Human Gastric Biopsies Research Articles

Lipopolysaccharide Diversity Evolving in Helicobacter pylori Communities through Genetic Modifications in Fucosyltransferases

Helicobacter pylori persistently colonizes the gastric mucosa of half the human population. It is one of the most genetically diverse bacterial organisms and subvariants are continuously emerging within an H. pylori population. In this study we characterized a number of single-colony isolates from H. pylori communities in various environmental settings, namely persistent human gastric infection, in vitro bacterial subcultures on agar medium, and experimental in vivo infection in mice. The lipopolysaccharide (LPS) O-antigen chain revealed considerable phenotypic diversity between individual cells in the studied bacterial communities, as demonstrated by size variable O-antigen chains and different levels of Lewis glycosylation. Absence of high-molecular-weight O-antigen chains was notable in a number of experimentally passaged isolates in vitro and in vivo. This phenotype was not evident in bacteria obtained from a human gastric biopsy, where all cells expressed high-molecular-weight O-antigen chains, which thus may be the preferred phenotype for H. pylori colonizing human gastric mucosa. Genotypic variability was monitored in the two genes encoding α1,3-fucosyltransferases, futA and futB, that are involved in Lewis antigen expression. Genetic modifications that could be attributable to recombination events within and between the two genes were commonly detected and created a diversity, which together with phase variation, contributed to divergent LPS expression. Our data suggest that the surrounding environment imposes a selective pressure on H. pylori to express certain LPS phenotypes. Thus, the milieu in a host will select for bacterial variants with particular characteristics that facilitate adaptation and survival in the gastric mucosa of that individual, and will shape the bacterial community structure.

Isolation of lactobacilli with probiotic properties from the human stomach

Recent evidence suggests that the human gastric microbiota is much more diverse than previously thought. The aim of this study was to assess the potential for isolating lactobacilli from the human stomach. Lactobacilli were selectively cultured from gastric biopsies from 12 patients undergoing routine endoscopy. Lactobacilli were present in four of 12 biopsies. We isolated, in total 10 different strains representing five species (Lactobacillus gasseri, L. fermentum, L. vaginalis, L. reuteri and L. salivarius). The 10 isolates varied greatly in their ability to inhibit the growth of two Gram-positive bacteria and two Gram-negative bacteria. Furthermore, the acid and bile resistance profiles of the 10 isolates spanned a wide range. Five different Lactobacillus species were cultured from human gastric biopsies for the first time. Diverse Lactobacillus species are more prevalent in the human stomach than previously recognized, representing an untapped source of bacteria with beneficial probiotic and/or biotechnological properties.

Co-culture of human gastric endoscopic biopsies with Helicobacter pylori: A simple method for studying early phases of bacteria–host interaction

Gastric biopsies obtained through endoscopy from patients uninfected by Helicobacter pylori were co-cultured with an H. pylori strain. According to tissue and H. pylori viability, interleukin 8 was increased in biopsy homogenate and supernatant after 12–36h culture. This simple method is suitable to investigate early phases of bacteria–host interaction.

Evaluation of a Nested-PCR assay based on the phosphoglucosamine mutase gene ( glmM) for the detection of Helicobacter pylori from raw milk

Helicobacter pylori is an organism widespread in the human population and sometimes responsible for serious illnesses. Since H. pylori has been detected in Italy from an high percentage of sheep milk samples, it has been hypothesized that contaminated milk, may act as a vehicle of transmission of the microorganism to humans. In this work, a Nested Polymerase Chain Reaction approach has been used to detect H. pylori phosphoglucosamine mutase gene ( glmM) from sheep, goat and cow milk artificially contaminated with wild H. pylori strains isolated from human gastric biopsies and the reference strain ( H. pylori ATCC 43504). The technique showed a high sensitivity of 3 CFU/ml and proved to be both specific and rapid, The authors suggest that it could be used as a sensitive method for a rapid screening of sheep, goat and cow milk samples during the microbiological control of these large consumed foods.

