Šaltenienė, Violeta

Įvadas. Žmogaus žarnyno epitelio barjero vientisumas ir jo sąveika su uždegiminiais procesais yra gyvybiškai svarbūs veiksniai paciento sveikatai, palaikantys žarnyno homeostazę ir prisidedantys prie uždegiminių žaryno ligų (UŽL) prevencijos. Remiantis naujausių mokslinių tyrimų duomenimis tulžies rūgščių metabolitas, litoholio rūgšties darinys, 7-ketolitoholio rūgštis (7-keto-LCA), identifikuojamas kaip potencialus terapinis agentas dėl savo gebėjimo veikti kaip farnezoido (angl. farnesoid) X receptoriaus (FXR) antagonistas. Dėl šio poveikio 7-keto-LCA gali skatinti žarnyno epitelio atsistatymą ir sustiprinti Wnt signalinio kelio atsaką, taip palengvindama žarnyno kamieninių ląstelių atsinaujinimą (Li ir kt., 2023). Metodai. Pradinis 7-keto-LCA (5–50 µM koncentracijų) tyrimas buvo atliktas naudojant Caco-2 (ATCCTM HTB-37) ląsteles. Metabolitas toliau tirtas pasitelkiant epitelinius 3D storosios žarnos organoidus, asmens nesergančio UŽL (kontrolė, n = 1) ir opiniu kolitu (OK, n = 1) sergančio paciento. Uždegimas moduliuotas ląsteles kultivuojant su INF-γ (10 ng/ml) ir TNF-α (10 ng/ml) citokinų mišiniu 24 valandas. Taikininių genų (TNF-α, CXCL9, OCLN, TJP1, FGF19 ir NR0B2) raiška buvo analizuojama naudojant kTL-PGR. Grupių skirtumams įvertinti naudotas Stjudento t kriterijus. Rezultatai laikomi statistiškai reikšmingais, kai p ≤ 0,05. Rezultatai. Pradinis tyrimas atskleidė, kad 7-keto-LCA sumažino uždegiminius citokinus koduojančių genų (TNF-α, CXCL9; p < 0,05) raišką ir padidino FXR signalinio kelio žymens (FGF19; p < 0,05) raišką. Pasirinkta 10 µM koncentracija toliau buvo analizuojama su epiteliniais 3D storosios žarnos organoidais. Tačiau, 7-keto-LCA nesumažino uždegimo žymenų raiškos nei OK, nei kontrolės organoiduose. Paciento uždegiminiame 3D organoidų modelyje 7-keto-LCA reikšmingai sumažino OCLN geno raišką (p < 0,05) ir padidino NR0B2 geno raišką UŽL nesergančio asmens organoiduose (p < 0,05). Išvados. Stebima tendencija, kad 7-keto-LCA geba palengvinti žarnyno homeostazės atkūrimą per NR0B2 geno raiškos atstatymą, lyginant OK paciento ir UŽL nesergančio asmens NR0B2 geno raiškos pokyčius.

Pratarmė. Mieli studentai, Gastroenterologija yra viena įdomiausių ir greičiausiai besiplėtojančių medicinos sričių. Dėl mokslo ir technologijų pažangos XXI a. gastroenterologija apima vis platesnį kepenų ir virškinamojo kanalo ligų, gydymo metodų spektrą. Tai neabejotinai viena įvairiapusiškiausių specialybių, kur akivaizdi klinikinių įgūdžių, molekulinių ir ultragarsinių tyrimų bei sudėtingų endoskopinių intervencijų sąsaja. Uždegiminės žarnyno ligos, retos virškinamojo kanalo ligos, mikrobiotos ir kepenų transplantacija yra tik nedidelė dalis sričių, kurios yra patikėtos gydytojams gastroenterologams. Šis Lietuvos sveikatos mokslų universiteto Medicinos akademijos Medicinos fakulteto Gastroenterologijos klinikos (toliau - Gastroenterologijos klinika) kolektyvo parengtas vadovėlis yra skirtas studentams, siekiantiems susipažinti su gastroenterologijos pagrindais. Jau beveik 30 metų Gastroenterologijos klinika aktyviai dalyvauja rengiant tarptautines diagnostikos ir gydymo gaires, yra prestižinių tarptautinių mokslo projektų ir konsorciumų dalyvė, o 2020 m. klinika tapo ir asocijuota Europos retų kepenų ligų tinklo nare. Mūsų klinikos mokslinių tyrimų rezultatai yra publikuojami prestižiniuose mokslo leidiniuose: Nature, Lancet, New England Journal of Medicine, Nature Genetics ir kituose. Rengdami šį vadovėlį stengėmės perteikti visą klinikos sukauptą ilgametę pedagoginę ir klinikinę patirtį bei naujausius mokslo pasiekimus. Tikimės, kad šis vadovėlis ne tik suteiks Jums pagrindinių žinių apie gastroenterologiją, bet ir paskatins rinktis šią specialybę rezidentūros studijose. Autorių vardu prof. Juozas Kupčinskas

