Small Extracellular Vesicles Loaded with Immunosuppressive miRNAs Leads to an Inhibition of Dendritic Cell Maturation.

In particular conditions, inhibition of an immune response is required to prevent tissue damage. Among these conditions are diseases caused by an over-reactive immune response, such as autoimmune or allergic disorders, or imminent organ rejection after transplantation. To avoid tissue damage, drug-mediated systemic immune suppression is an option, but it comes with high costs in the form of susceptibility to viral and bacterial infections. Thus, the induction of antigen-specific tolerance is preferable. Extracellular vesicles (EVs) are capable of delivering antigen together with immunosuppressive signals and may be used to specifically induce antigen-specific tolerance. However, naturally occurring EVs are heterogeneous and not all of them show immunosuppressive character. In our trials to engineer cell culture derived EVs to increase their tolerogenic potential, we equipped them with immunosuppressive miRNA mimics. Small EVs (sEVs) were isolated and purified from the human monocytic THP-1 cell line or from healthy donor peripheral blood mononuclear cells, and electroporated with miR-494 and miR-146a mimics. The acquired immunosuppressive potential of the modified sEVs was demonstrated by their ability to alter the major histocompatibility complex molecules and co-stimulatory receptors present on dendritic cells (DCs). To avoid allogeneic responses, the same cells that produced the sEVs served also as recipient cells. In contrast to the treatment with unmodified sEVs, the tolerogenic sEVs impeded lipopolysaccharide-induced maturation and kept DCs in a more immature developmental stage. Our experiments show that simple manipulations of sEVs using immunosuppressive cargo can lead to the inhibition of DC maturation.

U6/miR-211 expression ratio as a purity parameter for HEK293 cell-derived exosomes.

Small extracellular vesicles (sEVs) including exosomes are produced by all cell types and can be isolated from biological fluids and cell culture supernatants. The separation of exosomes with high purity from protein-rich media remains challenging. Besides contaminating proteins, small microvesicles (MVs) and apoptotic bodies are usually co-isolated with exosomes. The optimization of exosome separation and purification depends on reliable methods for the determination of the purity of the preparation, but no standard measurement has been defined so far. We tried to advance purity assessment. sEVs were isolated from HEK293 cell culture supernatants by various combinations of centrifugation, precipitation and size exclusion chromatography. sEVs with a diameter within the size range of 30-150 nm, typical for exosomes, were obtained with all tested isolation methods as shown by electron microscopy. To estimate the levels of protein contamination, flow cytometric analysis of the obtained vesicles was used. Based on the controlled preferential loading and enrichment of miR-211 into exosomes, a novel approach for the estimation of the fraction of HEK293 derived exosomes as opposed to MVs and apoptotic bodies in sEV mixtures was developed. This novel approach represents a simple qRT-PCR-based approach to improve the precise characterization of sEV isolates that is necessary for the usage of exosomes as carriers for therapeutic nucleic acids. Compared to the precipitation and size exclusion chromatography, the differential ultracentrifugation turned out to give sEVs with fairly intact shape and the highest purity according to the novel qRT-PCR-based approach, as well as to other established methods for purification assessment.

Methods for the Determination of the Purity of Exosomes.

BACKGROUND: Exosomes open exciting new opportunities for advanced drug transport and targeted release. Furthermore, exosomes may be used for vaccination, immunosuppression or wound healing. To fully utilize their potential as drug carriers or immune-modulatory agents, the optimal purity of exosome preparations is of crucial importance. METHODS: Articles describing the isolation and purification of exosomes were retrieved from the PubMed database. RESULTS: Exosomes are often separated from biological fluids containing high concentrations of proteins, lipids and other molecules that keep vesicle purification challenging. A great number of purification protocols have been published, however, their outcome is difficult to compare because the assessment of purity has not been standardized. In this review, we first give an overview of the generation and composition of exosomes, as well as their multifaceted biological functions that stimulated various medical applications. Finally, we describe various methods that have been used to purify small vesicles and to assess the purity of exosome preparations and critically compare the quality of these evaluation protocols. CONCLUSION: Combinations of various techniques have to be applied to reach the required purity and quality control of exosome preparations.

Melanoma-Derived Extracellular Vesicles Bear the Potential for the Induction of Antigen-Specific Tolerance.