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

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. 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%). The multiplex PCR assay developed herein was able to genotype H. pylori isolates based on the main virulence genes. 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.

Isolation and characterization of human gastric cell lines with stem cell phenotypes

The aim of this study was to develop an in vitro human gastric stem and/or progenitor cell model that may be used to study the mechanism of gastric carcinogenesis induced by Helicobacter pylori infection. Human gastric biopsy was minced and digested with collagenase and dispase and cultured in a low-calcium medium (serum-free keratinocyte medium; keratinocyte-SFM) supplemented with N-acetyl-L-cysteine and L-ascorbic acid 2-phosphate. Actively proliferating epithelial colonies with sustained growth were isolated and characterized for karyotype and phenotypes related to stem cell characteristics including proliferation and differentiation potential, ability of anchorage-independent growth (AIG), gap junctional intercellular communication (GJIC) and the expression of Oct-4, a transcription factor previously shown to be expressed in embryonic stem cells, adult stem cells and undifferentiated tumor cells. To study the carcinogenic effect of H. pylori infection, gastric stem and/or progenitor cells were incubated with H. pylori culture products and/or N-methyl-N-nitro-N-nitrosoguanidine (MNNG), a chemical carcinogen, to see the telomerase activation. Multiple cell lines with stem cell features were isolated by this new cell culture method. The results based on detailed characterization of one cell clone, KMU-GI2, revealed stem cell features of these cells. The initial clone contained mostly undifferentiated epithelial-like cells, which, upon subculture and propagation, gave rise to a heterogeneous cell population. Single cell-derived subclones, similar to the parental population, retained high differentiation potential and were capable of giving rise to many morphologically different cell types (i.e. epithelial-like, glial or neuron-like, round and various peculiar-shaped cells). Although these cells were normal in karyotype and competent in GJIC, they had the ability to grow in soft agar. Cells expressing epithelial membrane antigen (EMA), mucin 5AC, glial fibrillary acidic protein (GFAP), cytokeratin-18 (CK-18), trefoil factor 1 (TFF-1) and Oct-4 were found in the cell culture, but not E-cadherin-, gastrin- or telomerase-expressing cells. Furthermore, spontaneously immortalized non-tumorigenic clones could be derived from the cell population. After treating these cell cultures with the chemical carcinogen, MNNG and H. pylori culture products for 5 days, telomerase activity and telomerase mRNA expression were significantly elevated, while treatment with either of them showed no effect. The new cell culture method can be used to develop gastric epithelial cell clones with sustained growth from endoscopic biopsy. The gastric cell clone showed several stem and/or progenitor cell phenotypes (i.e. the ability of AIG, high differentiation capacity, high susceptibility to spontaneous immortalization and the expression of Oct-4). The telomerase expression in these gastric stem and/or progenitor cells can be upregulated by exposure to H. pylori culture products and MNNG, an important step in neoplastic transformation. These results show that putative human gastric stem and/or progenitor cell clones can be developed by our method and these cells could be useful for studying the mechanisms of human gastric carcinogenesis including the mechanism of action of H. pylori, as well as the regulation of the proliferation and differentiation of human gastric mucosa.

