WFDC2 Promotes Spasmolytic Polypeptide-Expressing Metaplasia Through the Up-Regulation of IL33 in Response to Injury.

BACKGROUND & AIMS: WAP 4-disulfide core domain protein 2 (WFDC2), also known as human epididymis protein 4, is a small secretory protein that is highly expressed in fibrosis and human cancers, particularly in the ovaries, lungs, and stomach. However, the role of WFDC2 in carcinogenesis is not fully understood. The present study aimed to investigate the role of WFDC2 in gastric carcinogenesis with the use of preneoplastic metaplasia models. METHODS: Three spasmolytic polypeptide-expressing metaplasia (SPEM) models were established in both wild-type and Wfdc2-knockout mice with DMP-777, L635, and high-dose tamoxifen, respectively. To reveal the functional role of WFDC2, we performed transcriptomic analysis with DMP-777-treated gastric corpus specimens. RESULTS: Wfdc2-knockout mice exhibited remarkable resistance against oxyntic atrophy, SPEM emergence, and accumulation of M2-type macrophages in all 3 SPEM models. Transcriptomic analysis revealed that Wfdc2-knockout prevented the up-regulation of interleukin-33 (IL33) expression in the injured mucosal region of SPEM models. Notably, supplementation of recombinant WFDC2 induced IL33 production and M2 macrophage polarization, and ultimately promoted SPEM development. Moreover, long-term treatment with recombinant WFDC2 was able to induce SPEM development. CONCLUSIONS: WFDC2 expressed in response to gastric injury promotes SPEM through the up-regulation of IL33 expression. These findings provide novel insights into the role of WFDC2 in gastric carcinogenesis.

EGF and BMPs Govern Differentiation and Patterning in Human Gastric Glands.

BACKGROUND & AIMS: The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organized inside glands and crypts. Regeneration depends on Wnt/ß-catenin pathway activation, but to understand homeostasis and its dysregulation in disease, we need to identify the signaling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen-, and gastric acid-producing cells. METHODS: We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief, and parietal cells. We localized the source of the growth factors in the tissue of origin. RESULTS: We show that epidermal growth factor is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with bone morphogenetic factor/Noggin signals, epidermal growth factor controls the differentiation of foveolar cells vs parietal or chief cells. We also show that epidermal growth factor is likely to underlie alteration of the gastric mucosa in the precancerous condition atrophic gastritis. CONCLUSIONS: Use of our recently established mucosoid cultures in combination with analysis of the tissue of origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signaling microenvironment in the human gastric glands.

Giardia lamblia and Helicobacter pylori coinfection in gastrointestinal biopsies: A retrospective single-center analysis from Switzerland.

BACKGROUND: The protozoan Giardia lamblia (GL) and the bacterium Helicobacter pylori (HP) are common causes of gastrointestinal disease. Coinfection is common and has been reported in studies from Africa, Europe, North America and Asia, but data for Switzerland are scarce. AIM: To investigate GL and HP prevalence and coinfection rate in gastrointestinal biopsies from the Zurich area of Switzerland. METHODS: Cases were retrieved from the laboratory information system (Medica Institute of Clinical Pathology, Zurich, Switzerland). Histological slides of cases with GL were reviewed, as were the concurrent gastric biopsies, where available. RESULTS: Between January 1, 2013 and December 31, 2020, GL was found in 88 (0.14%) of 62,402 patients with a small intestine biopsy and HP in 10,668 (15.5%) of 68,961 patients with a gastric biopsy. 74/88 (84.1%) of patients with GL had unremarkable small intestine biopsies, 13/88 (14.8%) had increased intraepithelial lymphocytes, 5/88 (5.7%) showed villous atrophy and 2/88 (2.3%) acute inflammation. 71/88 patients (80.7%) with GL had an available gastric biopsy, of which 12/71 (16.9%) were unremarkable, 28/71 (39.4%) had HP-associated gastritis, 11/71 (15.5%) showed reactive gastropathy and 1/71 (1.4%) had autoimmune gastritis. CONCLUSION: Coinfection with HP is common in patients with GL in gastrointestinal biopsies from the Zurich area of Switzerland. Therefore, gastroenterologists should consider sampling the stomach when GL is suspected for evaluation of possible concurrent HP-associated gastritis. Likewise, pathologists should scrutinize any small intestine biopsy for the presence of GL when HP-associated gastritis is seen, and vice versa.

Analyzing the Integrin Adhesome by In Situ Proximity Ligation Assay.