BACKGROUND: Cancer-induced immunosuppression is antigen-specific rather than systemic and the mechanisms for the antigen specificity are incompletely understood. Here we explore the option that tumor-associated antigens (TAAs) may be transferred to antigen-presenting cells (APCs), together with immunosuppressive molecules, through cancer-derived small extracellular vesicles (sEVs), such as exosomes. Stimulation of a suppressive phenotype in the very same APCs that take up TAAs may yield antigen-specific tolerance. METHODS: sEVs isolated from patient-derived or well-established melanoma cell lines were used to demonstrate the transfer of major histocompatibility complex (MHC) molecules to the surface of APCs. The immunosuppressive influence of sEVs was assessed by flow cytometry analysis of activation markers, cytokine expression, and mixed lymphocyte reactions. RESULTS: MHC class I molecules were transferred from melanoma cells to the cell surface of APCs by sEVs. Concomitantly, CD86 and CD40 co-stimulatory molecules were down-regulated and IL-6 production was strongly induced. TGF-ß transported by sEVs contributed to the promotion of a suppressive phenotype of APCs. CONCLUSION: The presented results indicate the existence of a hitherto undescribed mechanism that offers an explanation for antigen-specific tolerance induction mediated by cancer-derived sEVs.

Analysis of the miRNA Profiles of Melanoma Exosomes Derived Under Normoxic and Hypoxic Culture Conditions.

BACKGROUND/AIM: MicroRNAs (miRNAs) transported in melanoma-derived exosomes function as intercellular messengers supporting tumor survival and progression. Hypoxia increases melanoma phenotypic plasticity, drug resistance, and metastasis. MATERIALS AND METHODS: We determined the miRNA profiles in exosomes derived from melanoma cells grown under hypoxic and normoxic conditions by microarray analyses and reverse transcription-polymerase chain reaction (RT-PCR) in order to analyze the potential influence of vesicle-transported miRNAs on cancer-related pathways and transcriptional programs. RESULTS: Despite phenotypical differences of the four cell lines used, their exosomes shared the majority of miRNAs. The levels of three miRNAs were higher in normoxic exosomes, whereas 15 miRNAs were significantly more abundant under hypoxic conditions. Pathway analysis pointed at several cellular processes contributing to proliferation, drug resistance, and modification of the tumor microenvironment, including immunosuppression. CONCLUSION: The miRNA-expression profiles of exosomes from patient-derived melanoma cells are modified by oxygen concentration and reflect the phenotypic changes of melanoma cells under different growth conditions.

The role of selected adipokines and ghrelin in the prognosis after myocardial infarction in a 12-month follow-up in the presence of metabolic syndrome.

INTRODUCTION: The aim of this study was to evaluate the predictive value of selected adipokines in the improvement in the ejection fraction and in the development of adverse cardiac remodeling during 12 months of follow-up among patients with an ST-segment elevation acute myocardial infarction (STEMI) in the presence of metabolic syndrome (MeS). MATERIAL AND METHODS: The study population consisted of 69 patients (49 male; mean age: 59 ±10 years) with a first STEMI that was treated with a primary percutaneous coronary intervention (pPCI). In this group, 36 patients (18 male; mean age: 60 ±15 years) had MeS according to the definition of the International Diabetes Federation. The baseline clinical evaluation included a clinical examination and evaluation of the blood levels of C-reactive protein, ghrelin, resistin, and fasting glucose. Within 72 h after the STEMI, an echocardiographic examination was performed. A complete clinical evaluation was repeated after 12 months. Adverse cardiac remodeling was defined as an increase in the left ventricular end-diastolic volume of ≥ 8%. An improvement of the ejection fraction (EF) was defined as an increase of more than 5% in the EF. RESULTS: A concentration of ghrelin ≤ 160.46 pg/ml (AUC = 0.71, p = 0.032) had a good predictive value for the occurrence of adverse left ventricular remodeling but only in the patients without MeS. Among the patients with MeS, a concentration of resistin ≤ 5196 pg/ml (AUC = 0.073, p = 0.024) had a good predictive value for the occurrence of left ventricular remodeling. A concentration of leptin > 52.18 pg/ml (AUC = 0.81, p < 0.0001) and resistin > 4419.27 ng/ml (AUC = 0.67, p = 0.049) had a good predictive value for improvement of the LVEF in the patients without MeS. CONCLUSIONS: The selected adipokines had a good predictive value for the development of adverse cardiac remodeling and for improvement of the ejection fraction among patients after a STEMI in the presence of metabolic syndrome.

