Comparison of quantum dots immunofluorescence histochemistry and conventional immunohistochemistry for the detection of caveolin-1 and PCNA in the lung cancer tissue microarray

Luminescent semiconductor quantum dots (QDs) are a new class of fluorescent label with wide ranges of applications in cell imaging. In this study, we evaluated the capability of QDs immunofluorescence histochemistry (QDs-IHC) for detecting antigens of caveolin-1 and PCNA in the lung cancer tissue microarray (TMA) in comparison with the conventional immunohistochemistry (IHC) technique. Both methods revealed consistent antigen localization and statistically non-significant detection rates of caveolin-1 and PCNA expressions in our study. However, the sensitivity of QDs-IHC was higher than IHC. The positive detection rates of caveolin-1 and PCNA by QDs-IHC were 57% (40/70) and 86% (60/70), respectively, which were higher than the detection rates of 47% (33/70) and 77% (54/70), respectively, by IHC. Moreover, QDs exhibited a much better photostability, a broader excitation spectrum and a longer fluorescence lifetime. We showed here the advantages of QDs-IHC over IHC for the detection of caveolin-1 and PCNA in lung cancer TMA.     

Diagnosis of Helicobacter pylori infection and determination of clarithromycin resistance by fluorescence in situ hybridization from formalin-fixed, paraffin-embedded gastric biopsy specimens

A reliable diagnostic test for Helicobacter pylori is important in clinical practice and research. The ideal diagnostic test for H. pylori should be sensitive, specific, and cost-effective. Helicobacter pylori resistance to clarithromycin is a common reason for failure of eradication therapy. The aim of this study was to evaluate the fluorescent in situ hybridization (FISH) method to detect H. pylori and determine clarithromycin resistance in formalin-fixed, paraffin-embedded gastric biopsy specimens. One hundred seventeen gastric biopsy specimens from patients with dyspepsia were examined for the presence of H. pylori by conventional culture, FISH, and histopathological methods. A set of fluorescent-labeled oligonucleotide probes binding to either H. pylori 16S rRNA or 23S rRNA sequences were used for FISH analysis. Phenotypic antibiotic susceptibilities of the isolates were tested using the Epsilometer test method (E test). Helicobacter pylori was detected in 70 of 117 biopsy specimens by histopathological examination and FISH, whereas it was detected in 47 specimens by culturing. Histopathology and FISH techniques failed to identify H. pylori in 1 biopsy sample isolated by culture. Clarithromycin resistance was found in 11 of 46 H. pylori isolates using the E test method. All of the phenotypic resistance measurements of isolates were correlated with genotypic clarithromycin resistance. Eleven clarithromycin-resistant strains were identified by FISH. The diagnosis of H. pylori infection and the determination of clarithromycin resistance in formalin-fixed, paraffin-embedded specimens using FISH is promising because it provides a rapid, reliable, and culture-independent diagnosis.     

Lectin histochemistry of the gastric mucosa in normal and Helicobacter pylori infected guinea-pigs

Helicobacter pylori attaches via lectins, carbohydrate binding proteins, to the carbohydrate residues of gastric mucins. Guinea-pigs are a suitable model for a H. pylori infection and thus the carbohydrate composition of normal and H. pylori infected gastric mucosa was investigated by lectin histochemistry. The stomach of all infected animals showed signs of an active chronic gastritis in their mucosa, whereas no inflammation was present in the control animals. The corpus–fundus regions of the controls showed heterogeneous WGA, SNA-I, UEA-I and HPA binding in almost all parts of the gastric glands. While these lectins labelled the superficial mucous cells and chief cells heterogeneously, the staining of the parietal cells was limited to WGA and PHA-L. Mucous neck cells reacted heterogeneously with UEA-I, HPA, WGA and PHA-L. In the antrum, the superficial mucous cells and glands were stained by WGA, UEA-I, HPA, SNA-I or PHA-L. WGA, UEA-I, SNA-I and HPA labelled the surface lining cells strongly. The mucoid glands reacted heterogeneously with WGA, UEA-I, HPA, SNA-I and PHA-L. In both regions, the H. pylori infected animals showed similar lectin binding pattern as the controls. No significant differences in the lectin binding pattern and thus in the carbohydrate composition between normal and H. pylori infected mucosa could be detected, hence H. pylori does not induce any changes in the glycosylation of the mucosa of the guinea-pig. This unaltered glycosylation is of particular relevance for the sialic acid binding lectin SNA-I as H. pylori uses sialic acid binding adhesin for its attachment to the mucosa. As sialic acid binding sites are already expressed in the normal mucosa H. pylori can immediately attach via its sialic acid binding adhesin to the mucosa making the guinea-pig particularly useful as a model organism.This work is dedicated to Professor B. Tillmann on the occasion of his 65th birthday     

