Genetic diagnosis of H. pylori strains

Helicobacter pylori strains are divided into two broad families, type I and type II, based on whether or not they possess the cag pathogenicity island (cagPAI). It has been suggested that cagPAI is inherited by horizontal transfer from an unknown microorganism, and the genes of cag are thought to be encoded by a putative type IV secretory system. In addition, CagA may be delivered from attached H. pylori into the host crytoplasm by this system and is tyrosine phosphorylated in gastric epithelial cells. The phosphorylated CagA may play a crucial role in promoting the inflammatory responses of gastric mucosa. These findings suggest that type I H. pylori is a pathogenic H. pylori.     

幽门螺杆菌血清分型与上消化道疾病的关系

目的 探讨幽门螺杆菌(helicobacter pylori,Hp or H.pylori)分型与消化道不同疾病的关系。方法 入选198例Hp阳性的胃镜检查患者，采用免疫印迹法进行Hp的血清学分型，并取胃窦黏膜经HE染色观察胃窦黏膜病理组织学变化。结果 198例患者中检出HpI型菌株173例(87．4％)，Ⅱ型菌株25例(12．6％)。I型较II型Hp感染者胃镜下消化性溃疡、胃癌的比例更高，P=0.012；与胃炎组比较，十二指肠球部溃疡组、胃癌组的I型感染者更高（P值分别为0.026、0.048），而与胃溃疡组无显著差别(P=0.125)。病理组织学改变I型较II型Hp感染者的结果更为严重（P=0.038）。结论 临床上消化道疾病患者Hp感染以I型菌株最为多见。Hp感染的分型诊断有助于对胃、十二指肠疾病类型及病情的判断，I型菌株感染者需要更为积极的治疗。     

Green Tea Polyphenols Reduce Gastric Epithelial Cell Proliferation and Apoptosis Stimulated by Helicobacter pylori Infection

Recently the finding of gastric cancer in Helicobacter pylori (H. pylori)-infected mouse models was reported. Studies of humans and animal models have shown that H. pylori infection stimulates gastric epithelial cell proliferation and apoptosis. Polyphenols contained in green tea and related compounds were reported to have a variety anti-tumor effects and bactericidal properties. We studied the effect of green tea polyphenols on gastric cell proliferation and apoptosis in an H. pylori-infected mouse model. This model was prepared by inoculating Balb/c mice with 10(8) cfu of H. pylori (NCTC 11637 strain) by gavage. Beginning 18 weeks after inoculation, 0.5% polyphenols were given in drinking water every day for 2 weeks. Mice were sacrificed 1 h after bromodeoxyuridine (BrdU) was given i.p. for preparation of paraffin-embedded specimens. Cell proliferation and apoptosis were examined by the avidin-biotin complex method using anti-BrdU antibody and the TUNEL method, respectively. H. pylori infection resulted in increased BrdU-labeled cells in both the antrum and the bodies. Administration of polyphenols suppressed this increased proliferation. H. pylori infection increased apoptotic cells in both the antrum and the corpus in comparison with controls. This increase was not seen in H. pylori-infected mice given polyphenols. We conclude the administration with polyphenols might suppress gastric carcinogenesis that is in part related to H. pylori infection.     

Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations on bacterial survival

Tubercle bacilli of the pathogenic human strain H37Rv had previously been shown to multiply, after ingestion by cultured mouse peritoneal macrophages, within phagosomes that tended to remain unfused with secondary lysosomes. Means were sought therefore for promoting experimentally a modification of the host response so as to attain a high level of phagolysosome formation, enabling tests to be made of any effects on the course and outcome of the intracellular infection. This was achieved by exposing viable bacilli to specific rabbit antiserum before their ingestion. Quantitative assessments, using electron microscopy, now showed that a majority of the phagosomes containing intact bacilli had fused with ferritin-labeled lysosomes, and frequently the fusion was massive. Bacterial viability studies established that the serum pretreatment was not itsel bactericidal. In the course of progressive infections with strain H37Rv, monitored by counts both of viable bacterial units and of intracellular acid-fast organisms, no appreciable difference was found between the intracellular growth rates of control and antiserum-treated bacilli. Concurrent electron microscopy showed that bacilli could remain intact and multiply both in phaagolysosomes and in unfused phagosomes, ruling out the possibility of selective growth of antiserum-pretreated bacilli within the minority of phagosomes that remained unfused. It was concluded that "turning on" phagosome-lysosome fusion in normal macrophages did not influence the outcome of infection with virulent M. tuberculosis; lysosome contents manifestly failed to exercise an antibacterial effect on this organism. Nevertheless, the possibility remains that the lysosomes of specific immune macrophages have antituberculous potentiality. In that case the experimental "turning on or off" of fusion could be a decisive factor in the outcome of a virulent challenge. Should it not be, the antibacterial capabilities of immune cells would need to be ascribed to factors other than lysosomal attack, the latter being essentially for disposal of the dead organisms.     

