adjuvants are currently approved for human use. Neisseria meningitidis B proteoliposome-derived cochleate (AFCo1 - Adjuvant Finlay Cochleate 1) has been demonstrated to be a potent mucosal adjuvant. The present work demonstrates that intranasal immunization of 3 doses of tetanus toxoid (TT) coadministered with AFCo1 in mice promotes high systemic and mucosal responses. The anti-TT IgG serum titers and the mucosal anti-TT IgA in saliva and vaginal wash were significantly higher than TT alone. The analysis of antibody subclasses showed that intranasal administration of AFCo1 + TT induced not only IgG1 but also IgG2a anti-TT antibodies at levels comparable to those obtained with TT vaccine (vax-TET). These data support the fact that AFCo1 is a potent mucosal adjuvant in nasal immunization to a coadministered protein antigen.     

Therapeutic immunization against Helicobacter pylori infection in the absence of antibodies

Helicobacter pylori is an important human pathogen. Prophylactic immunization with bacterial antigen plus an adjuvant protects mice against challenge with live H. pylori. Surprisingly, it was found that immunizations of mice already infected with Helicobacter also influenced bacterial colonization. This concept of therapeutic immunization is a novel phenomenon. Because H. pylori lives in the lumen of the stomach, it was initially hypothesized that the protective mechanism would involve induction of secretory IgA. However, work with knockout mice has demonstrated that prophylactic immunization is equally effective in mice deficient in IgA and even in microMT mice lacking B lymphocytes. Currently nothing is known about therapeutic vaccination and the effect of immunizing a host with an ongoing ineffective immune response. To address this, we infected B-cell deficient, microMT mice with H. pylori and therapeutically immunized them four times in 3 weeks with bacterial sonicate and cholera toxin adjuvant. These immunizations significantly reduced colonization by H. pylori. The antibody- negative status of the microMT mice was confirmed by ELISA. Thus, therapeutic immunization stimulates an immune response, which reduces H. pylori infection via a mechanism that is antibody independent. How this is achieved remains to be determined, but may well involve a novel immune mechanism.     

幽门螺杆菌疫苗诱导的免疫保护反应及临床试验研究进展

幽门螺杆菌(Helicobacter pylori,Hp)是一种微需氧、寄生于胃黏膜表面的革兰阴性致病菌,可导致各种不同的疾病,如慢性活动性胃炎、胃和十二指肠溃疡,甚至包括胃黏膜相关淋巴样组织(mucosa-associated lymphoid tissue,MALT)淋巴瘤和胃腺癌等恶性疾病。目前根除Hp主要依靠包括2种抗生素、铋剂和质子泵抑制剂(proton pumpinhibitors,PPI)在内的四联疗法。随着Hp耐药性的增强,研发Hp疫苗成为防治Hp感染的新途径。Hp疫苗能够诱导机体产生抗原特异性血清IgG和黏膜sIgA体液免疫反应以及抗原特异性CD4+T细胞免疫反应等的有效免疫保护反应。     

Evolution of the Helicobacter pylori vacuolating cytotoxin in a human stomach

We describe two subclones of Helicobacter pylori, isolated contemporaneously from a human stomach, which differ markedly in the vacuolating cytotoxin gene, vacA, but whose near identity in sequences outside this locus implies a very recent common origin. The differences are consistent with homologous recombination with DNA from another strain and result in a changed vacA midregion and, importantly, in changed toxicity.