HorB (HP0127) is a Gastric Epithelial Cell Adhesin

The Helicobacter pylori protein HorB (encoded by HP0127) is a member of a paralogous family that includes the adhesins BabA, AlpA, AlpB, and HopZ, which contribute to adhesion to gastric epithelial cells. Of the verified H. pylori porins, the HorB sequence is most similar to that of HopE, but the function of HorB is unknown. The aim of our study was to investigate the role of HorB in H. pylori gastric epithelial cell adhesion. We disrupted the horB gene in H. pylori and measured the adhesion to gastric epithelial cells (AGS cells). We then assessed the effect that HorB disruption had on lipopolysaccharide (LPS) O-chain production and Lewis x and Lewis y antigen expression. A HorB mutant in the mouse-adapted strain H. pylori SS1 was created by marker exchange and mouse stomach colonization was quantified. Using reverse transcription polymerase chain reaction, human gastric biopsy material from H. pylori-infected patients was then examined for expression of the horB gene. Disruption of the horB gene reduced H. pylori adhesion by more than twofold. Adhesion in the horB knockout strain was restored to wild-type levels by re-introduction of HorB into the chromosome. Disruption of HorB reduced production of LPS O-chains and lowered the level of expression of Lewis x and Lewis y antigens. Insertional mutagenesis of the horB gene in H. pylori SS1 reduced mouse stomach colonization threefold. Finally, expression of the horB gene was detected in human gastric biopsy material from H. pylori-infected patients. From these data we conclude that HorB has a role in H. pylori adhesion during infection.

Characterization of acid/base transporters in parietal cells isolated from human gastric biopsy samples

Background: The acid/base transporters of human parietal cells have not been characterized yet. However, these transporters may play a role in the mechanism of acid secretion. The aim of this study was to investigate the transporters of the parietal cells obtained from human biopsy samples and dissects the mechanisms relevant to human pathology. Methods: Gastric glands were isolated from human biopsy samples by enzymatic digestion and then cultured for 48h. Parietal cells were identified using FITC-conjugated Dolichos biflorus lectin. The intracellular pH (pHi) of parietal cells was measured by using the fluorescence dye BCECF-AM. Results: Removal of Na+ from the standard HEPES solution perfusing the parietal cells caused a rapid and marked intracellular acidosis. Re-introduction of Na+ to the solution caused a complete pHi recovery. Since the solution did not contain HCO3-, this finding confirms the presence of a Na+ dependent H+ efflux mechanism in parietal cells. The Na+/H+ exchangers could be partially blocked by amiloride. The administration of standard HCO3-/CO2 rapidly and greatly decreased pHi. pHi recovery was not detected after the acidification suggesting the marginal role of HCO3- efflux into the parietal cells. In the absence of HCO3-, Cl- removal caused a small reversible alkalinization in parietal cells suggesting the small availability of HCO3- ions in the cytoplasm. However, in HCO3- containing solution a significantly higher alkalinization was observed. These results confirm functionally active Cl-/HCO3- exchange mechanisms in parietal cells. We found that histamin could stimulate proton secretion which could be blocked by ranitidine and pantoprazole. Conclusions: The fluorescence measurements revealed that human parietal cells express functionally active Na+/H+ and Cl-/HCO3- exchangers and proton pumps. However, we could not identify functionally active Na+/HCO3- co-transporters. This method is also suitable to study the regulation of the proton pump. This work was supported by OTKA, MTA and OM

Quantitative real-time PCR for the clinical detection of Helicobacter pylori

Accurate diagnosis of Helicobacter pylori infection is very important in both clinical practice and research. We evaluated the sensitivity of real-time PCR (RT-PCR) for the detection and quantification of Helicobacter pylori using DNA from 91 human gastric biopsy samples divided into three groups: 46 biopsies from untreated patients who according to the references methods were considered H. pylori-negative (group A); 35 biopsies from patients previously treated against H. pylori and considered to be cured by "gold standard" tests (group B); and 10 biopsies from patients H. pylori-positive by all available methods (group C). The sensitivity of the RT-PCR assay was higher than that of standard methods. Of the 81 patients considered to be uninfected according to the references methods, 16 were H. pylori-positive by PCR, 10 of which were patients who had received H. pylori eradication therapy and 6 were untreated patients. Based on these findings we recommend that RT-PCR should be use in addition to standard methods in clinical studies to monitor the results of H. pylori eradication therapy.

