抗幽门螺杆菌血清IgE和胃粘膜肥大细胞在Hp致病中的作用

目的探讨机体免疫反应在幽门螺杆菌（Ｈｐ）致病中的作用。方法采用间接ＥＬＩＳＡ法检测了１４９例患者血清中抗ＨｐＩｇＥ,并用改良甲苯胺蓝染色法检测其胃粘膜中肥大细胞（ＭＣ）。结果①Ｈｐ阳性者血清抗ＨｐＩｇＥ含量、阳性率和胃粘膜中ＭＣ总数及脱颗粒比均显著高于Ｈｐ阴性者（Ｐ＜０．０１）;②不同胃部疾病之间血清抗ＨｐＩｇＥ含量、阳性率和胃粘膜中ＭＣ总数及脱颗粒比有显著差异,活动性胃炎显著高于非活动性胃炎和消化性溃疡（Ｐ＜０．０１）,中重度胃炎显著高于轻度胃炎（Ｐ＜０．００１）;③血清抗ＨｐＩｇＥ阳性者胃粘膜内ＭＣ总数及脱颗粒比均显著高于抗ＨｐＩｇＥ阴性者（Ｐ＜０．０１）,且血清抗ＨｐＩｇＥ含量与胃粘膜内ＭＣ脱颗粒比呈正相关（ｒ＝０．６０,Ｐ＜０．００１）。结论血清抗ＨｐＩｇＥ参与了Ｈｐ的致病过程,其机制可能为刺激ＭＣ脱颗粒,而致胃粘膜损伤。     

Human mast cell tryptase in biology and medicine

The most abundant prestored enzyme of human mast cell secretory granules is the serine-protease tryptase. In humans, there are four tryptase isoforms, but only two of them, namely the alpha and beta tryptases, are known as medically important. Low levels of continuous tryptase production as an immature monomer makes up the major part of the baseline serum tryptase levels, while transient release of mature tetrameric tryptase upon mast cell degranulation accounts for the anaphylactic rise of serum tryptase levels. Serum tryptase determination contributes to the diagnosis or monitoring of mast cell disorders including mast cell activation – induced anaphylaxis, mastocytosis and a number of myeloproliferative conditions with mast cell lineage involvement. Baseline serum tryptase levels are predictive of the severity risk in some allergic conditions.     

Correlation between serological and mucosal inflammatory responses to Helicobacter pylori.

In 82 patients who underwent gastroduodenoscopy, acute and chronic gastric mucosal inflammation was scored for severity, and systemic humoral immune responses to Helicobacter pylori antigens were assessed by enzyme-linked immunosorbent assays. On the basis of culture, gastric histology, and serologic evaluation, 33 patients were classified as H. pylori infected and 36 were classified as uninfected. Thirteen patients had negative cultures and stains but were seropositive and were analyzed separately from the other two groups. Specific serum immunoglobulin G (IgG) subclass responses to H. pylori whole-cell antigens and specific IgG responses to the 54-kDa heat shock protein homolog (Hp54K) and vacuolating cytotoxin were significantly greater in infected than in uninfected patients as were specific IgA responses to whole-cell antigens and cytotoxin (P < 0.001). Among the H. pylori-infected persons, serum IgG responses to Hp54K and to the vacuolating cytotoxin were correlated with acute mucosal inflammatory scores. In contrast, serum IgA responses to whole-cell sonicate and to vacuolating cytotoxin were inversely related to chronic inflammatory scores. By multivariant regression analysis, only specific serum IgG responses to Hp54K correlated with severity of inflammation (both acute and chronic; P < 0.001); these responses may be markers of inflammation or these antibodies could play a direct role in the pathogenesis of H. pylori-induced inflammation.     

Serum pepsinogen I and gastrin concentrations in children positive for Helicobacter pylori.