Detection of Helicobacter pylori and determination of clarithromycin susceptibility using formalin-fixed, paraffin-embedded gastric biopsy specimens by fluorescence in situ hybridization

BACKGROUND: Clarithromycin resistance and poor compliance to therapy are often responsible for Helicobacter pylori eradication therapy failure. AIM: To evaluate fluorescence in situ hybridization (FISH) as a nonculture method to simultaneously detect H. pylori and to identify clarithromycin resistance. METHODS: Fifty-four patients with dyspepsia (17 male, 37 female subjects; mean age, 46.5; range, 21-78 years) were studied. Two antrum and corpus biopsies were taken from each patient. Positive rapid urease test (RUT) and histopathologic examinations defined H. pylori positivity. A total of 108 formalin-fixed paraffin-embedded gastric mucosal biopsies were examined retrospectively by the FISH (seaFAST H. pylori Combi-Kit) method. RESULTS: Forty-five patients (83.3%) were H. pylori positive and 43 (95.5%) were also positive by FISH. There were two false-positive FISH results. Fourteen patients (31.1%) had clarithromycin-susceptible strains, 4 (8.9%) resistant strains, and 27 (60%) both susceptible and resistant strains. CONCLUSION: FISH results correlated well with H. pylori infection and were able to identify clarithromycin-susceptible and -resistant strains. This technique will be helpful in determining the bacterial density and the success of treatment where clarithromycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.     

胃微生物菌群与幽门螺杆菌感染的胃贲门腺癌发病相关性分析

目的 研究胃微生物菌群与胃贲门腺癌发病的相关性。方法 选择2016年4月—2018年4月在本院就诊的胃贲门腺癌（GCA）患者109例。患者均经病理科组织病理确诊为GCA。对照组为健康人群100例,根据有无H. pylori检出将对照组和GCA组患者分为有H. pylori和无H. pylor,其中对照组有H. pylori检出19例,无H. pylori检出81例,GCA组有H. pylori检出53例,无H. pylori检出56例。用胃镜采集标本,胃镜采集镜下正常的贲门胃黏膜。样本取材后置于无菌冻存管放入液氮中保存,使用16S rRNA的方法进行胃黏膜微生物种类检测。结果 GCA组患者幽门螺杆菌（Helicobacter pylori）感染比例明显高于对照组,无论有无H. pylori感染的对照组人群中,丰度较高的菌属是不动杆菌（Acinetobacter）,罗斯氏菌（Rothhia）,嗜血杆菌（Haemophilus）,拟杆菌（Bacteroides）,链球菌（Streptococcus）,韦荣球菌（Veillonella）,普氏菌（Prevotella）。无H. pylori组内,GCA组样本中普氏菌（Prevotella）、瓦氏菌（Shewanella）、盐单胞菌（Halomonas）丰度明显高于对照组,有H. pylori组内,GCA组样本中普氏菌（Prevotella）、瓦氏菌（Shewanella）、盐单胞菌（Halomonas）、卟啉单胞菌（Porphyromonas）、梭杆菌（Fusobacterium）丰度明显高于对照组,不动杆菌（Acinetobacter）明显低于对照组。有H. pylori的GCA组样本中普氏菌（Prevotella）、瓦氏菌（Shewanella）、盐单胞菌（Halomonas）、卟啉单胞菌（Porphyromonas）、梭杆菌（Fusobacterium）丰度明显高于无H. pylori的GCA组,不动杆菌（Acinetobacter）明显低于无H. pylori的GCA组。结果还发现GCA与普氏菌（Prevotella）、瓦氏菌（Shewanella）、盐单胞菌（Halomonas）、卟啉单胞菌（Porphyromonas）、梭杆菌（Fusobacterium）正相关,与不动杆菌（Acinetobacter）负相关。结论 GCA患者胃内菌群数量和结果与健康人群存在明显差异,H. pylori可能影响胃内菌群结构在GCA发病中发挥作用。     