Introduction: A compromised intestinal epithelial barrier plays a central role in inflammatory bowel diseases (IBD) by increasing intestinal permeability and promoting inflammation. Therefore, the restoration of intestinal barrier has become critical in IBD management 1.Methylxanthine caffeine and its metabolites are widely consumed bioactive compounds that are known to influence various physiological pro -cesses2.However, their impact on gut health, particularly in modulating inflammation and epithelial permeability, remains underexplored. Aims & Methods: The aim of this study was to investigate potential effects of caffeine (CAF) and its primary metabolites paraxanthine (PRX), theobromine (TBR) and theophylline (TPHL) on inflammatory responses and barrier integrity in colonic epithelial organoids, providing insights into their potential role in IBD management. Primary screening of different concentrations of CAF (1/2.5/5 mM) and its metabolites PRX (10/50/100 µM), TBR(10/20/50 µM) and TPHL (25/100/500 µM) was performed in Caco-2 cells. Selected metabolites and concentrations were further tested on inflamed3D intestinal epithelial organoids derived from ulcerative colitis patient(n = 1) and non-IBD individual (n = 1). Both Caco-2 cells and organoids were pre-treated with metabolites for 1 hour before inflammation induction and were further co-treated with TNF-α/IFN-γ cytokine mix (10 ng/ml each) and metabolites for 24 hours. The effects on inflammation status and epithelial barrier integrity were tested through targeted gene expression analysis using RT-PCR and FITC-Dextran (4kDa) assay, while the effects on generation of reactive oxygen species were investigated by Flow cytometry and DHE assay. The study was approved by the Kaunas Regional Biomedical Research Ethics Committee (protocol no. BE-2-31).Results: Primary screening revealed that both CAF and its metabolite PRX affected the expression of genes encoding inflammatory cytokines (TNF,CXCL9; p ≤ 0.05) and tight junction proteins (OCLN, TJP1, CLDN1; p ≤ 0.05)and were selected for further analysis in 3D intestinal organoids. Neither CAF, nor PRX reduced the expression of inflammatory cytokine-encoding genes in inflamed intestinal organoids. However, both CAF and PRX in-creased expression of tight junction proteins in inflamed intestinal organoids derived from control non-IBD (TJP1 – 3.16-fold by CAF and 1.33-fold by PRX (p ≤ 0.05); OCLN – 1.57-fold by CAF and 1.38-fold by PRX (p ≤ 0.05))and ulcerative colitis (TJP1 – 1.32-fold by PRX; OCLN - 1.14-fold by CAF and1.27-fold by PRX (p ≤ 0.05)) patient-derived organoids. Mean percentages of FITC-Dextran positive organoids were 43 %, 51 %, and 34 % in untreated, inflamed, and inflamed + PRX groups, respectively, revealing a trend in barrier restoration, though it did not reach statistical significance (p =0.16 in inflamed vs. inflamed + PRX). A tendency of reduction in reactive oxygen species production was also observed after exposure to caffeine or paraxanthine (by 17 % and 11 %, respectively (p > 0.05)).Conclusion: Methylxanthine paraxanthine might have a positive effect on gut epithelial barrier function through restoration of the expression of tight junction protein-coding genes.