Serum pepsinogen I, serum gastrin concentration, and inflammatory scores were measured in a population of 71 children undergoing upper gastrointestinal endoscopy for investigation of upper abdominal pain. Forty four were initially colonised with Helicobacter pylori. The indices were measured before treatment (in 71 children), one month (in 41 children), and six months (in 21 children) after stopping treatment. Before treatment there was a significant correlation between serum pepsinogen concentration, total inflammatory score, and H pylori state, but no correlation between serum gastrin concentrations and H pylori state. Similarly, the total inflammatory score and serum pepsinogen concentrations were significantly correlated. There was no such correlation in children negative for H pylori. After treatment the inflammatory score improved in those patients in whom H pylori had been eradicated. There was also a significant fall in serum pepsinogen I and serum gastrin concentration in those patients in whom H pylori had been eradicated. These results were similar to those found six months after treatment had been stopped. These findings suggest that the serum pepsinogen I concentration could be considered a useful marker for gastritis and can be used as an index of severity of gastritis in H pylori positive subjects. The measurement of serum gastrin concentrations does not give useful information.     

Diagnosis of Helicobacter pylori infection by specific gastric mucosal IgA and IgG pylori antibodies.

AIMS--To investigate the diagnostic value of mucosal IgA and IgG Helicobacter pylori antibodies. METHODS--The study population comprised 209 consecutive patients with severe dyspeptic complaints referred for upper gastrointestinal endoscopy. A positive culture or histological identification of H pylori in gastric biopsy specimens, or both, were used to confirm infection. Specific IgA and IgG H pylori antibodies were determined using a modified ELISA technique. RESULTS--Of the 209 patients, 137 were infected with H pylori. The diagnostic value of systemic IgA and IgG H pylori antibodies was confirmed. Systemic IgA antibodies had a sensitivity of 76.6% (95% confidence interval 69.5-83.7) and a specificity of 94.4% (89.1-99.7). The sensitivity and specificity for systemic IgG antibodies were, respectively, 97.1% (94.3-99.9) and 98.6% (95.9-100). A moderate but clinically important correlation was found between local and systemic IgA and IgG. Mucosal IgA H pylori antibodies had a sensitivity of 98.5% (96.5-100) and a specificity of 91.7% (85.3-98.1), while for IgG these figures were, respectively, 88.3% (82.9-93.7) and 98.6% (95.9-100). As a diagnostic test mucosal IgA H pylori antibodies were comparable with culture and histology. CONCLUSION--Determination of local IgA and IgG H pylori antibody levels is a highly sensitive and specific test for the diagnosis of H pylori infection.     

Circulating cytokines and gastrin levels in asymptomatic subjects infected by Helicobacter pylori (H. pylori)

The pathophysiology of hypergastrinemia in H. pylori infection has been largely investigated and different reports clearly show that the infected antrum has a marked inflammatory response with a suggestive local production of cytokines. Notwithstanding, a few data are available on the circulating levels of cytokines and gastrin in the asymptomatic people carrying H. pylori infection. Thus, aim of the study was to evaluate circulating proinflammatory cytokines [Interleukin (IL)-8, Interleukin (IL)-10, Interferon (IFN)-gamma, and Tumor Necrosis Factor (TNF)-alpha] and gastrin levels in H. pylori positive asymptomatic subjects vs. H. pylori negative ones. To this end, thirty healthy volunteers with no digestive symptoms or systemic disease were enrolled and H. pylori infection was identified by a 13C-urea breath test. Plasma levels of gastrin were determined using the RIA kit whereas IL-8, TNF-alpha, IL-10, and IFN-gamma levels in serum were measured with a solid-phase ELISA. Fifteen infected people showed significantly higher gastrin and TNF-alpha levels than uninfected subjects. On the contrary, IL-8 levels were significantly higher in the uninfected subjects than in H. pylori positive ones (P < 0.0422). IFN-gamma and IL-10 circulating levels were not affected by H. pylori presence, being not significantly different in the two groups.     