Ultrastructure of Helicobacter pylori in human gastric mucosa and H. pylori-infected human gastric mucosa using transmission electron microscopy and the high-pressure freezing–freeze substitution technique

Conventional ultrastructural analyses of Helicobacter pylori and H. pylori-infected gastric mucosa by transmission electron microscopy (TEM) have limitations because of structural artifacts introduced during fixation. We used high-pressure freezing (HPF) followed by freeze substitution for TEM to investigate the ultrastructure of H. pylori and H. pylori-infected gastric mucosa. For HPF-freeze substitution, human gastric biopsy specimens were placed in the HPF instrument at a pressure of approximately 2,000 atm at the temperature of liquid nitrogen. Specimens were then transferred to an instrument for freeze substitution. Specimens were first placed in acetone containing 2% OsO4 at -85 degrees C. The temperature was increased to -3 degrees C, followed by embedding in Quetol 812. Ultrathin sections were double-stained by uranium acetate/lead nitrate. HPF and conventionally prepared samples were examined by TEM. The H. pylori envelope was clearly seen to consist of an outer membrane, periplasmic space, and plasma membrane. The periplasmic space was filled with electron-dense materials. A peptidoglycan layer was only occasionally visible. A thick, very fine filamentous or reticular fringe corresponding to the bacterial glycocalyx was seen surrounding the H. pylori cells. At the adhesion loci of H. pylori to gastric epithelial cells, H. pylori was connected to the epithelial cells by very fine, thickly arranged filaments or more closely, with a contact zone. The epithelial cells showed indentations or pedestals. The well-developed, thick bacterial glycocalyx of H. pylori appears to strongly interact with external cellular components and may play an important role in the adhesion of H. pylori to epithelial cells.

Gastric Epithelial Expression of Macrophage Migration Inhibitory Factor is not Altered by Helicobacter pylori Infection in Humans

Recent reports have shown an upregulation of macrophage migration inhibitory factor (MIF) during gastric ulcer development in a rat model and elevated counts of MIF-positive cells in biopsies from Helicobacter pylori-infected patients. H. pylori infection is a proven cofactor in humans causing gastritis and gastric ulcers. The aim of this study was to characterize MIF expression in human gastric epithelial cells in response to H. pylori. MIF mRNA and MIF protein expression was detected in human gastric epithelial cell lines after stimulation with proinflammatory cytokines or infection with H. pylori (cagA+/vacA+) using real-time reverse transcriptase-polymerase and enzyme-linked immunosorbent assay. Interleukin-8 secretion was measured as positive control. MIF mRNA and MIF protein expression was assessed in H. pylori-positive and -negative human gastric biopsy samples. While interleukin-8 mRNA expression and interleukin-8 secretion were upregulated in gastric epithelial cells in vitro after H. pylori infection, no changes in MIF mRNA expression and MIF secretion could be detected. We found no significant differences in MIF expression in total RNA extracted from gastric biopsy tissue when comparing H. pylori-positive to control patients. Likewise, MIF protein expression in gastric epithelium was unaffected by H. pylori infection as compared to uninfected tissue. While an increased MIF expression and positive effects of MIF blockade in ulcer healing have been shown in a rodent model and elevated numbers of MIF-positive cells have been found in H. pylori-infected human tissue, we herein could not confirm any differences in human gastric epithelial MIF expression and secretion after H. pylori infection in vitro and in vivo.

Expression profile of mucins (MUC2, MUC5AC and MUC6) in Helicobacter pylori infected pre-neoplastic and neoplastic human gastric epithelium.