Introduction: Cholesterol is a key lipid involved in maintaining intestinal epithelial cell membrane structure and function. Recent studies suggest that microbiome-derived cholesterol metabolites, like ursodeoxycholicacid (UDCA) and 7-ketolithocholic acid (7-keto-LCA) promote intestinal epithelial repair and enhance Wnt signalling, thereby facilitating the self-renewal of intestinal stem cells (Li et al., Cell Host & Microbes 2024).Aims & Methods: This study aimed to evaluate the effects of cholesterol-derived microbial metabolites—specifically lithocholic acid (LCA),deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), and 7-keto-LCA—on intestinal epithelial barrier integrity, inflammatory responses, and homeostasis. Primary screening was conducted in Caco-2 monolayers cotreated with pro-inflammatory cytokines IFN-γ and TNF-α for 24 hours to mimic an inflammatory environment. Metabolites were tested at the following concentrations: LCA (10 µM, 50 µM, 75 µM), DCA (10 µM, 100 µM,200 µM), UDCA (100 µM, 250 µM, 500 µM), and 7-keto-LCA (5 µM, 10 µM, 50 µM, 100 µM). Selected compounds showing significant effects were further validated in 3D intestinal epithelial organoid cultures. Gene expression analysis of key markers—TNF-α, CXCL9 (inflammation), OCLN, TJP1 (barrier integrity), and FGF19, NR0B2 (homeostasis)—was performed using quantitative RT-PCR. Statistical significance was determined using Student’st-test with a threshold of p ≤ 0.05.Results: Primary screening on Caco-2 cells revealed that 7-keto-LCA(10µM, 50µM) and LCA (75µM) downregulated the expression of genes encoding inflammatory cytokines (TNF-a and/or CXCL9; p < 0.05) and upregulated the expression of tight junction proteins (OCLN and TPJ1; p < 0.05)and FXR downstream marker (FGF19, p < 0.05). The tested concentrations of DCA and UDCA did not reduce the expression of inflammatory cytokine coding genes and reinstate the expression of tight junction proteins coding genes. For further analysis in 3D intestinal organoid model we selected7-keto-LCA (10µM). 7-keto-LCA significantly upregulated the expression ofNR0B2 (p < 0.05) and showed a tendency to reduce the expression of TNF-αin intestinal organoids (p = 0.12). Conclusion: 7-keto-LCA showed a trend to reduce inflammation and re -store homeostasis in intestinal organoid model.

Background and Objectives: Helicobacter pylori, classified as a Group I carcinogen, is the main risk factor for gastric cancer, one of the leading causes of cancer mortality globally. Lithuania reports one of the highest gastric cancer rates in Europe, yet recent large-scale epidemiological data on H. pylori prevalence are lacking. This study aimed to assess the current seroprevalence of H. pylori in Lithuanian adults and its associations with sociodemographic, environmental factors, and dyspeptic symptoms. Materials and Methods: A cross-sectional study was conducted between 2020 and 2023 at the Lithuanian University of Health Sciences in Kaunas city. Randomly selected adults aged 25–69 years underwent venous blood sampling for H. pylori IgG antibody testing (Serion ELISA) and completed a questionnaire on demographic–environmental factors and dyspeptic symptoms in the past 30 days. Subjects previously treated for H. pylori were excluded from seroprevalence analysis. Seroprevalence was compared across age groups using χ2 and Z-tests with Bonferroni correction. Multivariable logistic regression identified factors associated with H. pylori seropositivity. The selected level of statistical significance was p < 0.05. Results: A total of 1046 adults (mean age 47.2 years, SD = 11.5; 50% males) participated in the study. The overall age-standardized H. pylori seroprevalence was 63.1% (95% CI 60.4–66.7). Seropositivity increased with age, peaking at 80.3% in males aged 55–69. Higher seroprevalence was observed among those with basic education and those lacking access to municipal or heated water during childhood. Regression analysis revealed that male sex, aging, and lower education were significantly associated with H. pylori seropositivity. No significant link was found between H. pylori seroprevalence and gastrointestinal complaints. Conclusions: H. pylori seroprevalence remains high in Lithuanian adults, highlighting the need for ongoing surveillance and consideration of screening strategies. H. pylori infection was linked to sociodemographic and environmental factors but not dyspeptic complaints.