The in situ proximity ligation assay (PLA) is capable of detecting single protein events such as protein protein-interactions and posttranslational modifications (e.g., protein phosphorylation) in tissue and cell samples prepared for analysis by immunofluorescent or immunohistochemical microscopy. The targets are detected using two primary antibodies which must be from different host species. A pair of secondary antibodies (PLA probes) conjugated to complementary oligonucleotides is applied to the sample, and a signal is generated only when the two PLA probes are in close proximity by their binding to the two primary antibodies that have bound to their targets in close proximity. The signal from each pair of PLA probes is visualized as an individual fluorescent spot. These PLA signals can be quantified (counted) using image analysis software (ImageJ), and also assigned to a specific subcellular location based on microscopy image overlays. In principle, in situ PLA offers a relatively simple and sensitive technique to analyze interactions among any proteins for which suitable antibodies are available. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes consisting of more than 150 proteins, also known as the integrin adhesome, which link the extracellular matrix (ECM) to the actin cytoskeleton and regulate the functioning of mechanosignaling pathways. The in situ PLA approach is well suited for examining the spatiotemporal aspects of protein posttranslational modifications and protein interactions occurring in dynamic multiprotein complexes such as integrin mediated focal adhesions.

Muscularis macrophages establish cell-to-cell contacts with telocytes/PDGFRα-positive cells and smooth muscle cells in the human and mouse gastrointestinal tract.

BACKGROUND AND AIM: Muscularis macrophages (MMs) not only mediate the innate immunity, but also functionally interact with cells important for gastrointestinal motility. The aim of this study was to determine the spatial relationship and types of contacts between the MMs and neighboring cells in the muscularis propria of human and mouse stomach, small intestine, and large intestine. METHODS: The distribution and morphology of MMs and their contacts with other cells were investigated by immunohistochemistry and transmission electron microscopy. KEY RESULTS: Immunohistochemistry showed variable shape and number of MMs according to their location in different portions of the muscle coat. By double labeling, a close association between MMs and neighboring cells, that is, neurons, smooth muscle cells, interstitial cells of Cajal (ICCs), telocytes (TCs)/PDGFRα-positive cells, was seen. Electron microscopy demonstrated that in the muscle layers of both animal species, MMs have similar ultrastructural features and have specialized cell-to-cell contacts with smooth muscle cells and TCs/PDGFRα-positive cells but not with ICCs and enteric neurons. CONCLUSION & INFERENCES: This study describes varying patterns of distribution of MMs between different regions of the gut, and reports the presence of distinct and extended cell-to-cell contacts between MMs and smooth muscle cells and between MMs and TCs/PDGFRα-positive cells. In contrast, MMs, although close to ICCs and nerve elements, did not make contact with them. These findings indicate specialized and variable roles for MMs in the modulation of gastrointestinal motility whose significance should be more closely investigated in normal and pathological conditions.

The potential function of IKKα in gastric precancerous lesion via mediating Maspin.

OBJECTIVE: To know the potential role of IKKα (an NF-κB noncanonical pathway) in gastric precancerous lesion via mediating Maspin. METHODS: Gastric cancer, precancerous lesion and control tissues (chronic non-atrophic gastritis) were collected for determining the expression of IKKα and Maspin by immunohistochemistry. Thereafter, gastric precancerous models were established and divided into the Control group, Model group and Model + shIKKα group. All rats were subjected to observe the pathological changes and ultramicro structure of the gastric mucosa by HE staining or electron microscope, and to measure the serum levels of inflammatory cytokines by ELISA, the expression of apoptosis-related proteins by immunohistochemistry, as well as the expression of IKKα and Maspin by quantitative real-time PCR and Western blotting. RESULTS: Precancerous lesion and gastric cancer tissues manifested significant upregulation of IKKα positive expression, concomitant with downregulation of the positive expression of Maspin, and these changes were more evident in the gastric cancer tissues. In comparison with the Control group, rats in the Model group had significant increases in serum levels of TNF-α, IL-1ß, IL-6 and COX-2, with up-regulations of Bcl-2, CyclinD1, IKKα and p-IKKα, and down-regulations of Bax, Caspase-3 and Maspin. shIKKα treatment attenuate inflammation and apoptosis in gastric precancerous lesion (GPL) rat, with the downregulation of IKKα and p-IKKα, and upregulation of Maspin. CONCLUSION: Inhibiting IKKα, via upregulating Maspin, can mitigate the inflammation and promote cell apoptosis in precancerous rats, thereby delaying the development of the precancerous lesions.