The differences in the relationship between diastolic dysfunction, selected biomarkers and collagen turn-over in heart failure patients with preserved and reduced ejection fraction.

BACKGROUND: The aim of the study was to assess the correlation of the selected biomarkers and collagen turn-over indices with advanced echocardiographic parameters among patients with preserved and reduced ejection fraction (EF). METHODS: We included 62 patients with the symptomatic heart failure. The patients were divided in to two groups according to the evaluated ejection fraction (EF - Simpson method): heart failure with reduced ejection fraction (HFrEF) group - 30 patients with low EF - 35-50% (16 male, mean age 54.9 ± 12.6), heart failure with preserved ejection fraction (HFpEF) group - 32 patients with EF > 50% (16 male, mean age 62.3 ± 7.6). Clinical evaluation included 6-min walk test, biochemistry, procollagen type I N-terminal propeptide (PINP), procollagen type III N-terminal propetide (PIIINP), matrix metaloproteinase-2 (MMP2), ghrelin, and galectin-3 levels measurements. Echocardiographic examination was performed with analysis of diastolic function and global longitudinal strain (GLS). RESULTS: The GLS in the HFrEF group was significantly lower than in the HFpEF group at the baseline (GLS: 9.56 vs. 16.03, p < 0.01). There was a strong negative correlation of the PIIINP and GLS in HFrEF group (r = -0.74, p = 0.005), but only a moderate negative correlation in HFpEF (r = -0.55, p = 0.02). In the HFrEF group, there was a moderate negative correlation between the baseline level of galectin-3 and GLS (r = -0.59, p = 0.03). The correlation of ghrelin and tissue inhibitor of matrix metalloproteinase-1 with EF in the HFrEF group was moderate and statistically significant (r = 0.62, p = 0.02 and r = -0.63, p = 0.02, respectively). CONCLUSIONS: Procollagen type III peptide has a strong negative correlation with left ventricular GLS. Galectin-3 relationship with strain may indicate novel pathophysiological pathways and requires further investigation.

The influence of Helicobacter pylori eradication on the expression and methylation status of the FHIT gene in non-cancerous gastric mucosa of dyspeptic patients.

OBJECTIVE: To investigate the effect of Helicobacter pylori (H. pylori) eradication on the expression level of the FHIT gene and its methylation status in the gastric mucosa of dyspeptic patients with or without a family history of gastric cancer (FHGC). METHODS: In all, 31 patients with H. pylori infection including 13 with FHGC were enrolled in the study. The effectiveness of H. pylori eradication were confirmed by UBT, RUT and multiplex PCR (the presence of selected H. pylori strains) for biopsy samples from the antrum and corpus. Histopathological assessment was also performed. The expression of FHIT mRNA was determined by quantitative reverse transcription-polymerase chain reaction and the methylation status of the FHIT promoter was assessed by methylation-specific polymerase chain reaction. RESULTS: After H. pylori eradication, the improvement of inflammation from superficial gastritis to normal mucosa (G → N) was observed in 39% of the patients without FHGC and in 54% of those with FHGC. FHIT mRNA expression was increased in patients without FHGC after H. pylori eradication (P < 0.05), while there was no statistically significant change in gene methylation status after H. pylori eradication (P > 0.05). For the samples from those with FHGC, the FHIT mRNA expression was not significantly changed and the methylation status fluctuated evenly. CONCLUSIONS: H. pylori eradication results in the improvement of gastric mucosal inflammation and histopathological non-atrophic changes. The FHIT gene expression is increased in patients without FHGC, which may contribute to the prevention of GC development.

Downregulation of striatin leads to hyperphosphorylation of MAP2, induces depolymerization of microtubules and inhibits proliferation of HEK293T cells.