The Effect of Class II Major Histocompatibility Complex Expression on Adherence of Helicobacter pylori and Induction of Apoptosis in Gastric Epithelial Cells: A Mechanism for T Helper Cell Type 1–mediated Damage

Helicobacter pylori infection is associated with gastric epithelial damage, including apoptosis, ulceration, and cancer. Although bacterial factors and the host response are believed to contribute to gastric disease, no receptor has been identified that explains how the bacteria attach and signal the host cell to undergo apoptosis. Using H. pylori as “bait” to capture receptor proteins in solubilized membranes of gastric epithelial cells, class II major histocompatibility complex (MHC) molecules were identified as a possible receptor. Signaling through class II MHC molecules leading to the induction of apoptosis was confirmed using cross-linking IgM antibodies to surface class II MHC molecules. Moreover, binding of H. pylori and the induction of apoptosis were inhibited by antibodies recognizing class II MHC. Since type 1 T helper cells are present during infection and produce interferon (IFN)-γ, which increases class II MHC expression, gastric epithelial cell lines were exposed to H. pylori in the presence or absence of IFN-γ. IFN-γ increased the attachment of the bacteria as well as the induction of apoptosis in gastric epithelial cells. In contrast to MHC II–negative cell lines, H. pylori induced apoptosis in cells expressing class II MHC molecules constitutively or after gene transfection. These data describe a novel receptor for H. pylori and provide a mechanism by which bacteria and the host response interact in the pathogenesis of gastric epithelial cell damage.     

Phagolysosome formation in normal and colchicine-treated macrophages

Intracellular lysosomal fusion has been evaluated in cultivated mouse peritoneal macrophages by measurement of transfer of acid phosphatase to polyvinyltoluene (PVT)-containing phagolysosomes. Enzyme transfer was found to be directly and significantly related to the uptake of PVT and to be independent of time allowed for phagolysosome formation over time periods of 15 min to 18 h. In addition, the extent of transfer of lysosomal enzyme to phagolysosomes was unaffected by treatment of the cells with 10(-6) M colchicine, a dose which eradicates morphologically identifiable microtubules in this cell type within 2 h. The data indicate that intracellular fusion of lysosomes with phagosomes in the macrophage does not require formed microtubules and suggest that fusion occurs promptly after interiorization of inert particles.     

Suppressive Effects of Selected Food Phytochemicals on CD74 Expression in NCI-N87 Gastric Carcinoma Cells

Helicobacter pylori (H. pylori) is one of the most widespread human pathogens, and plays major roles in chronic gastritis and gastric cancer. CD74 of gastric epithelial cells has recently been identified as an adhesion molecule to urease in H. pylori. In this study, we found that CD74 is highly expressed in a constitutive manner in NCI-N87 human gastric carcinoma cells at both the protein and mRNA levels as compared with Hs738St./Int fetal gastric cells. Subsequently, a novel cell-based ELISA able to rapidly screen the suppressive agents of CD74 expression was established. NCI-N87 cells were treated separately with 25 different food phytochemicals (4-100 microM) for 48 h and subjected to our novel assay. From those results, a citrus coumarin, bergamottin, was indicated to be the most promising compound with an LC(50)/IC(50) value greater than 7.1, followed by luteolin (>5.4), nobiletin (>5.3), and quercetin (>5.1). Our findings suggest that these CD74 suppressants are unique candidates for preventing H. pylori adhesion and subsequent infection with reasonable action mechanisms.     