Distribution of lanthanum carbonate in the gastric mucosa confirmed by electron microscopy with a magnified endoscopy: a case report and literature review.

We describe the case of a 70-year-old man with diabetic nephropathy undergoing hemodialysis. Four years following hemodialysis, he started taking lanthanum carbonate 1500 mg/day and lansoprazole 30 mg/day. Nine years following hemodialysis, he underwent screening esophagogastroduodenoscopy, which demonstrated the presence of the whitish cobblestone-like mucosa in the gastric corpus and multiple reddish depressed lesions with annular whitish mucosa in the antrum. With magnified narrow-band imaging endoscopy, a yellowish-white substance was observed in the villous structure, and subepithelial vessels were observed on the yellowish-white substance. Biopsies were taken from the whitish cobblestone-like mucosa of the upper corpus, a reddish depressed part of the antrum. Histologically, aggregates of cells containing amphophilic fine granular material were found in the mucosal interstitium. These cells stained positive for CD68 and were identified as histiocytes. Since he had been taking lanthanum carbonate for 5 years, we considered the possibility of histiocyte-mediated phagocytosis of lanthanum. Digital mapping via scanning electron microscopy with energy-dispersive X-ray spectrometry showed the presence of lanthanum and phosphorus in the interstitium and cytoplasm of histiocytes. The white, rough mucosa in the gastric body appeared 6 months following the commencement of lanthanum administration and still exists 3 years and 5 months after discontinuation of lanthanum.

The lipoprotein HP1454 of Helicobacter pylori regulates T-cell response by shaping T-cell receptor signalling.

Helicobacter pylori (HP) is a Gram-negative bacterium that chronically infects the stomach of more than 50% of human population and represents a major cause of gastric cancer, gastric lymphoma, gastric autoimmunity, and peptic ulcer. It still remains to be elucidated, which HP virulence factors are important in the development of gastric disorders. Here, we analysed the role of the HP protein HP1454 in the host-pathogen interaction. We found that a significant proportion of T cells isolated from HP patients with chronic gastritis and gastric adenocarcinoma proliferated in response to HP1454. Moreover, we demonstrated in vivo that HP1454 protein drives Th1/Th17 inflammatory responses. We further analysed the in vitro response of human T cells exposed either to an HP wild-type strain or to a strain with a deletion of the hp1454 gene, and we revealed that HP1454 triggers the T-cell antigen receptor-dependent signalling and lymphocyte proliferation, as well as the CXCL12-dependent cell adhesion and migration. Our study findings prove that HP1454 is a crucial bacterial factor that exerts its proinflammatory activity by directly modulating the T-cell response. The relevance of these results can be appreciated by considering that compelling evidence suggest that chronic gastric inflammation, a condition that paves the way to HP-associated diseases, is dependent on T cells.

Transcellular stomach absorption of a derivatized glucagon-like peptide-1 receptor agonist.

Oral administration of therapeutic peptides is hindered by poor absorption across the gastrointestinal barrier and extensive degradation by proteolytic enzymes. Here, we investigated the absorption of orally delivered semaglutide, a glucagon-like peptide-1 analog, coformulated with the absorption enhancer sodium N-[8-(2-hydroxybenzoyl) aminocaprylate] (SNAC) in a tablet. In contrast to intestinal absorption usually seen with small molecules, clinical and preclinical dog studies revealed that absorption of semaglutide takes place in the stomach, is confined to an area in close proximity to the tablet surface, and requires coformulation with SNAC. SNAC protects against enzymatic degradation via local buffering actions and only transiently enhances absorption. The mechanism of absorption is shown to be compound specific, transcellular, and without any evidence of effect on tight junctions. These data have implications for understanding how highly efficacious and specific therapeutic peptides could be transformed from injectable to tablet-based oral therapies.

Characterization of Gastrospheres Using 3D Coculture System.