BackgroundHelicobacter pylori (H. pylori) causes gastritis and intestinal metaplasia (IM) that may evolve to gastric carcinoma. The objective of this study was to compare the profile of mucins in the progressive stages of H. pylori infected pre-neoplastic and neoplastic human gastric epithelium. We used a panel of monoclonal antibodies with well-defined specificities of MUC2, MUC5AC and MUC6 to characterize the expression pattern of mucins by immunohistochemistry.MethodsRUT and ELISA were down for H. pylori confirmation. Human gastric biopsy sections were stained using immunohistochemistry with MUC2, MUC5AC and MUC6 antibodies.ResultsMUC5AC was expressed in the superficial epithelium and the upper part of the gastric pits. MUC6 expression was detected in the lower part of the gastric glands. MUC2 was expressed in intestinal metaplasia, mostly in goblet cells. The mucin expression profile in the progressive stages of H. pylori infected human gastric epithelium allows the identification of intestinal metaplasia, which is characterized by a decreased expression of the gastric mucins (MUC5AC and MUC6) and de novo expression of MUC2.ConclusionIn conclusion, our results suggest that there is altered expression of MUC5AC and MUC6 together with the aberrant expression of MUC2 in intestinal metaplasia, during the process of gastric carcinogenesis. The present study indicates that the MUC2 mucin expression pattern is a reliable marker of intestinal metaplasia, which appears in the context of H. pylori infected individuals.

Helicobacter pylori regulates the expression of inhibitors of DNA binding (Id) proteins by gastric epithelial cells

Id transcription factors control proliferation, differentiation and apoptosis by inhibiting the DNA binding of basic helix-loop-helix transcription factors. Increased expression of Id proteins promotes proliferation, inhibits differentiation, and is associated with intestinal tumorigenesis. We aimed to determine how Helicobacter pylori may alter the expression of Id proteins by gastric epithelial cells: it was hypothesised that H. pylori, a known carcinogen, would result in increased expression of one or more Id family members. In vitro and in vivo models of infection were employed, including treatment of AGS gastric epithelial cells with wild-type H. pylori strains, 60190 and SS1, and Mongolian gerbils infected with H. pylori SS1. A small cohort of human gastric mucosal biopsies was also examined. Treatment of AGS cells with H. pylori resulted in down-regulation of Id1 and Id3. Unexpectedly, expression of the main target of Id proteins, the basic helix-loop-helix transcription factor E2A, was also suppressed, with an associated decrease in E-box binding activity. In contrast, H. pylori induced the expression of the CDK inhibitor p21 WAF-1/cip1, and the homeobox transcription factor, Cdx2, an early marker of intestinal metaplasia of the stomach epithelium. Gastric epithelium from H. pylori-infected gerbils demonstrated similar changes, with decreased Id2, Id3 and E2A, and elevated p21 WAF-1/cip1 expression. In human gastric epithelium also, H. pylori infection was associated with reduced Id and E2A expression. In conclusion, H. pylori alters the expression of Id proteins, in vitro and in vivo; it is hypothesised that these changes contribute to H. pylori-associated pathologies.

Multiplex-Touchdown PCR Assay for the Detection and Genotyping of Helicobacter pylori from Artificially Contaminated Sheep Milk

Helicobacter pylori (Hp) is an organism commonly present worldwide in the human population, sometimes causing serious illnesses such as duodenal and gastric ulcers, adenocarcinoma of the stomach, and low-grade B-cell mucosa-associated lymphoid tissue lymphoma of the stomach. This article describes a multiplex-touchdown PCR method for the identification and genotyping (vacA—s1/m1, s1/m2, and s2/m2—and cagA genes) of Hp directly from sheep milk artificially contaminated with Hp strains from human gastric biopsies and with Hp ATCC 43504. The strains from humans carried s1/m2 cagA+ and s2/m2 cagA− allelic combinations, while the ATCC strains carried an s1/m1 cagA+ allelic combination. The technique showed a sensitivity of 15 CFU/ml for species identification and of 1,500 CFU/ml for the detection of genes encoding for VacA and CagA. It has proven to be specific and rapid, and the authors suggest that it be used as a rapid screening method to ensure that sheep milk is uncontaminated with this organism.