Microtubules are tubular polymers of α/ß-tubulin that are involved in the maintenance of cell shape, motility, and intracellular transport and in the segregation of chromosomes during cell division. Microtubules are dynamic structures, and their assembly is regulated by phosphoproteins called microtubule-associated proteins (MAPs). We propose that striatin, a protein belonging to the striatin family of proteins, is involved in regulation of microtubules. In HEK293T cells, striatin colocalizes with microtubules and stably associates with PP2Ac. Inhibition of striatin expression results in hyperphosphorylation of MAP2 and destabilizes microtubules. Striatin-induced destabilization of microtubules inhibited the proliferation of HEK293T cells and caused the accumulation of cells in the G0/G1 phase of the cell cycle. These results suggest that the PP2A/striatin complex modulates microtubule dynamics by regulating MAP2 phosphorylation.

Influence of hypoxia inducible factors on the immune microenvironment in ovarian cancer.

BACKGROUND: Ovarian tumors remain immunogenic even at advanced stages, but cancer-induced immunosuppression abrogates immune surveillance. The composition of the immune microenvironment in ovarian tumors was characterized by analyzing selected immunosuppressive factors in specimens from cancer patients. The influence of the hypoxia inducible factors on the immune microenvironment was also addressed. MATERIALS AND METHODS: Tumor tissue was collected from 21 ovarian cancer patients immediately following tumor excision during surgery. The mRNA expression of selected genes was quantified, and tumor infiltrating leukocytes were characterized by flow cytometry to identify regulatory T-cells, myeloid-derived suppressor cells, and type-2 macrophages. RESULTS: Overall, a pronounced heterogeneity was found among the analyzed samples. Nevertheless, statistical analysis revealed that the expression of hypoxia inducible factors correlated with the transcription levels of several immunosuppressive molecules. CONCLUSION: The activity of hypoxia inducible factors contributes to cancer immunosuppression in ovarian cancer patients.

The heterogeneous immune microenvironment in breast cancer is affected by hypoxia-related genes.

The immune system constitutes an important first-line defence against malignant transformation. However, cancer mediated immunosuppression inactivates the mechanisms of host immune surveillance. Cancer cells shut down anti-cancer immunity through direct cell-cell interactions with leukocytes and through soluble factors, establishing an immunosuppressive environment for unimpeded cancer growth. The composition of the immunosuppressive microenvironment in breast tumours is not well documented. To address this question, selected immunosuppressive factors were analyzed in tumour specimens from 33 breast cancer patients after surgery. The mRNA expression of selected genes was quantified in fresh tumour samples. Tumour infiltrating leukocytes were characterized by flow cytometry to identify regulatory T cells, myeloid derived suppressor cells, and type 2 macrophages. Statistical analysis revealed several interesting correlations between the studied parameters and clinical features. Overall, a surprisingly high degree of heterogeneity in the composition of the immunosuppressive environment was found across all breast cancer samples which adds to the complexity of this disease. The influence of the hypoxia inducible factors (HIFs) on the immune microenvironment was also addressed. The level of HIFs correlated with hormone receptor status and the expression of several immunosuppressive molecules. Targeting HIFs might not only sensitize breast tumours for radiation and chemotherapies but also interfere with cancer immunosuppression.

[The clinical significance of GastroPanel in diagnostics of Helicobacter pylori eradication efficiency in patients with dyspepsia with correlation of family history of gastric cancer]. / Kliniczne znaczenie GastroPanelu w diagnostyce skutecznej eradykacji Helicobacter pylori u chorych z dyspepsja w zaleznosci od rodzinnego wystepowania raka zoladka.