Background Methylxanthine caffeine and its metabolites are widely consumed bioactive compounds that are known to influence various physiological processes 1. However, their impact on gut health, particularly in modulating inflammation and epithelial permeability, remains underexplored. In this study we aimed to investigate the effects of caffeine and its primary metabolites paraxanthine, theobromine and theophylline on inflammatory responses and barrier integrity in gut epithelial models, providing insights into their potential role in gastrointestinal health in the context of Inflammatory bowel disease (IBD).

Methods Primary screening of different concentrations of caffeine and its metabolites paraxanthine, theobromine and theophylline was performed in Caco-2 cells. Selected metabolites were further tested on inflamed 3D intestinal epithelial organoids derived from ulcerative colitis patient (n = 1) and non-IBD individual (n = 1). Inflammation was induced for 24 h using TNF-α and IFN-γ cytokine mix. The effects on inflammation status and epithelial barrier integrity were tested through targeted gene expression analysis using RT-PCR, while the effects on generation of reactive oxygen species were investigated by Flow cytometry and DHE assay.

Results Primary screening revealed that both caffeine and its metabolite paraxanthine affected the expression of genes encoding inflammatory cytokines (TNF-α, CXCL9; p < 0.05) and tight junction proteins (OCLN, ZO-1, CLDN1; p < 0.05) and were selected for further analysis in 3D intestinal organoids. Neither caffeine nor paraxanthine reduced the expression of inflammatory cytokine-encoding genes in inflamed intestinal organoids. However, both metabolites significantly restored the expression of tight junction protein-coding genes OCLN, ZO-1 and CLDN1 (p < 0.05). A tendency of reduction in reactive oxygen species production (by 11 %) was also observed after exposure with paraxanthine.

Conclusion Methylxanthine caffeine and its primary metabolite paraxanthine might have a positive effect on gut epithelial barrier function through restoration of the expression of tight junction protein-coding genes.

Introduction: Helicobacter pylori (H.pylori) infection is the most prevalent type of bacterial infection in the world. Despite significant reduction of prevalence of infection globally, H.pylori remains the main cause of chronic gastritis and gastric cancer. Previous studies on H.pylori prevalence in Lithuania was performed on small size selective population groups. Aims & Methods: The aim of the current study was to investigate H.pylori seroprevalence in general adult population. The study “Chronic Diseases and their Risk Factors in the Adult Population” involved Kaunas city residents aged 25-64 years, who were randomly selected from the Lithuanian population register lists. The study started in 2020, but was terminated after a quarantine due to COVID-19 infection. It was resumed in 2023. Invitations were sent by post to the selected population to come for a health check-up at Kaunas Clinics, Lithuanian University of Health Sciences Hospital. By 23 June 2023, 1074 people had taken part in the study. An ELISA(SERION ELISA® Classic H.pylori IgG, Serion, Germany) was used for the detection of human IgG antibodies to H.pylori in blood serum. All subjects were administered a questionnaire that included questions about factors possibly associated with the prevalence of H.pylori infection.Results: H.pylori antibodies were tested in the blood serum of 1046 participants, representing 97.4% of the total population. The mean age was 47.2± 11.5 years. Standardization of the prevalence of H.pylori antibodies according to the age structure of the population of Kaunas city showed that the seroprevalence of H.pylori antibodies in males was 66.0%, in females- 59.3%, and in all the subjects - 61.8%. Seroprevalence increased with age in groups of 25-34, 35-44, 45-54, 55-64 yrs and was 49,2%, 62,5%, 71.1%;67,5%, and 64%, respectively. H.pylori antibodies were significantly (p<0.05) more common in subjects with secondary and lower education compared to those with higher and university education. Place of residence during childhood did not influence the prevalence of H.pylori infection.Subjects who consumed drinking tap water during childhood were less likely to be infected with H.pylori. The prevalence of H.pylori antibodies was found to be higher in homes without hot water when growing up. A survey of the participants revealed that previously 17.6 % of them were tested for H.pylori, infection was detected in 56,5 %. Medication to eradicate H.pylori was used by 132 (84.6%) subjects with a history of infection.In the current study, H.pylori antibodies were found in 58.3% of subjects who had previously taken medication to eradicate the infection, but sero -prevalence significantly (p<0,05) decreased with increasing the time since eradication, especially in women. Conclusion: H.pylori IgG antibodies have been detected in a large proportion of adult Kaunas residents aged 25-64 years. High seroprevalence of H.pylori in Lithuania (a country with moderate incidence of gastric cancer)raise a question on possible introducing of screen-and-test strategies for H.pylori to diminish gastric cancer incidence and mortality.