Phospholipases A2 in gastric juice of Helicobacter pylori--positive and negative individuals.

Gastric juice is known to have phospholipase A2 catalytic activity. Helicobacter pylori (H. pylori) has been reported to produce phospholipase A2, which is believed to hydrolyse the protective layer of gastric mucosal phospholipids and to promote mucosal damage. The current study aimed at identifying secretory phospholipase A2 subtypes (pancreatic group I phospholipase A2 and synovial-type group II phospholipase A2) in gastric juice and their relation to the presence of H. pylori in gastric mucosal biopsies in the same individuals. Gastric juice was collected from 29 individuals during gastroscopy. Biopsies were taken from the antrum and body of the stomach to determine the H. pylori status. We found catalytically active phospholipase A2 and both group I and group II phospholipases A2 in the gastric juice samples. The catalytic activity and the mass concentrations of group I and group II phospholipases A2 correlated significantly with the pH value in gastric juice. The gastric juice of H. pylori positive individuals did not contain higher amounts of phospholipases A2 than the juice of H. pylori negative individuals. Rather, the mass concentration of group II phospholipase A2 in gastric juice seemed to be somewhat lower in individuals with H. pylori infection than in uninfected individuals. The results of the current study show that both group I and group II phospholipases A2 are present in gastric juice. The main sources of phospholipases A2 in gastric juice are probably other than H. pylori.     

Virulence gene of H. pylori

H. pylori is a well-recognized pathogen that infects up to 50% of humans in the world. H. pylori lives for decades in the hostile environment of the human stomach. H. pylori is closely associated with histologic gastritis, gastric ulceration, duodenal ulceration, gastric cancer and MALT lymphoma. These various clinical outcomes are considered by 1) different virulence, 2) host response, 3) other environmental factors, and their interactions. Since the whole genome was sequenced in 1997, the virulence genes have been investigated in molecular genetic aspects. The cag pathogenicity island (cagPAI) is a complex of virulence genes, which code approximately 30 proteins. The cagPAI acquired by horizontal transfer and is coding for type 4 secretion machinery system. Via this system, many virulence gene products or other interactive proteins are transferred into the host cells.     

Processing of Mycobacterium tuberculosis antigen 85B involves intraphagosomal formation of peptide-major histocompatibility complex II complexes and is inhibited by live bacilli that decrease phagosome maturation.

Mycobacterium tuberculosis (MTB) inhibits phagosomal maturation to promote its survival inside macrophages. Control of MTB infection requires CD4 T cell responses and major histocompatibility complex (MHC) class II (MHC-II) processing of MTB antigens (Ags). To investigate phagosomal processing of MTB Ags, phagosomes containing heat-killed (HK) or live MTB were purified from interferon-gamma (IFN-gamma)-activated macrophages by differential centrifugation and Percoll density gradient subcellular fractionation. Flow organellometry and Western blot analysis showed that MTB phagosomes acquired lysosome-associated membrane protein-1 (LAMP-1), MHC-II, and H2-DM. T hybridoma cells were used to detect MTB Ag 85B(241-256)-I-A(b) complexes in isolated phagosomes and other subcellular fractions. These complexes appeared initially (within 20 min) in phagosomes and subsequently (>20 min) on the plasma membrane, but never within late endocytic compartments. Macrophages processed HK MTB more rapidly and efficiently than live MTB; phagosomes containing live MTB expressed fewer Ag 85B(241-256)-I-A(b) complexes than phagosomes containing HK MTB. This is the first study of bacterial Ag processing to directly show that peptide-MHC-II complexes are formed within phagosomes and not after export of bacterial Ags from phagosomes to endocytic Ag processing compartments. Live MTB can alter phagosome maturation and decrease MHC-II Ag processing, providing a mechanism for MTB to evade immune surveillance and enhance its survival within the host.     