UNLABELLED: Gastric cancer remains a significant medical and social problem. Familial, hereditary, social, and demographic factors increase the susceptibility of subjects to cancer development, especially those infected with Helicobacter pylori (H. pylori). Apart from genetic studies, there are ongoing biochemical studies of possible practical value in assessment of the risk of gastric cancer development. The GastroPanelBiohit test, that include determination of the levels of gastrin (G-17), pepsinogen I (PGI), pepsinogen II (PGII) and antibodies IgG/IgA against H. pylori in serum, allowed us to determine whether there are any abnormal changes in the gastric mucosa. The aim of the study was to determine whether GastroPanel parameters, identified in patients with dyspeptic symptoms (with or without history of gastric cancer in first degree relatives) before and after successful eradication of H. pylori, have any clinical value, especially in gastric cancer development. MATERIAL AND METHODS: The study comprised 61 patients aged 18-56 years with symptoms of dyspepsia. In all patients, the preliminary urea breath test (UBT) for the presence of H. pylori was performed and the positive result qualified for further study. For final analysis, 42 patients were approved, who were divided into two groups: group I (a control group) - 22 patients with negative family history of gastric cancer among the relatives of first degree, group II - 20 patients with positive history of gastric cancer among the relatives of first degree. All the patients had the gastroscopy with the biopsy of gastric mucosa for the histopathological evaluation. Additionally, the GastroPanel test was performed. RESULTS: In the blood serum of the patients with H. pylori infection, the concentrations of gastrin (G-17), pepsinogen I (PGI) and pepsinogen II (PGII) did not depend on family history of gastric cancer (p > 0.05). Successful eradication of H. pylori decreases the levels of G-17, PGI and PGII (statistical significance p < 0.05), and this correlates with the histopathological changes of gastric mucosa. The patients with positive family history of gastric cancer had more intense H. pylori colonization of gastric mucosa (IV degree of insensitivity of infection in UBT; group I - 22% vs group II - 69%) as compared to the control group. After effective eradication of H. pylori, statistically significant decreases of IgG H. pylori antibodies and of the level of gastrin (p < 0.05) in blood serum were seen (in a 3 months follow up) only in the control group. CONCLUSIONS: Independently of the history of familial gastric cancer, the GastroPanelBiohit test provides important clinical data useful for diagnosis, for assessment of effectiveness of H. pylori eradication therapy and in evaluation of the degree of the inflammatory changes in gastric mucosa.

[Effect of Helicobacter pylori eradication on the expression level of SATB1 and c-Myc genes in gastric mucosa of patients with family history of gastric cancer]. / Wplyw eradykacji Helicobacter pylori na poziom ekspresji genów SATB1 i c-Myc w blonie sluzowej zoladka u pacjentów z dyspepsja w zaleznosci od rodzinnego obciazenia wystepowaniem raka zoladka.

UNLABELLED: Helicobacter pylori (H. pylori) is a class 1 gastric carcinogen with the proved influence on gastric cancer development. The products of SATB1 and c-Myc genes play important role in cancer development and their levels are elevated in gastric cancer tissues. The aim of the study was to analyze an effect of H. pylori eradication on the expression of the SATB1 and c-Myc genes in the gastric mucosa of dyspeptic patients with family history of gastric cancer. MATERIAL AND METHODS: Twenty patients enrolled to the studies were divided into two groups: nine patients (group I) without the family history of gastric cancer, and eleven patients with the family history of gastric cancer (group II). Endoscopic biopsies of gastric mucosa were taken from the antrum and corpus of H. pylori-infected subjects before and after bacteria eradication. The corresponding levels of expression were determined by analysis of the respective mRNA levels with the use of the real-time RT-PCR method. The level of each mRNA was normalized to the levels of mRNA of two reference genes, RPL29 and GAPDH. RESULTS: Independently of stomach topography, the antrum versus corpus, in the group I patients the levels of mRNA of SATB1 and c-Myc after eradication were higher in the following cases: SATB1/ GAPDH p = 0.017914 (antrum); SATB1/RPL29 p = 0.046400 (corpus); SATB1/GAPDH p = 0.027709 (corpus). For group II patients no statistically significant increase of the level of the c-Myc and SATB1 genes was observed. CONCLUSIONS: Patients with the family history of gastric cancer and H. pylori infection, with reversible histopathological changes of the gastric mucosa, have significantly higher levels of SATB1 and c-Myc genes expression as compared to the patients without family history of gastric cancer, regardless of the topography of the stomach. After successful eradication, the SATB1 mRNA level in samples of patients with the family history of gastric cancer did not increase, in contrast to the control group of patients. Presumably, the observed effect is associated with hypermethylation of the promoter of that gene. However, the level of c-Myc gene expression was not significantly different before and after removal of the bacteria, for both groups of patients.

Family history of gastric cancer correlates with decreased expression of HINT1 tumor suppressor gene in gastric mucosa of dyspeptic patients.