To understand the molecular mechanisms involved in gastric disorders and regeneration, we need an in vitro tridimensional (3D) culture model, which can mimic the in vivo gastric microenvironment. A 3D coculture system named gastrosphere is proposed herein, composed of primary human gastric epithelial and stromal cells. The primary cultures were obtained from endoscopic gastric biopsies, and after mechanical and enzymatic dispersion, epithelial (HGE3) and stromal (HGS12) cells were expanded. After extensive immunocytochemical characterization, cells were seeded onto 96-well round bottom plates previously covered with 1% agarose. Cells were cultured in KM-F12 culture medium with 10% fetal bovine serum (FBS), antibiotics, and antimycotics, in humidified air at 37 °C and atmosphere containing 5% CO2 for 72 h or until spheres formation. Then gastrospheres were carefully transferred to a rotary cell culture system (RCCS-4), and maintained for 07, 14, 21, and 28 days. Gastrospheres were morphologically characterized by immunocytochemistry [cytokeratins (CK), vimentin, α-smooth muscle actin (α-SMA), laminin (LN), fibronectin (FN), and type IV collagen (CIV), proliferating cell nuclear antigen (PCNA)], and electron microscopy. In gastrospheres, the cytokeratin-positive epithelial cells were found in the outer layer, while vimentin-positive stromal cells were localized in the center of the gastrospheres. PCNA+ cells were mainly seen at the peripheral and in the intermediary region while nestin+ cells were also depicted in the latter zone. Scanning electron microscopy revealed groups of cohesive gastric cells at the periphery, while transmission electron microscopy demonstrated some differentiated mucous-like or zymogenic-like cells in the periphery and stromal structures located at the center of the 3D structures. Extracellular matrix was deposed between cells. Our data suggest that in vitro gastrospheres recapitulate the in vivo gastric microenvironment.

Cytochemical features of the digestive tract mucosa of Hemisorubim platyrhynchos (Siluriformes: Pimelodidae).

Membranous organelles, acid glycoconjugates and lipids were characterized in the digestive tract mucosa of Hemisorubim platyrhynchos by cytochemistry techniques. Two types of mucous-secreting cells were observed in the digestive tract epithelium: goblet cells in the oesophagus and intestine and epithelial cells in the stomach. These cells had a Golgi apparatus more developed than the other cell types. The cytochemical analysis revealed that secretory granules are reactive to acid glycoconjugates, varying in reaction intensity according to the region of the digestive tract. Acid glycoconjugate reactions were also observed in oesophageal epithelial cell microridges and in enterocyte microvilli. In the digestive tract, acid glycoconjugates act to protect the epithelial surface, increasing mucous viscosity, which facilitates the passage of food, prevents the binding of parasites and facilitates their removal. Through lipid staining, a coated membrane was observed around each secretory granule of the oesophageal and intestinal goblet cells, while gastric epithelial cells granules were fully reactive. Oxynticopeptic cells of the gastric glands showed lipid droplets in the cytoplasm and also in the mitochondrial matrix, which act as an energy reserve for these cells that have a high energy demand. Enterocytes showed a well-developed smooth endoplasmic reticulum, especially in the apical region of the cell, being related to absorption and resynthesis of lipids.

Clinical Translation of Tethered Confocal Microscopy Capsule for Unsedated Diagnosis of Eosinophilic Esophagitis.

Esophagogastroduodenoscopy (EGD) is a widely used procedure, posing significant financial burden on both healthcare systems and patients. Moreover, EGD is time consuming, sometimes difficult to tolerate, and suffers from an imperfect diagnostic yield as the limited number of collected biopsies does not represent the whole organ. In this paper, we report on technological and clinical feasibility of a swallowable tethered endomicroscopy capsule, which is administered without sedation, to image large regions of esophageal and gastric mucosa at the cellular level. To demonstrate imaging capabilities, we conducted a human pilot study (n = 17) on Eosinophilic Esophagitis (EoE) patients and healthy volunteers from which representative cases are presented and discussed. Results indicate that, compared to endoscopic biopsy, unsedated tethered capsule endomicroscopy obtains orders of magnitude more cellular information while successfully resolving characteristic tissue microscopic features such as stratified squamous epithelium, lamina propria papillae, intraepithelial eosinophils, and gastric cardia and body/fundic mucosa epithelia. Based on the major import of whole organ, cellular-level microscopy to obviate sampling error and the clear cost and convenience advantages of unsedated procedure, we believe that this tool has the potential to become a simpler and more effective device for diagnosing and monitoring the therapeutic response of EoE and other esophageal diseases.

Lanthanum-Induced Mucosal Alterations in the Stomach (Lanthanum Gastropathy): a Comparative Study Using an Animal Model.