VacA and HP-NAP, Ying and Yang of Helicobacter pylori-associated gastric inflammation

Helicobacter pylori is a Gram-negative bacterium which infects almost half of the population worldwide and represents the major cause of gastroduodenal pathologies, such as duodenal and gastric ulcer, gastric cancer, B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) and autoimmune gastritis. H. pylori colonization is followed by infiltration of the gastric mucosa by polymorphonuclear cells, macrophages and lymphocytes. Two of the major H. pylori virulence factors are the vacuolating cytotoxin (VacA) and the H. pylori neutrophil-activating protein (HP-NAP). VacA has been proposed as a modulator of immune cell function because of its capacity to interfere with antigen presentation and to inhibit T-cell activation. HP-NAP was designated as neutrophil-activating protein because it stimulates high production of oxygen radicals from neutrophils. We have recently demonstrated that HP-NAP is able to recruit leukocytes in vivo and to stimulate either neutrophils or monocytes to release IL-12, a key cytokine for the differentiation of naive Th cells into the Th1 phenotype. Altogether these evidences indicate that both VacA and HP-NAP play a major role in generating and maintaining the gastric inflammatory response associated with the H. pylori infection.     

Salmonella stimulate macrophage macropinocytosis and persist within spacious phagosomes

Light microscopic studies of phagocytosis showed that Salmonella typhimurium entered mouse macrophages enclosed in spacious phagosomes (SP). Viewed by time-lapse video microscopy, bone marrow-derived macrophages exposed to S. typhimurium displayed generalized plasma membrane ruffling and macropinocytosis. Phagosomes containing Salmonella were morphologically indistinguishable from macropinosomes. SP formation was observed after several methods of bacterial opsonization, although bacteria opsonized with specific IgG appeared initially in small phagosomes that later enlarged. In contrast to macropinosomes induced by growth factors, which shrink completely within 15 min, SP persisted in the cytoplasm, enlarging often by fusion with macropinosomes or other SP. A Salmonella strain containing a constitutive mutation in the phoP virulence regulatory locus (PhoPc) induced significantly fewer SP. Similar to Yersinia enterocolitica, PhoPc bacteria entered macrophages in close-fitting phagosomes, consistent with that expected for conventional receptor-mediated phagocytosis. These results suggest that formation of SP contributes to Salmonella survival and virulence.     

Inhibition of phagosome-lysosome fusion in macrophages by certain mycobacteria can be explained by inhibition of lysosomal movements observed after phagocytosis

We have investigated the mechanism of the inhibition of phagosome-lysosome (P-L) fusion in macrophages known to occur after infection by Mycobacterium tuberculosis and by the mouse pathogen Mycobacterium microti. We have used an M. microti infection and have studied, first, the saltatory movements of periphagosomal secondary lysosomes by means of visual phase-contrast microscopy (a similar use of the method having been previously supported by computer analyses). The movements became slow or static after ingestion of live but not of heat-killed M. microti. They were unaffected by a fusiogenic mycobacterium M. lepraemurium. Second, we studied the behavior of a normally fusiogenic unrelated organism, Saccharomyces cerevisiae, after its phagocytosis by cells already containing live M. microti ingested 18 h previously. We observed, using a fluorescent assay of fusion, that many of these yeast phagosomes now also failed to fuse with the lysosomes; in contrast, when the host M. microti had been heat killed the yeast phagosomes fused normally. These observations were extended by ultrastructural quantitative analyses of P-L fusion, which confirmed the nonfusion of phagosomes of live M. microti and, more particularly, the change to nonfusion from the normal fusion behavior of the separate phagosomes of accompanying yeasts. Third, we have assembled evidence against the likelihood that these M. microti-induced phenomena are nonspecific, i.e., secondary to a general depression of activity of heavily infected host cells. The evidence includes the feasibility of adjusting the degree of infection so as to facilitate visual assessment of organelle movements without the presence of detectable damage to the cells studied; the absence of lysosomal stasis after comparable infection with another mycobacterium of comparable virulence (M. lepraemurium); and the reversibility of the stasis. We conclude that inhibition of lysosome saltatory movements (and consequently its secondary effect on the associated yeasts) is a significant, specifically induced phenomenon. From these observations and considerations, therefore, in conjunction with the analogous inhibition of lysosomal movements in normal macrophages by some chemical inhibitors of P-L fusion, and our suggestion that this association is causally related, we now suggest that M. microti-induced focal lysosomal stasis is also the main means by which the inhibition of P-L fusion is brought about by this organism. This concept is strengthened by the observations on S. cerevisiae, which provide strong evidence that stasis can cause suppression of fusion.     