Identification of Non- Helicobacter pylori Spiral Organisms in Gastric Samples from Humans, Dogs, and Cats

Tightly coiled bacteria are a rare cause of gastric pathology in humans and represent a mixture of species for which a zoonotic origin is suspected. Similar organisms are common inhabitants of the gastric mucosae of carnivores and pigs. It was the goal of the present study to determine the actual occurrence of each individual Helicobacter species in human, canine, and feline stomachs in order to better understand the possible zoonotic significance. Gastric biopsy samples from humans with histological evidence of non-Helicobacter pylori spiral bacteria (n = 123) and samples from the gastric antrum, corpus, and cardia from dogs (n = 110) and cats (n = 43) were subjected to a multiplex PCR, enabling the identification of Helicobacter felis, Helicobacter bizzozeronii, Helicobacter salomonis, and "Candidatus Helicobacter suis." A PCR for detecting H. pylori was applied to all human samples. Single infections with "Candidatus Helicobacter suis," H. felis, H. bizzozeronii, H. salomonis, a hitherto unknown genotype of a non-H. pylori spiral organism (Helicobacter-like organism 135 [HLO135]), and H. pylori were identified in 30.9%, 8.9%, 2.4%, 11.4%, 7.3%, and 8.9% of the human biopsy samples, respectively. Mixed infections (16.3%) with two or even three of these were also found. In the canine stomach, H. bizzozeronii (70.0%) was encountered as the main spiral organism, while H. felis (62.7%) and HLO135 (67.4%) were the predominant Helicobacter species found in the feline gastric mucosa. Although the majority of human non-H. pylori organisms are Helicobacter species naturally occurring in the stomachs of pigs, cats, and dogs, the frequent identification of H. salomonis in human gastric biopsy samples is in contrast to its rare identification in pet carnivore samples, urging us to suspect other sources of infection.

Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells

Enterotoxigenic Escherichia coli (ETEC) is a prevalent cause of traveler's diarrhea and infant mortality in third-world countries. Heat-labile enterotoxin (LT) is secreted from ETEC via vesicles composed of outer membrane and periplasm. We investigated the role of ETEC vesicles in pathogenesis by analyzing vesicle association and entry into eukaryotic cells. Fluorescently labeled vesicles from LT-producing and LT-nonproducing strains were compared in their ability to bind adrenal and intestinal epithelial cells. ETEC-derived vesicles, but not control nonpathogen-derived vesicles, associated with cells in a time-, temperature-, and receptor-dependent manner. Vesicles were visualized on the cell surface at 4 degrees C and detected intracellularly at 37 degrees C. ETEC vesicle endocytosis depended on cholesterol-rich lipid rafts. Entering vesicles partially colocalized with caveolin, and the internalized vesicles accumulated in a nonacidified compartment. We conclude that ETEC vesicles serve as specifically targeted transport vehicles that mediate entry of active enterotoxin and other bacterial envelope components into host cells. These data demonstrate a role in virulence for ETEC vesicles.

Nitric oxide-an endogenous inhibitor of gastric acid secretion in isolated human gastric glands.

BackgroundEndothelial nitric oxide synthase (eNOS) has previously been detected in the glandular part of the human gastric mucosa. Furthermore, nitric oxide (NO) has been shown to influence gastric secretion in various animal models. The present study was conducted to investigate the influence of exogenously and endogenously derived NO on histamine- and cAMP-stimulated gastric acid secretion in isolated human oxyntic glands.MethodsOxyntic glands were isolated from human gastric biopsies and were subsequently pre-treated with NO donors and nitric oxide synthase inhibitors and then exposed to histamine or dibutyryl-cAMP (db-cAMP). The secretory response of the glands was determined as accumulation of [14C]aminopyrine.ResultsThe histamine- or db-cAMP-induced acid secretion was attenuated by L-arginine, a known source of endogenous NO, and also by the NO-donors sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP). Pre-treatment with either of the NOS inhibitors NG-nitro-L-arginine methyl ester (L-NAME) or NG-nitro-L-arginine (L-NNA) enhanced the secretory response.ConclusionOur results show that NO inhibits gastric acid secretion in isolated human gastric glands, and that there is endogenous formation of NO within the glandular epithelium in the vicinity of the parietal cells.