Gastric cancer (GC) is the fourth most common cancer and the second most common cause of cancer-associated mortality worldwide. Approximately 10% of gastric cancers are hereditary and a small percentage of these cases (1-3%) have been classified as a single hereditary syndrome (hereditary diffuse gastric cancer). We previously demonstrated that a family history of gastric cancer (FHGC) contributes to a predisposition towards the development of gastric cancer. Our data revealed that for dyspeptic patients whose first-degree relative(s) succumbed to GC, the levels of the fragile histidine triad pro-apoptotic protein in gastric mucosa were decreased. Another member of the histidine triad protein superfamily is histidine triad nucleotide-binding protein 1 (HINT1), a novel tumor suppressor that plays an inhibitory role in the control of gene transcription. The study comprised 38 ethnically homogeneous patients with dyspeptic symptoms without concomitant chronic diseases (18 controls/20 patients with FHGC). The results showed that the samples from the control patients predominantly exhibited non-atrophic changes (approximately 90%), whereas atrophic changes occurred more frequently in patients with FHGC. Notably, the expression levels of the HINT1 gene were markedly higher in the samples with atrophy taken from the antrum of FHGC patients compared to the non-atrophic samples. Moreover, the levels of HINT1 mRNA in samples obtained from the antrum of patients with FHGC were lower compared to analogous samples from the control individuals. The decreased levels of HINT1 mRNA in the antrum samples of patients with the FHGC indicate that it is a factor predisposing those patients to the development of gastric cancer.

Gene silencing activity of siRNA molecules containing phosphorodithioate substitutions.

Chemically synthesized small interfering RNAs (siRNAs) have been widely used to identify gene function and hold great potential in providing a new class of therapeutics. Chemical modifications are desired for therapeutic applications to improve siRNA efficacy. Appropriately protected ribonucleoside-3'-yl S-[ß-(benzoylmercapto)ethyl]pyrrolidino-thiophosphoramidite monomers were prepared for the synthesis of siRNA containing phosphorodithioate (PS2) substitutions in which the two non-bridging oxygen atoms are replaced by sulfur atoms. A series of siRNAs containing PS2 substitutions have been strategically designed, synthesized, and evaluated for their gene silencing activities. These PS2-siRNA duplexes exhibit an A-form helical structure similar to unmodified siRNA. The effect of PS2 substitutions on gene silencing activity is position-dependent, with certain PS2-siRNAs showing activity significantly higher than that of unmodified siRNA. The relative gene silencing activities of siRNAs containing either PS2 or phosphoromonothioate (PS) linkages at identical positions are variable and depend on the sites of modification. 5'-Phosphorylation of PS2-siRNAs has little or no effect on gene silencing activity. Incorporation of PS2 substitutions into siRNA duplexes increases their serum stability. These results offer preliminary evidence of the potential value of PS2-modified siRNAs.

Gastric ghrelin in relation to gender, stomach topography and Helicobacter pylori in dyspeptic patients.

AIM: To investigate the level of gastric ghrelin in stomach mucosa of dyspeptic patients in relation to Helicobacter pylori (H pylori) infection, bacterial cytotoxicity, topography and gender. METHODS: The study comprised 40 premenopausal women (19 H pylori positive) and 48 men (17 H pylori positive) with functional dyspepsia. All gastric biopsy specimens revealed normal mucosa or non-atrophic gastritis. Gastric ghrelin concentration was determined by Enzyme linked immunosorbent assay. The cagA and vacA strains of bacterial DNA were identified by multiplex polymerase chain reaction. RESULTS: In general, infection with H pylori caused an increase in gastric ghrelin level regardless of gender and stomach topography. Significantly more hormone was present in both, non-infected and H pylori positive female samples, as compared to males. The distribution of bacterial strains showed cagA(+) vacA s1m1 and cagA(-) vacA s2m2 genotypes as the most common infections in the studied population. A tendency to higher ghrelin levels was observed in less cytotoxic (cagA negative) strain-containing specimens from the antrum and corpus of both gender groups (without statistical significance). CONCLUSION: An increase in gastric ghrelin levels at the stage of non-atrophic gastritis in H pylori positive patients, especially in those infected with cagA(-) strains, can exert a gastroprotective effect.

Helicobacter pylori infection and family history of gastric cancer decrease expression of FHIT tumor suppressor gene in gastric mucosa of dyspeptic patients.