Introduction: Ulcerative colitis (UC) is an inflammatory bowel disease marked by chronic inflammation of the colon’s inner lining. Its pathogenesis involves a multifactorial interplay between genetic and environmental factors, dysregulated immune responses, and alterations in the gut microbiome. MicroRNAs (miRNAs) have emerged as critical regulators of gene expression implicated in UC pathogenesis.Understanding the differential expression of miRNAs in colonic epithelial cells and their extracellular vesicles (EVs) during bacterial exposure could offer potential insights into the interplay between gut microbiota and epithelial cell-mediated inflammatory responses.Aims & Methods: The objective of this study was to investigate the expression of UC-associated miRNAs in colonic epithelial cells and their EVs,using UC patients and control subjects-derived colonic epithelial cells co-cultures with Escherichia coli and Phocaeicola vulgatus bacteria.Colonic stem cells from healthy controls (HC) (n=10) and patients with active UC (n=10) were used to generate 3D colonic epithelial organoids,which subsequently served as a cell source for constructing polarized monolayers of colonic epithelial cells. These monolayers were co-cultured with commensal E. coli or P. vulgatus bacteria for 2 hours, washed, replenished with fresh culture medium, and further incubated for an additional 24 hours. EVs from cell-conditioned media were isolated using precipitation method and characterized by ELISA (CD63, HSP70, Apo-A1), Dynamic Light Scattering and cryo-TEM. RNAs from colonic epithelial cells and precipitated EVs were isolated using silica-column based method. MiRNAs expression was evaluated by TaqMan qPCR. Statistical analysis and visualization of the results were performed using R studio.Results: Although statistically insignificant, but the expression of miR-183-5p and miR-135b-5p in epithelial cells of HC group was tended to de -crease after contact with E. coli compared to untreated cells (resp. FC=0.84(p=0.574) and FC=0.87 (p=0.798)), while miR-146a-5p increased (FC=1.37(p=0.798)). In the EVs samples of HC, the same trends were observed.Interestingly, in the UC patients’ cells, after incubation with E. coli, the expression of all studied miRNAs - miR-183-5p, miR-135b-5p, miR-146a-5p - tended to increase (resp. FC=1.53 (p=0.730), FC=1.71 (p=0.666), andFC=2.39 (p=0.258)).Similar expression changes were observed in the EVs of colonic epithelial cells from UC patients following stimulation with E. coli (resp. FC=1.32(p=0.730) (miR-183-5p), FC=1.32 (p=0.666) (miR-135b-5p), and FC=2.07(p=0.258) (miR-146a-5p)).After culturing colonic epithelial cells with P. vulgatus the expression of all investigated miRNAs, compared to unstimulated cells, remained almost unchanged, both in HC and in UC patients’ monolayers. Meanwhile, in both the HC and UC groups, a decrease in miR-183-5p and miR-135b-5p expression was observed in EVs following contact with P. vulgatus, compared to control conditions (in the HC group resp. FC=0.59 (p=0.382) andFC=0.51 (p=0.234); in the UC group resp. FC=0.70 (p=0.605) and FC=0.73(p=0.489)). However, the expression of miR-146a showed a decreasing trend only in the HC group compared to unstimulated cells (FC=0.53(p=0.161)).Conclusion: Due to the considerable variation in the response between study subjects, the observed expression changes did not reach statistical significance. The experimental study revealed a tendency that the expression of miRNAs associated with UC can be modulated by commensal gut bacteria, particularly E. coli, both in colonic epithelial cells and their EVs.