Comparison of localized gastric mucosa-associated lymphoid tissue (MALT) lymphoma with and without Helicobacter pylori infection

BACKGROUND: The clinical features and clinical course of Helicobacter pylori-negative gastric mucosa-associated lymphoid tissue (MALT) lymphoma are unclear and a treatment strategy has not yet been established. AIM: To clarify the clinical differences between H. pylori-positive and H. pylori-negative gastric MALT lymphoma, we compared these two types of gastric MALT lymphoma. MATERIALS AND METHODS: Fifty-seven patients with localized gastric MALT lymphoma were studied. H. pylori infection was present in 41 and absent in 16. Treatment consisted of antibiotic therapy and/or 30 Gy radiation therapy. Response assessment was performed every 3-6 months by esophagogastroduodenoscopy including gathering biopsy samples, endoscopic ultrasonography, clinical examination, and various imaging procedures. The median follow-up period was 37 months. RESULTS: There were no significant differences between H. pylori-positive and H. pylori-negative gastric MALT lymphoma patients in terms of sex, age, stage, gross phenotype, affected area of the stomach, or the presence of monoclonality. Complete regression was achieved with antibiotic therapy against H. pylori-negative gastric MALT lymphoma in one of nine patients (11.1%), compared to 28 of 38 patients (73.7%) with H. pylori-positive gastric MALT lymphoma (p < .001). Radiation therapy showed high effectiveness for the local control of H. pylori-negative or antibiotic-resistant gastric MALT lymphoma (92.9%), although distant recurrence was recognized in three of 14 patients (21.4%). Two of 16 patients (12.5%) with H. pylori-negative gastric MALT lymphoma died because of the transformation of the disease into diffuse large B-cell lymphoma. There was a significant difference in both the overall and cause-specific survival rate between the two groups (p = .038). CONCLUSION: Radiation therapy is the effective treatment for H. pylori-negative or antibiotic-resistant localized gastric MALT lymphoma. However, careful systemic follow-up for distant involvement should be required. Transformation into diffuse large B-cell lymphoma is thought to be the important cause of death in patients with gastric MALT lymphoma.     

Helicobacter pylori infection and gastric cardia cancer in Chaoshan region

Helicobacter pylori (H. pylori) infection represents the most important risk factor for gastric cancer, while its association with gastric cardia cancer (GCC) has not been recognized yet. In this current study, we aim to investigate the status of H. pylori infection in the gastric cardia tissue samples from high-risk populations in Chaoshan littoral region, and the relationship between H. pylori infection and chronic inflammation as well as the proliferative activity of the gastric cardia epithelial cells. A total of 706 gastric cardia biopsy specimens were obtained from 372 GCC cases and 334 tumor-free controls in Chaoshan littoral, a high-risk region for esophageal and gastric cardia cancer. Immunohistochemistry and Giemsa staining were employed for the verification of H. pylori infection. H. pylori infection rate was significantly higher in GCC (81.5%, P < 0.01) and gastric carditis (80.1%, P < 0.01) in comparison with that in the healthy group (34.8%). A significant higher prevalence of chronic inflammation was found in H. pylori+ samples (96.9%) than that in H. pylori− specimens (80.5%) (P < 0.01). To explore the possible role of H. pylori infection-related chronic inflammation in the GCC, we found that the expression of Ki-67 was progressively increased in tissues with chronic inflammation degrees from normal to severe inflammation (P < 0.01). Collectively, these results suggest that persistent H. pylori infection and the related chronic inflammation may contribute to the high incidence of GCC in Chaoshan littoral.     