Lanthanum (La) carbonate (LC) is one of the most potent phosphate binders that prevents the elevation of serum phosphate levels in patients with end-stage renal diseases undergoing dialysis. LC binds strongly to dietary phosphate and forms insoluble complexes that pass through the gastrointestinal tract. La deposition in patients treated with LC is a recently documented finding particularly observed in gastric mucosa. We herein describe the detailed gastric mucosal lesions in 45 LC-treated patients and address the potential underlying pathologic mechanism using oral LC administration in rats. Microscopically, La deposition, as shown by subepithelial collections of plump eosinophilic histiocytes or small foreign body granulomas containing coarse granular or amorphous inclusion bodies, was found in the gastric mucosa of 44 (97.8%) of the 45 dialysis patients in the study cohort, which was most frequently associated with foveolar hyperplasia (37.8%). Using oral administration of rats with 1000 mg/day LC for 2 or more weeks, La deposition was consistently detectable in the gastric mucosa but not in other organs examined. In addition, various histologic alterations such as glandular atrophy, stromal fibrosis, proliferation of mucous neck cells, intestinal metaplasia, squamous cell papilloma, erosion, and ulcer were demonstrated in the rat model. Thus, orally administered LC can induce mucosal injury, designated here as La gastropathy, which may alter the local environment and result in La deposition in the gastric mucosa, thereby potentially inducing abnormal cell proliferation or neoplastic lesions.

Effect of allantoin on experimentally induced gastric ulcers: Pathways of gastroprotection.

Gastric ulcer affects people worldwide, and its inefficacy and recurrence have fueled the search for new therapeutic strategies. Despite the well-known use of allantoin in medicines and cosmetic products, its effect has not yet been studied with regard to gastric ulcer. Hence, the aim of the present study was to explore the pharmaco-mechanistic efficacy of allantoin against commonly harmful agents that cause injuries to the stomach. Ethanol, indomethacin, and stress-induced gastric ulcer models were adopted, in addition to pylorus ligature, a quantification of vascular permeability, glutathione (GSH), gastric adhered mucus, prostaglandin (PGE2), pro-inflammatory cytokines levels, myeloperoxidase (MPO), and catalase (CAT) activities. The gastric lesions were examined by gross, histological, and ultrastructural features. The results showed that treatment with allantoin (60mg/kg, per oral) reduced the gastric ulcer formation in all models. Furthermore, allantoin reduced the parameters of gastric acid secretion and attenuated both the vascular permeability and MPO activity. The levels of pro-inflammatory cytokines were also reduced, accompanied by a restoration of CAT activity and GSH levels. Notably, allantoin treatment preserved the gastric-adhered mucus and PGE2 levels after ethanol administration. Microscopic and ultrastructural analysis revealed that allantoin maintained tissue integrity and prevented morphological changes in cells caused by ethanol. In summary, we demonstrated for the first time that allantoin possesses gastroprotective activity through anti-inflammatory, anti-oxidative, antisecretory, and cytoprotective mechanisms. The antisecretory and cytoprotective mechanisms are probably associated with an increase in PGE2 levels.

Potential malignant transformation in the gastric mucosa of immunodeficient mice with persistent Mycoplasma penetrans infection.

Mycoplasma infection has been reported in immunocompromised cancer patients; nevertheless, it is not clear if persistent Mycoplasma infection could facilitate the proliferation of cancer cells in immunocompromised organisms. The aim of this study was to examine the relationship between persistent Mycoplasma infection and malignant transformation in an immunodeficient host model. Immunodeficient mouse model was established using cyclophosphamide and mice gastric mucosal cells were infected with Mycoplasma penetrans (Mpe). After 18 weeks, mice were sacrificed and gastric mucosal Mpe infected cells were identified by fluorescence in situ hybridization (FISH). Moreover, pathological and ultrastructural changes in mice gastric mucosa were evaluated and the expression of multiple proto-oncogenes was examined by Western blot. Our data show that Mpe infection was detected in the blood of immunodeficient mice and Mpe persistent infection in mice gastric mucosa was confirmed by FISH. There were pathological and ultrastructural malignant transformation occurred in the gastric mucosa of infected mice compared to control mice. Mpe infected mice showed lower expression of p53 and p21 and higher H-ras expression compared to the control group. Moreover, expression of NF-κB p65 subunit increased in Mpe infected mice, similar to the TNF-α expression. Bax expression in gastric mucosa of Mpe infected mice was lower while Bcl-2 expression was higher than in the uninfected control group. Collectively these data demonstrate that persistent Mpe infection is associated with aberrant expression of multiple proto-oncogenes in gastric mucosa of immunodeficient mice which potentially facilitate the malignant transformation.