BACKGROUND: The expression of a fragile histidine triad (FHIT) protein is lost in stomach tumors. The study aimed at determining whether FHIT expression is affected by Helicobacter pylori infection, strain virulence (vacA and cagA genes) and histopathological changes in the gastric mucosa of patients with functional dyspepsia having first-degree relatives with gastric cancer. MATERIALS AND METHODS: Eighty-eight never-smoking patients with functional dyspepsia were selected for the study, and 48 of them had first-degree relatives with gastric cancer. Bacterial DNA amplification was used to identify H. pylori colonization. The level of FHIT gene expression was determined by qRT-PCR (mRNA) and Western blot (FHIT protein) analyses. RESULTS: For patients having first-degree relatives with gastric cancer FHIT expression was lower (mRNA by ca. 40-45% and protein by 30%) compared with the control patients (p < .05). H. pylori infection decreased the FHIT mRNA level by 10-35% and the protein level by 10-20%. Bacterial strain vacA(+)cagA(+) lowered FHIT mRNA by ca. 30-35% in the antrum samples of both groups and in corpus samples of patients with first-degree relatives with gastric cancer (p < .05). The FHIT mRNA level was twice as high in control H. pylori-negative patients with intestinal metaplasia, compared with those with non-atrophic gastritis. CONCLUSIONS: The decreased FHIT gene expression associated with hereditary factors and with H. pylori infection, especially with vacA(+)cagA(+)-positive strains, may be related to gastric carcinoma development.

Expression of somatostatin receptor subtype 3 in the gastric mucosa of dyspeptic patients in relation to Helicobacter pylori infection and a family history of gastric cancer.

BACKGROUND AND AIM: The cytotoxic activity of Helicobacter pylori contributes significantly to the pathogenesis of gastric carcinoma. A preliminary study suggested that somatostatin receptor subtype 3 (SSTR3) might play a role in cell apoptosis and the growth of gastric cancer. The aim of the present study was to determine the influence of H. pylori infection and a family history of gastric cancer on the expression of SSTR3 in the gastric mucosa of non-cancer patients with dyspepsia. METHODS: The expression of the SSTR3 gene in the gastric mucosa of the stomach antrum and corpus of 53 patients was determined by the use of quantitative reverse transcription-polymerase chain reaction. RESULTS: The SSTR3 mRNA level was lower in the H. pylori-infected patients, as compared to the non-infected patients, independently of a family history of gastric cancer and stomach topography. The greatest decrease of approximately 40% and 35% (P < 0.05) was observed for the antrum of the H. pylori-positive patients without and with a family history of gastric cancer, respectively. In the corpus, these differences were much smaller, regardless of a family history of gastric cancer. Interestingly, for H. pylori-negative patients, the density (at the mRNA level) of the SSTR3 receptor in the antrum was higher than in the corpus mucosa. CONCLUSIONS: A decrease in the density of SSTR3 (especially in the antrum) in individuals with H. pylori infection and particularly with a family history of gastric cancer may point to an environmental and inherited predisposition in the development of distal gastric cancer.

[Somatostatin receptor subtype 3 (SSTR3) mRNA level in gastric mucosa of patients with dyspepsia]. / Poziom mRNA receptora somatostatyny typu 3 (SSTR3) w blonie sluzowej zoladka u osób z dyspepsja.

UNLABELLED: Somatostatin (SST) inhibits cellular processes related to secretion, motor activity and cell proliferation. It operates through SSTR1-5 receptors. Density of the SSTR3 receptor is decreased in gastric adenocarcinoma. AIM: Determination of the SSTR3 mRNA level in gastric mucosa of patients with dyspepsia, in respect to stomach topography, H. pylori infection, patient gender and the type of histopathological changes was aimed in these studies. MATERIALS AND METHODS: A real time RT-PCR method was used to determine the SSTR3 mRNA level in samples collected from the stomach antrum and corpus of 27 patients with dyspepsia (18-59 years old) without family history of cancer. RESULTS: Among Hp(-) patients, the level of the SSTR3 mRNA in samples taken from the antrum was by ca. 65% higher (p < 0.05) than from the stomach corpus. Infection with H. pylori significantly decreased the SSTR3 level in antrum (ca. 50%, p < 0.05), especially in females. Among the Hp(+) patients, the development of histopathological changes in that part of stomach was accompanied by decrease of the expression of SSTR3 receptor (p > 0.05). CONCLUSIONS: H. pylori infection related reduction of the SSTR3 density in the antrum mucosa speaks for the need of eradication of these bacteria in the prevention of distal gastric cancer.