湖南地区幽门螺杆菌CagA基因3’端多态性与胃小二指肠疾病的关系

目的 探讨湖南地区幽门螺杆菌（Helifcobacter Hpylori）细胞毒素相关基因（CagA基因）3＇端可变区序列特征及其与胃十二指肠疾病的关系.方法 本地区有明显上消化道症状患者235例,其中慢性胃炎（CG）57例,胃溃疡（GU）62例,十二指肠溃疡（DU）70例,胃癌（GC）46例.于胃镜检查时用灭菌活检钳取胃窦组织1块,分离培养出H.pylori 89株,用PCR法对上述菌株的CagA基因扩增及测序,并通过生物信息学软件进行多重序列比对和相似性分析.结果 H.pylori培养阳性率为37.9%（89/235）,其中H.pylori CagA阳性者占91.7%（77/84）,GU组、DU组和GC组CagA阳性率高于CG组,其差异有统计学意义（P〈0.05）.77株H.pylori CagA基因3＇端均具有3个EPIYA重复序列,其中第2个EPIYA序列存在3种突变型,占18.2%（14/77）.H.pylori CagA基因3＇端序列特征以东亚型为主,占88.3%（68/77）,东亚型的CagA阳性菌株在GU组、DU组及GC组高于CG组（P〈0.05）.所有东亚型CagA阳性菌株CagA序列特征类似于Yamaoka报道的A型.结论 湖南地区H.pylori CagA阳性菌株以东亚型为主,均具有3个EPIYA重复序列,其中第2个序列存在3种突变型,其与消化性溃疡和胃癌发生有关.     

幽门螺杆菌感染与胃粘膜林巴滤泡关系探讨

目的：为了探讨含有细胞毒素相关基因A（cagA）菌株感染与胃粘膜淋巴滤泡形成之间的关系,并对胃粘膜淋巴滤泡的发生与幽门螺杆菌（Hp）感染状况的关系等进行观察。方法：我们对655例慢性胃炎,消化性溃疡的患者进行胃镜检查,取胃窦粘膜组织作Hp检测和组织病理检查,并选择70份Hp培养阳性的临床分离菌,用PCR扩增法进行cagA基因的检测。结果：Hp感染患者中胃粘膜淋巴滤泡的发生率（60.14%）显著高于非Hp感染者（17.06%）;胃粘膜淋巴滤泡在活动性胃炎比非活动性胃炎中更易检测到;在Hp相关性胃肠病中,慢性胃炎、胃溃疡、十二指肠球溃这三者之间胃粘膜淋巴滤泡的发生率无显著性差异;另外,还观察到含cagA基因的Hp菌株与胃粘膜淋巴滤泡的增生两者之间无相关性。结论：胃粘膜淋巴滤泡的发生直接与Hp感染相关,并可作为一种Hp感染相关性胃肠病中一个较为恒定的形态特征;Hp作为一种抗原刺激胃粘膜产生淋巴滤泡的作用与Hp菌株的毒力（cagA基因）无关,任何Hp感染均可刺激胃粘膜产生淋巴滤泡。     

Association between Helicobacter pylori cytotoxic type I CagA-positive strains and migraine with aura