Clinicopathological characteristics of invasive gastric Helicobacter pylori.

Helicobacter pylori organisms have been observed deep within the stomach mucosa with an "intracellular" appearance, although the clinicopathological characteristics of such cases remain poorly understood. We analyzed 18 cases of deep mucosal H pylori and associated clinical (sex, age, history of H pylori infection, or proton pump inhibitor [PPI] use, medications, smoking, alcohol use, comorbidities, treatment response) and pathological (presence of lymphoid aggregates, intestinal metaplasia, PPI effect, active and/or chronic inflammation, quantity of invasive versus surface H pylori) characteristics. Electron microscopy was performed on 6 cases with the highest burden of invasive H pylori. Within our sample, 3 of 16 had a history of H pylori infection, 10 of 15 were receiving PPIs at the time of biopsy, and 12 of 13 had a negative posttreatment follow-up. Histology revealed that invasive H pylori were more commonly associated with chronic inflammation, in both the antrum (15/15 chronic, 8/15 acute) and fundus (17/18 chronic, 8/18 acute). Electron microscopy showed organisms within intercellular and luminal spaces, but no intracellular organisms. Deep mucosal H pylori often have an intracellular appearance but are contained within intercellular and luminal spaces and are responsive to standard therapy.

Acid and alkaline phosphatase localization in the digestive tract mucosa of the Hemisorubim platyrhynchos.

This cytochemical study investigated the acid and alkaline phosphatase of the digestive tract of Hemisorubim platyrhynchos. Acid phosphatase was detected in the lining epithelium throughout the digestive tract, whereas alkaline phosphatase was only observed in the intestine. In the esophagus, an acid phosphatase reaction occurred in the apical cytoplasm of the epithelial cells and was related to epithelial protection and freeing of superficial cells for sloughing. Similar results were also observed in epithelial cells of gastric epithelium. In the gastric glands, acid phosphatase occurred in lysosomes of the oxynticopeptic cells acting in the macromolecule degradation for use as an energy source, whereas in the vesiculotubular system, its presence could be related to secretion processes. Furthermore, acid phosphatase in the intestine occurred in microvilli and lysosomes of the enterocytes and was correlated to absorption and intracellular digestion. However, no difference was reported among the regions of the intestine. However, alkaline phosphatase reaction revealed a large number of reaction dots in the anterior intestine, with the number decreasing toward the posterior intestine. This enzyme has been related to several functions, highlighting its role in the nutrient absorption primarily in the anterior intestine but also being essential in pH regulation because this is a carnivorous species with many gastric glands with secretions that could damage the intestine.

Mir-30d increases intracellular survival of Helicobacter pylori through inhibition of autophagy pathway.

AIM: To determine if mir-30d inhibits the autophagy response to Helicobacter pylori (H. pylori) invasion and increases H. pylori intracellular survival. METHODS: The expression of mir-30d was detected by quantitative polymerase chain reaction (PCR), and autophagy level was examined by transmission electron microscopy, western blot, and GFP-LC3 puncta assay in human AGS cells and GES-1 cells. Luciferase reporter assay was applied to confirm the specificity of mir-30d regulation on the expression of several core molecules involved in autophagy pathway. The expression of multiple core proteins were analyzed at both the mRNA and protein level, and the intracellular survival of H. pylori after different treatments was detected by gentamicin protection assay. RESULTS: Autophagy level was increased in AGS and GES-1 cells in response to H. pylori infection, which was accompanied by upregulation of mir-30d expression (P < 0.05, vs no H. pylori infection). In the two gastric epithelial cell lines, mimic mir-30d was found to repress the autophagy process, whereas mir-30d inhibitor increased autophagy response to H. pylori invasion. mir-30d mimic decreased the luciferase activity of wild type reporter plasmids carrying the 3' untranslated region (UTR) of all five tested genes (ATG2B, ATG5, ATG12, BECN1, and BNIP3L), whereas it had no effect on the mutant reporter plasmids. These five genes are core genes of autophagy pathway, and their expression was reduced significantly after mir-30d mimic transfection (P < 0.05, vs control cells without mir-30d mimic treatment). Mir-30d mimic transfection and direct inhibition of autophagy increased the intracellular survival of H. pylori in AGS cells. CONCLUSION: Mir-30d increases intracellular survival of H. pylori in gastric epithelial cells through inhibition of multiple core proteins in the autophagy pathway.