The nontoxic CTA1-DD adjuvant enhances protective immunity against Helicobacter pylori infection following mucosal immunization

Safe and efficacious adjuvants are much needed to facilitate the development of mucosal vaccines. Here, we have asked whether our nontoxic vaccine adjuvant, CTA1-DD, can enhance protective immunity against Helicobacter pylori infection. Intranasal immunizations with H. pylori lysate together with CTA1-DD-adjuvant induced significant protection in C57Bl/6 mice, almost as strong as similar immunizations using cholera toxin (CT)-adjuvant. Protection remained strong even at 8 weeks postchallenge and the bacterial colonization was reduced by 20-fold compared to lysate-immunized controls. Although CTA1-DD was designed to bind to B cells, microMT mice developed significant, but lower, level of protection following immunization. Intranasal immunizations with CT adjuvant in C57Bl/6 mice resulted in the development of severe postimmunization gastritis at 2 and 8 weeks postchallenge, whereas the degree of gastritis was substantially lower in the CTA1-DD-immunized mice. Protection induced by both CTA1-DD- and CT adjuvant was associated with a strong local infiltration of CD4(+) T cells in the gastric mucosa, and recall responses to specific Ag elicited substantial IFN-gamma production, indicating Th1-dominance. These findings clearly demonstrate that CTA1-DD adjuvant is a promising candidate to be further exploited in the development of a mucosal vaccine against H. pylori infection.     

Purification of tryptase from a human mast cell line

The neutral protease tryptase has been isolated from a human mast cell line, HMC-1. The HMC-1 line was established from the peripheral blood of a patient with mast cell leukemia and maintained as continuously proliferating clones in vitro and as solid mast cell tumors in nude mice. HMC-1-derived tryptase was purified by sequential chromatography on Dowex 1, DEAE 5 PW, and heparin-agarose. Purified tryptase has an apparent molecular weight of 150,000, as determined by molecular sieve HPLC, but migrates as a doublet of bands of 32/35,000 on SDS-PAGE gels. Maximal enzymatic activity was observed at pH 8.5. Cleavage of tosyl-L-arginine methyl ester by purified tryptase was inhibited by dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone 2 HCl, HgCl2, tosyl-L-lysine chloromethyl ketone, leupeptin, and PMSF but not by benzamidine, aprotinin, tosyl-L-phenyl-alanine chloromethyl ketone, soybean trypsin inhibitor, human plasma, ovomucoid inhibitor, or lima bean trypsin inhibitor. Microsequencing of purified tryptase yielded an amino terminal sequence that was identical to that previously reported for human pituitary-derived tryptase.     

Relationship of CagA to serum gastrin concentrations and antral G, D cell densities in Helicobacter pylori infection.

The purpose of this study was to investigate whether the densities of antral gastrin and somatostatin-immunoreactive cells in Helicobacter pylori (H. pylori) infection were related to the bacterial expression of cytotoxin-associated gene A (CagA). 32 patients who had underwent diagnostic esophagogastroduodenoscopy were studied. On the histologic examination all patients had antral gastritis. We divided the subjects into three groups. Group I consisted of 6 patients who had chronic superficial gastritis, group II, 9 patients who had H. pylori-associated gastritis but with no expression of CagA, and group III, 17 patients who had H. pylori-associated gastritis with the expression of CagA. In group I and II, serum gastrin levels, and antral G cell and D-cell were measured. In group III, serum gastrin levels, and antral G cell and D-cell were measured, before and after the eradication of H. pylori. The results were as follows. Firstly, serum gastrin concentrations were significantly higher in the patients with H. pylori infection than in the negative controls. Nextly, there was no correlation between the changes in antral G or D-cell density and H. pylori infection. Thirdly, group III had a significant increase in serum gastrin concentrations and a significant decrease in antral D-cell density than group I. Forthly, eradication of H. pylori in group III showed a significantly increased antral D-cell density. Our results suggest that hypergastrinemia in H. pylori-associated gastritis is relevant to the presence of CagA, and the possible mechanism of hypergastrinemia may be related to antral D-cell deficiency, which is caused by H. pylori infection with the expression of CagA.     