Helicobacter pylori infection induces interleukin-8 receptor expression in the human gastric epithelium.

The gastric pathogen Helicobacter pylori is known to activate multiple proinflammatory signaling pathways in epithelial cells. In this study, we addressed the question of whether expression of the interleukin-8 receptors IL-8RA (CXCR1) and IL-8RB (CXCR2) is upregulated in H. pylori-infected human gastric biopsy samples. Biopsy samples from patients infected with H. pylori strains harboring the cag pathogenicity island (PAI) expressed larger amounts of both receptors. In addition, IL-8RB expression was induced in the gastric epithelial cell line AGS upon infection with a clinical isolate containing the cag PAI, while a strain lacking the cag PAI did not. Our finding suggests that gastric epithelial cells express IL-8R in response to H. pylori infection.

Kinetic differentiation mode chromatography using 8-quinolinol and fluorimetric detection for sensitive determination of aluminum adhering to the gastric mucosa

A highly sensitive method of kinetic differentiation (KD) mode high-performance liquid chromatography (HPLC) with fluorimetric detection was established using 8-quinolinol to measure aluminum adhering to the gastric mucosa. After sucralfate was hydrolyzed by 1 mol/l hydrochloric acid, an 8-quinolinolate–aluminum complex was produced by reacting aluminum with an 8-quinolinol solution. Then contaminants in the gastric mucosa and sucralfate were removed by liquid–liquid extraction with chloroform. Next, the 8-quinolinolate–aluminum complex was separated on a reversed-phase column that was specifically designed to detect aluminum (50×4.6-mm I.D.). Separation was done at a flow-rate of 0.8 ml/min, using BES buffer containing sodium dodecyl sulfate (pH 7.0) as the mobile phase. Fluorescence was detected at 370 nm (excitation) and 504 nm (emission). The sensitivity of this method was more than 1000 times greater than that of absorptiometry using 8-quinolinol. The detection and quantitation limits were 1.68 and 5.11 ng/ml, respectively. When tested with aluminum solutions of 10, 30, and 90 ng/ml, the intra-assay and inter-assay coefficients of variation were below 7.1%, with an error of less than 8.3%. Aluminum adhering to the gastric mucosa was determined by HPLC and absorptiometry after administration of sucralfate to rats. The HPLC method showed that aluminum levels were higher at sites of ulceration than in the normal mucosa at all times after sucralfate administration. When the values above zero obtained for absorptiometry were assessed, there was a significant correlation ( r=0.993, P<0.0001) between the aluminum concentrations measured by the two methods. This new HPLC method could be applied to the determination of aluminum in small samples, such as human gastric mucosal biopsy specimens.

Helicobacter pylori: an invading microorganism? A review

In this review we evaluate the pros and cons of Helicobacter pylori invasion of epithelial cells as part of the natural history of H. pylori infection. H. pylori is generally considered an extracellular microorganism. However, a growing body of evidence supports the controversial hypothesis that at least a subset of H. pylori microorganisms has an intracellular (intraepithelial) location. Most significant is the fact that H. pylori invades cultured epithelial cells with invasion frequencies similar to Yersinia enterocolitica and better than Shigella flexneri; furthermore, studies of invasion mechanisms suggest that H. pylori invasion of and survival within epithelial cells is not merely a passive event, but requires active participation of the microorganism. Although many studies of human gastric biopsy specimens have failed to demonstrate any intracellular H. pylori, some studies have revealed a minor fraction of H. pylori inside gastric epithelial cells, with possible linkage to peptic ulceration and epithelial cell damage. In conclusion, these data encourage further research to establish whether intracellular H. pylori does play a role in H. pylori colonization of the human stomach and in peptic ulcer pathogenesis.