The place and role of serologic methods in detecting Helicobacter pylori infection

The aim of the study was to determine the place and role of serologic methods in detecting Helicobacter pylori (H. pylori) infection, on the basis of estimated enzyme-linked immunosorbent assay (ELISA) and complement fixation test (CFT) sensitivity and specificity. A total of 549 patients were included in the study. ELISA and CFT as serologic methods were compared with invasive methods (rapid urease test--CLO test, culture, histology). The sensitivity of serologic methods was above 90%, and their specificity was around 80%. Study results confirmed the value, reliability and usefulness of serologic methods in the detection of H. pylori infection.     

胃黏膜定植乳酸杆菌对幽门螺杆菌感染的影响

目的探讨胃黏膜定植乳酸杆菌对幽门螺杆菌感染及胃黏膜菌群数量的影响。方法收集130例慢性胃炎患者胃窦黏膜组织,病理学检测幽门螺杆菌,提取胃黏膜基因组DNA,采用荧光定量PCR法检测乳酸杆菌和总细菌数。结果幽门螺杆菌阳性组和阴性组的乳酸杆菌检出率和乳酸杆菌数(Log)差异均无统计学意义(P0.05);乳酸杆菌阳性者和阴性者之间胃黏膜总细菌数(Log)差异无统计学意义(P0.05);乳酸杆菌细菌数(Log)与胃黏膜总细菌数(Log)无显著相关性(P0.05);不同炎症程度胃炎患者乳酸杆菌检出率差异无统计学意义(P0.05);肠化组和未肠化组乳酸杆菌检出率差异无统计学意义(P0.05);但是重度胃炎组幽门螺杆菌感染率和总细菌数量均显著高于轻度(P0.05)和中度胃炎组(P0.05)。结论胃黏膜定植乳酸杆菌对幽门螺杆菌的感染无影响,其存在与否及细菌数量对胃黏膜总细菌数亦无明显影响,并与胃炎炎症程度及肠化无关。乳酸杆菌不能通过抑制幽门螺杆菌定植、调节胃黏膜菌群而减轻炎症反应。     

Immunoproteomics of Helicobacter pylori infection and relation to gastric disease

The Gram negative bacterium Helicobacter pylori is a human pathogen which infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer. A systematic, proteome based approach was chosen to detect candidate antigens of H. pylori for diagnosis, therapy and vaccine development and to investigate potential associations between specific immune responses and manifestations of disease. Sera from patients with active H. pylori infection (n = 24), a control group with unrelated gastric disorders (n = 12) and from patients with gastric cancer (n = 6) were collected and analyzed for the reactivity against proteins of the strain HP 26695 separated by two-dimensional electrophoresis. Overall, 310 antigenic protein species were recognized by H. pylori positive sera representing about 17% of all spots separated. Out of the 32 antigens most frequently recognized by H. pylori positive sera, nine were newly identified and 23 were confirmed from other studies. Three newly identified antigens which belong to the 150 most abundant protein species of H. pylori, were specifically recognized by H. pylori positive sera: the predicted coding region HP0231, serine protease HtrA (HP1019) and Cag3 (HP0522). Other antigens were recognized differently by sera from gastritis and ulcer patients, which may identify them as candidate indicators for clinical manifestations. The data from these immunoproteomic analyses are added to our public database (http://www.mpiib-berlin.mpg.de/2D-PAGE). This platform enables one to compile many protein profiles and to integrate data from other studies, an approach which will greatly assist the search for more immunogenic proteins for diagnostic assays and vaccine design.     