Recent studies have suggested an association between Helicobacter pylori infection and migraine. However, various strains of the bacterium are present, some endowed with greater pathogenicity. In particular, H. pylori type I CagA-positive strains induce a higher release of proinflammatory substances by the gastric mucosa that could trigger systemic vasospasms. The aim of the present study was to assess the prevalence of H. pylori CagA-positive strains in subjects with migraine. One hundred and seventy-five patients affected by migraine (49 with aura, 126 without aura) were consecutively enrolled and matched for sex, age, social background and geographical origin with 152 controls. Helicobacter pylori infection was assessed through 13C-urea breath test. Specific serological IgG against CagA were detected through ELISA. The prevalence of H. pylori infection was similar in migraine patients and in controls (40% vs. 39%, respectively). Among migraine patients, prevalence of infection was not related to presence or absence of aura (45% vs. 37%, respectively). However, among infected subjects, a significantly higher prevalence of CagA-positive strains was observed in patients affected by migraine with aura when compared with those affected by migraine without aura (41% vs. 19%, P < 0.01) and with controls (41% vs. 17%, P < 0.01). CagA-positive H. pylori strains were found to be strongly associated with migraine with aura. A higher inflammatory response of the gastric mucosa to more virulent strains could release substances that may act as triggers of vasospasm in peculiar cerebral arterial districts, probably implicated in the 'aura' phenomenon.     

Changes in Haemophilus influenzae capsule locus: possible emergence of novel variants in Brazil

A total of 28 strains of Haemophilus influenzae (Hi) a and b isolated from clinical samples before and after the introduction of the Hib conjugate vaccine in Brazil were analyzed to determine variants of the capsular gene. Our results suggest the occurrence of new variants closely related to types I and II previously described elsewhere. Eleven Hib strains belonged to type I, 8 were type II, and 3 Hia strains were type II. Six strains showed negative results after polymerase chain reaction targeting capsule locus; the variable regions were sequenced and compared with types I and II. Phylogenetic analysis showed that 5 Hib strains were actually subtypes of type I (type I-A), whereas 1 Hia strain was a subtype of type II (type II-A). Types I and II strains were present in both periods of vaccination. This study suggests that a gradual change in the capsule genes of H. influenzae is probably occurring, and novel variants might be emerging among Brazilian isolates.     

幽门螺杆菌的根治对肝硬化患者血氨的影响

目的评价肝硬化患者Hp感染状况对血氨浓度的影响。方法对46例肝硬化伴高血氨患者的血液以及胃液中氨浓度进行分析,所有患者给予低蛋白饮食、卡那霉素、乳果糖以及丰富的支链氨基酸溶液治疗,其中24例患者仍有高血氨,根据Hp感染分布的状况,把24例患者分成三组：Hp在胃中弥漫性分布的患者为Ⅰ组,Hp在胃中局部分布的患者为Ⅱ组,Hp阴性的患者为Ⅲ组。24例患者均给予口服10mg雷贝拉唑,1000mg阿莫西林以及400mg克拉霉素或500mg甲硝唑2周进行Hp的根治。结果第Ⅰ组Hp根治后血液以及胃液中血氨浓度明显下降,Hp根治12周后血氨浓度明显降低（P〈0．05）,第Ⅱ组、Ⅲ组进行Hp根治后血氨浓度未见明显降低。结论胃中Hp弥漫性感染是导致肝硬化患者血氨升高的原因之一,针对Hp弥漫性分布的肝硬化患者,必须进行有效的Hp根治。     

Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses

Helicobacter pylori enhances the risk for ulcer disease and gastric cancer, yet only a minority of H. pylori-colonized individuals develop disease. We examined the ability of two H. pylori isolates to induce differential host responses in vivo or in vitro, and then used an H. pylori whole genome microarray to identify bacterial determinants related to pathogenesis. Gastric ulcer strain B128 induced more severe gastritis, proliferation, and apoptosis in gerbil mucosa than did duodenal ulcer strain G1.1, and gastric ulceration and atrophy occurred only in B128+ gerbils. In vitro, gerbil-passaged B128 derivatives significantly increased IL-8 secretion and apoptosis compared with G1.1 strains. DNA hybridization to the microarray identified several strain-specific differences in gene composition including a large deletion of the cag pathogenicity island in strain G1.1. Partial and complete disruption of the cag island in strain B128 attenuated induction of IL-8 in vitro and significantly decreased gastric inflammation in vivo. These results indicate that the ability of H. pylori to regulate epithelial cell responses related to inflammation depends on the presence of an intact cag pathogenicity island. Use of an H pylori whole genome microarray is an effective method to identify differences in gene content between H. pylori strains that induce distinct pathological outcomes in a rodent model of H. pylori infection.