Possible circadian variation of serum mast cell tryptase concentration

BACKGROUND: A temporarily elevated level of serum mast cell tryptase (ST) indicates mast cell activation and occurs in systemic anaphylactic reactions (SAR). We measured ST following a sting challenge in vespid venom-allergic patients treated with venom immunotherapy (VIT) and in healthy controls, respectively. AIM OF THE STUDY: To assess changes of ST over time in vespid venom-allergic patients at the occasion of a re-sting and in healthy controls. METHODS: A sting challenge was performed in 20 patients on vespid VIT to monitor efficacy of VIT. ST was measured between 9.00 and 10.00 a.m. (baseline). Sting challenge was performed at 2.00 p.m., and ST was determined again 20 min, 90 min and 18 h later. Measurements at corresponding times of the day were done in nine healthy controls. RESULTS: One patient developed a mild SAR to the sting challenge which was associated with a temporary increase of ST. In the other 19 patients who tolerated the sting challenge without SAR ST decreased significantly by 18.0% (median, range 8.3-36.7%). Twenty minutes after the sting when compared with baseline levels (P < 0.001), a significant decrease of ST was still present after 90 min (median 13.7%) (P < 0.001), but not after 18 h (P = 0.57). A comparably significant temporary decline was found in controls. CONCLUSIONS: The temporary decline of ST in patients and in controls suggests a circadian variation of ST concentration. A normal diurnal pattern of ST concentration after sting challenge is associated with successful treatment.     

幽门螺杆菌毒素相关蛋白CagA上调胃泌素基因表达

目的 对幽门螺杆菌分泌的cagA蛋白能否调控胃泌素基因表达及作用机制进行研究.方法 用含有cagA基因的真核表达载体——pcDNA3.1ZEO(-)/cagA7转染AGS和SGC-7901细胞;同时培养幽门螺杆菌NCTC11637后感染AGS和SGC-7901细胞;加入JAK2信号通路抑制剂AG490和ERK信号通路抑制剂U0126,实时荧光定量PCR检测转染和感染细胞中胃泌素mRNA的表达.结果 用PeDNA3.1ZEO(-)/eagA7转染和NCTC11637感染胃癌细胞AGS和SGC-7901后胃泌素mRNA表达最显著增加(P＜0.05),但加入AG490和U0126后胃泌素mRNA的表达量显著降低(P＜O.05).结论 cagA上调胃泌素基因的表达,ERK/MAPK和JAK/STAT信号通路参与了CagA对胃泌素表达的调控.     

绝经前后子宫内膜息肉中肥大细胞类胰蛋白酶及类糜蛋白酶的表达及意义

目的探讨肥大细胞类胰蛋白酶(MCT)及类糜蛋白酶(MCC)在绝经前、后子宫内膜息肉组织(EP)中的表达及临床意义。方法收集120例患者标本,其中绝经前EP组和绝经后EP组各35例,绝经前正常增生期子宫内膜组和绝经后正常萎缩性子宫内膜组各25例。采用Max VisionTM/HRP免疫组织化学染色法,检测各组中MCT及MCC阳性的肥大细胞(MCs)数量并分析临床意义。结果绝经前、后EP组和正常子宫内膜组中MCT和MCC阳性的MCs计数均值分别为高倍镜下(11.82±5.24)个和(2.94±2.20)个、(4.18±2.32)个和(2.18±1.52)个、(2.19±1.80)个和(0.49±0.60)个以及(0.35±0.32)个和(0.19±0.26)个。两两比较结果显示,绝经前、后EP组MCT和MCC阳性的MCs数量明显高于同期正常子宫内膜组(P0.05);在EP组中,MCT阳性的MCs数量在绝经前高于绝经后(P0.05),而MCC阳性的MCs数量没有明显差异(P0.05);在正常子宫内膜组中,MCT和MCC阳性的MCs数量在绝经前均高于绝经后(P0.05)。结论肥大细胞(MCs)的过度活化以及伴随的炎症性损害可能是绝经前、后子宫内膜息肉形成及发展的原因。     