Objective: Previous studies on H. pylori in Lithuania were performed on small-size selective population groups. Therefore, the study’s aim was to investigate H. pylori seroprevalence in the general adult population. Patients and Methods: The study involved Kaunas residents aged 25-64 who were randomly selected from population register lists. The classic H. pylori IgG (Serion, Germany) test was used to detect IgG antibodies. All participants filled out a questionnaire about factors possibly associated with H. pylori infection. Results: H. pylori antibodies were tested in the blood serum of 1,046 participants. The mean age was 47.2±11.5 years. Seroprevalence of H. pylori in males was 66.0%, in females 59.3%, and in all subjects 61.8%. Seroprevalence increased with age in groups of 25-34, 35-44, 45-54, and 55-64 years and was 49.2%, 62.5%, 71.1%, 67.5%, and 64%, respectively. H. pylori antibodies were more common (p<0.05) in subjects with secondary and lower education compared to those with higher education. The prevalence of H. pylori antibodies was found to be higher in homes without hot water when growing up. 17.6% of participants were previously tested for H. pylori, and infection was detected in 56.5% of them. Medication for eradication was used by 132 (84.6%) participants with a history of infection. In the current study, H. pylori antibodies were found in 58.3% of subjects who previously underwent eradication, but seroprevalence decreased (p<0.05) with increasing time since eradication, especially in women. Conclusions: The high seroprevalence of H. pylori in Lithuania (a country with a moderate incidence of gastric cancer) raises a question on the possible introduction of screen-and-test strategies for H. pylori to diminish gastric cancer incidence and mortality.

Objective: Ulcerative colitis (UC) is characterized by disrupted mucosal barrier and microbial composition. Yet, it remains uncertain if commensal gut bacteria can influence the maintenance of the colon’s protective lining. Therefore, we aimed to assess how colonic epithelial cells from UC patients respond to commensal bacteria. Materials and Methods: Co-culture model of colonic epithelial organoid-derived monolayers of UC patients (n=9) and non-IBD controls (n=8) and Escherichia coli (ATCC25922) and Phocaeicola vulgatus (ATCC8482) bacteria type strains were used for evaluation of epithelial barrier integrity (ZO1), pathogen recognition (TLR4) and stress induction (HSPA1A, HSPB1) gene expression, as well as miRNA (miR183-5p, miR-135b-5p, miR-146a-5p) expression in host epithelial cells and extracellular vesicles (EVs). Results: E. coli and P. vulgatus did not trigger pathogen-pattern recognition or stress responses in colonic epithelial cells but showed a tendency to increase ZO1 expression in non-IBD cells (FC = 3.59 and FC = 2.27, respectively, p ≥ 0.05), while decreasing it in UC cells (FC = 0.67 and FC = 0.75, respectively, p ≥ 0.05). The studied miRNAs – miR-183-5p, miR-135b-5p, miR-146a-5p – tended to increase in the EVs of UC colonic epithelial cells after E. coli stimulation (FC=1.32, FC=1.32, and FC=2.07, respectively, p ≥ 0.05). Conversely, cultivation with P. vulgatus tended to decrease miR-183-5p (FC=0.70, FC=0.59, respectively, p ≥ 0.05) and miR-135b-5p (FC=0.73, FC=0.51, respectively, p ≥ 0.05) expression in both UC and non-IBD groups. Conclusions: E. coli and P. vulgatus contibute in compromising mucosal barrier integrity and influencing miRNA expression in extracellular vesicles.