Efficacy of immunohistochemical staining in detecting Helicobacter pylori in Saudi patients with minimal and atypical infection

Gastric Helicobacter pylori infection is diagnosed based on histopathological evaluation of gastric mucosal biopsies, urease test, urea breath test, H. pylori culturing, or direct detection using polymerase chain reaction (PCR). This study aimed to evaluate the efficacy of immunohistochemical (IHC) staining in detecting H. pylori in gastric biopsies from patients with chronic gastritis and minimal or atypical infection. Gastric biopsies from 50 patients with chronic gastritis were subjected to routine haematoxylin and eosin (H&E), modified Giemsa, and IHC staining. The results of staining were compared with those of quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis identified 32 (64%) H. pylori-positive cases, whereas IHC, H&E, and modified Giemsa staining identified 29 (58%), 27 (54%), and 21 (42%) positive cases. The sensitivity of IHC staining (87.50%) was higher than that of H&E (59.38%) and modified Giemsa (43.75%) staining. The specificity of H&E, modified Giemsa, and IHC staining was 55.56%, 61.11%, and 94.44%, respectively. IHC staining exhibited the highest diagnostic accuracy (90%), followed by H&E (58%) and modified Giemsa (50%) staining. Active gastritis, intestinal metaplasia, and lymphoid follicles were detected in 32 (64%), 4 (8%), and 22 (44%) cases, respectively, and all of these cases were H. pylori positive. In contrast to routine H&E and modified Giemsa staining, IHC allows for the accurate H. pylori detection in cases with minimal or atypical infection. Moreover, IHC can be an alternative diagnostic method to qRT-PCR for detection of H. pylori in such cases.Key words: Helicobacter pylori, immunohistochemistry, modified Giemsa stain, real-time polymerase chain reaction, chronic gastritis     

Helicobacter pylori genotyping and sequencing using paraffin-embedded biopsies from residents of colombian areas with contrasting gastric cancer risks

Background:   cagA -positive and vacA s1 and m1 genotypes of Helicobacter pylori are associated with an elevated risk of gastric cancer (GC). We determined these genotypes using paraffin-embedded gastric biopsy specimens harvested from infected individuals and compared genotype distributions in two Colombian populations residing in geographic regions with a high and low incidence of GC.
Methods:   DNA from paraffin-embedded gastric biopsies from 107 adults was amplified using primers specific for cagA , for the cag 'empty site', for the s and m alleles of vacA , and for H. pylori 16S rRNA.
Results:   H. pylori infection was detected by molecular assays in 97 (90.7%) biopsies. Complete genotyping of cagA and vacA was achieved in 94 (96.9%) cases. The presence of cagA was detected in 78 of 97 cases (80.4%); when considered separately, cagA and vacA s regions were not significantly associated with a particular geographic area. The vacA m1 allele and s1m1 genotypes were more common in the area of high risk for GC ( p =  .037 and p  = .044, respectively), while the vacA m2 allele and s2m2 genotypes were more prevalent in the low-risk area. The prevalence of the combination of cagA -positive, vacA s1m1 genotypes was 84.3% and 60.5% for high and low risk areas, respectively ( p =  .011).
Conclusions:  H. pylori cagA and vacA genotyping from paraffin-embedded gastric biopsies permitted reliable typability and discrimination. The more virulent cagA- positive s1m1 strains, as well as vacA m1 genotype, were more prevalent in high risk than in low risk areas, which may contribute to the difference in GC risk between those two regions.     