Effect of human mast cell tryptase on human plasma proenzymes

The effect of human skin mast cell tryptase on human plasma proenzymes (prothrombin, coagulation factor XII, complement C1s, protein C and plasminogen) was investigated. Tryptase had no effect on these proenzymes, when incubated with them at 37 degrees C for up to 90 min, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the ability to hydrolyze specific peptide p-nitroanilide substrates. After prolonged treatment with tryptase, proenzymes could be fully activated with their specific activators. The results indicate that tryptase neither activates these plasma proenzymes nor inactivates the corresponding active enzymes. As a positive control, the tryptase preparation was also incubated with human fibrinogen and rat thymus histones. Prolonged treatment with tryptase increased the thrombin-induced clotting time of fibrinogen. Tryptase also efficiently hydrolyzed histone H1 from rat thymus. Histones H3/H2B and H2A were hydrolyzed less efficiently than H1, and no hydrolysis of histone H4 by tryptase was detected under the experimental conditions.     

Helicobacter pylori cytotoxin induces vacuolation of primary human mucosal epithelial cells.

We investigated whether Helicobacter pylori cytotoxin induces vacuolation in primary epithelial cells from normal human mucosa. Epithelial cells purified by enzyme digestion and elutriation were evaluated for vacuolation in a blinded protocol by light and electron microscopy before and after incubation with culture supernatant (CS) from H. pylori 60190, which has vacuolating activity for HeLa cells (Tox+), and isogenic H. pylori mutant 60190-v1, which lacks this activity (Tox-). Primary epithelial cells (>98% pure) exposed to CS from Tox+ H. pylori exhibited marked vacuolation (52% +/- 5% of cells) compared with epithelial cells exposed to either CS from Tox- H. pylori (23% +/- 3.2%) or uninoculated control broth (23% +/- 3.7%) (P < 0.05) by light microscopy, which was confirmed by electron microscopy and antibody inhibition studies. These are the first data to show that H. pylori cytotoxin causes vacuolation of primary human mucosal epithelial cells.     

Helicobacter pylori.

Helicobacter pylori is a gram-negative bacterium which causes chronic gastritis and plays important roles in peptic ulcer disease, gastric carcinoma, and gastric lymphoma. H. pylori has been found in the stomachs of humans in all parts of the world. In developing countries, 70 to 90% of the population carries H. pylori. In developed countries, the prevalence of infection is lower. There appears to be no substantial reservoir of H. pylori aside from the human stomach. Transmission can occur by iatrogenic, fecal-oral, and oral-oral routes. H. pylori is able to colonize and persist in a unique biological niche within the gastric lumen. All fresh isolates of H. pylori express significant urease activity, which appears essential to the survival and pathogenesis of the bacterium. A variety of tests to diagnose H. pylori infection are now available. Histological examination of gastric tissue, culture, rapid urease testing, DNA probes, and PCR analysis, when used to test gastric tissue, all require endoscopy. In contrast, breath tests, serology, gastric juice PCR, and urinary excretion of [15N]ammonia are noninvasive tests that do not require endoscopy. In this review, we highlight advances in the detection of the presence of the organism and methods of differentiating among types of H. pylori, and we provide a background for appropriate chemotherapy of the infection.     

Evaluation of Helicobacter pylori vacA genotype in Japanese patients with gastric cancer.

AIM: To examine the vacA genotypes of Helicobacter pylori strains in Japan and to define whether any specific genotype was associated with gastric cancer. METHODS: The allelic variation of vacA gene was studied using a recently introduced polymerase chain reaction based vacA genotyping system. RESULTS: 80 H pylori strains were isolated from gastric biopsies of 40 patients with gastric cancer and 40 control subjects in a Japanese population. All strains were s1/m1 subtype and 79 of 80 strains were classified as s1a subtype. CONCLUSIONS: The recently proposed vacA genotyping system is applicable to Japanese H pylori strains and most strains have the s1a genotype, associated with increased virulence. While the high frequency of s1a/m1 vacA genotype might play a role in the increased incidence of atrophic gastritis and gastric cancer in Japanese subjects, it precludes its use as a predictor of clinical outcome of H pylori infection in Japan.