A simple multiplex PCR assay for diagnosing virulent Helicobacter pylori infection in human gastric biopsy specimens from subjects with gastric carcinoma and other gastro-duodenal diseases

AIM: To evaluate and develop a multiplex polymerase chain reaction (PCR) assay for diagnosing and specific identification of virulent Helicobacter pylori strains and their main virulence genes cagA, cagE, cagT, vacA and hrgA. METHODS AND RESULTS: Genomic DNA from 82 gastric tissues was screened. A master pool of all the ingredients of multiplex reaction was prepared for amplification. Amplicons were sequenced to confirm the amplification of each target genes. Multiplex PCR assay was able to detect all the five target genes in 81.7% and deletions in one or more loci among 18.3%. Genotype cagT +ve/hrgA +ve/cagA +ve/cagE +ve/vacAs1 +ve was more predominant in this study population (67.07%). hrgA, cagT, cagE and cagA genes were present in 100%, 92.7%, 85.4% and 81.7% of the subjects, respectively. The vacAs1 subtype had higher prevalence frequency in patients with overt gastrointestinal disease (78.57%) than with GERD (gastro-esophageal reflux disease) and NUD (non-ulcer dispepsia) (50%). CONCLUSIONS: The multiplex PCR assay developed herein was able to genotype H. pylori isolates based on the main virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify H. pylori and the majority of their virulence gene markers by multiplex PCR assay represents a considerable advancement over other PCR-based methods for genotyping H. pylori from large population, and can be explored to gain insights at the genotypic variability exhibited by this pathogen.     

Helicobacter pylori infection promotes methylation of WWOX gene in human gastric cancer

WW domain-containing oxidoreductase (WWOX), a tumor suppressor gene, was reported to be downregulated in gastric cancer and other tumors. However, the mechanism by which WWOX is inactivated remains unclear. In our study, methylation status of WWOX was determined by MSP and sequencing. Our results showed that WWOX hypermethylation was frequently detected in gastric cancer, and also significantly correlated with Helicobacter pylori (H. pylori) infection. Promoter methylation of WWOX was induced in BCG823 and AGS cells co-cultured with H. pylori. Finally, we found that expression of DNMT1 and DNMT3A were enhanced when cells were co-cultured with H. pylori. Our study indicated that H. pylori infection promoted methylation of WWOX gene in gastric cancer.     

Quantitative urease test for Helicobacter pylori infection in patients with gastric and duodenal ulcer

The monitoring for the presence of H. pylori carrier state in a group of patients with gastric and duodenal ulcer was carried out with subsequent determination of the relationship between the intensity of the urease activity of the bioptic specimen of the mucous membrane and the severity of the course of the disease. For this purpose we developed the scheme for the evaluation of the severity of the disease with quantitative criteria. The data obtained in this work showed no correlation between the severity of this disease and usease activity. Still the method of the quantitative determination of the urease activity of the bioptic specimen made it possible to evaluate the rate of the production of hydroxyl anions by a given H. pylori strain and thus quickly establish the presence of carrier state.     

Epithelial cell kinetics of the gastric mucosa during Helicobacter pylori infection

Helicobacter pylori is an important pathogen in major gastroduodenal diseases, including inflammation with ulceration and gastric malignancies. Alterations in H. pylori associated cell turnover in gastric epithelial cells are examined in relation to inflammatory activity, bacteria load and cytokines which may improve knowledge concerning the outcome of gastric diseases caused by H. pylori. Antral biopsies from 42 dyspeptic patients including 27 H. pylori-positive and 15 H. pylori-negative patients were tested for apoptotic activity by the TUNEL assay, and immuno-histochemically for p53 and the proliferative marker Ki-67. H. pylori infection, bacteria load and inflammatory activity were associated with increased cell turnover as judged by enhanced activities of TUNEL, p53 and Ki-67. Only p53 was significantly correlated to IFN-gamma, IL-8 and IL-10. The H. pylori-positive state was furthermore accompanied by varying degrees of altered distribution pattern of the markers studied, with occasional presence of apoptosis in the deeper pit zones, upward extension of Ki-67 and to a lesser degree of p53. Given a similar pattern of change in proliferation and apoptosis in some neoplastic lesions in other parts of the gastrointestinal tract, such studies in cell turnover may provide insights valuable in the investigations of potential precursors of gastric malignancies.