The microRNA expression in crypt-top and crypt-bottom colonic epithelial cell populations demonstrates cell-type specificity and correlates with endoscopic activity in ulcerative colitis.

BACKGROUND AND AIMS: Colonic epithelial barrier dysfunction is one of the early events in ulcerative colitis (UC) and microRNAs (miRNAs) participate in its regulation. However, cell type-specific miRNome during UC is still unknown. Thus, we aimed to explore miRNA expression patterns in colon tissue and epithelial cells at active and quiescent UC. METHODS: Small RNA-sequencing in colon tissue, crypt-bottom (CD44+), and crypt-top (CD66a+) colonic epithelial cells from two cohorts of UC patients (n=74) and healthy individuals (n=50) was performed. Data analysis encompassed differential expression, weighted gene co-expression network, correlation, gene-set enrichment analyses. RESULTS: Differentially expressed colonic tissue miRNAs showed potential involvement in regulation of interleukin-4 and interleukin-13 signalling during UC. As this pathway plays role in intestinal barrier regulation, consecutive analysis of spatially distinct colonic epithelial cell populations was performed. Cell-type (crypt-top and crypt-bottom) specific miRNA expression patterns were identified in both active and quiescent UC. Target genes of differentially expressed epithelial miRNAs at different disease activity were overrepresented in epithelial cell migration and therefore intestinal barrier integrity regulation. The pro-inflammatory miRNA co-expression module M1 correlated with endoscopic disease activity and successfully distinguished active and quiescent UC not only in both epithelial cell populations, but also in the colon tissue. The anti-inflammatory module M2 was specific to crypt-bottom cells and significantly enriched in the quiescent UC patients. CONCLUSIONS: miRNA expression was specific to colonic epithelial cell populations and UC state, reflecting endoscopic disease activity. Irrespective of the UC state, deregulated epithelial miRNAs were associated with regulation of intestinal barrier integrity.

Constituents of stable commensal microbiota imply diverse colonic epithelial cell reactivity in patients with ulcerative colitis.

BACKGROUND: Despite extensive research on microbiome alterations in ulcerative colitis (UC), the role of the constituent stable microbiota remains unclear. RESULTS: This study, employing 16S rRNA-gene sequencing, uncovers a persistent microbial imbalance in both active and quiescent UC patients compared to healthy controls. Using co-occurrence and differential abundance analysis, the study highlights microbial constituents, featuring Phocaeicola, Collinsella, Roseburia, Holdemanella, and Bacteroides, that are not affected during the course of UC. Co-cultivation experiments, utilizing commensal Escherichia coli and Phocaeicola vulgatus, were conducted with intestinal epithelial organoids derived from active UC patients and controls. These experiments reveal a tendency for a differential response in tight junction formation and maintenance in colonic epithelial cells, without inducing pathogen recognition and stress responses, offering further insights into the roles of these microorganisms in UC pathogenesis. These experiments also uncover high variation in patients' response to the same bacteria, which indicate the need for more comprehensive, stratified analyses with an expanded sample size. CONCLUSION: This study reveals that a substantial part of the gut microbiota remains stable throughout progression of UC. Functional experiments suggest that members of core microbiota - Escherichia coli and Phocaeicola vulgatus - potentially differentially regulate the expression of tight junction gene in the colonic epithelium of UC patients and healthy individuals.

Prolonged culturing of colonic epithelial organoids derived from healthy individuals and ulcerative colitis patients results in the decrease of LINE-1 methylation level.

Patient-derived human intestinal organoids are becoming an indispensable tool for the research of digestive system in health and disease. However, very little is still known about the long-term culturing effect on global genomic methylation level in colonic epithelial organoids derived from healthy individuals as well as active and quiescent ulcerative colitis (UC) patients. In this study, we aimed to evaluate the epigenetic stability of these organoids by assessing the methylation level of LINE-1 during prolonged culturing. We found that LINE-1 region of both healthy control and UC patient colon tissues as well as corresponding epithelial organoids is highly methylated (exceeding 60%). We also showed that long-term culturing of colonic epithelial organoids generated from stem cells of healthy and diseased (both active and quiescent UC) individuals results in decrease of LINE-1 (up to 8%) methylation level, when compared to tissue of origin and short-term cultures. Moreover, we revealed that LINE-1 methylation level in sub-cultured organoids decreases at different pace depending on the patient diagnosis (healthy control, active or quiescent UC). Therefore, we propose LINE-1 as a potential and convenient biomarker for reliable assessment of global methylation status of patient-derived intestinal epithelial organoids in routine testing of ex vivo cultures.

Comparison of genomic and transcriptional microbiome analysis in gastric cancer patients and healthy individuals.

BACKGROUND: Helicobacter pylori and the stomach microbiome play a crucial role in gastric carcinogenesis, and detailed characterization of the microbiome is necessary for a better understanding of the pathophysiology of the disease. There are two common modalities for microbiome analysis: DNA (16S rRNA gene) and RNA (16S rRNA transcript) sequencing. The implications from the use of one or another sequencing approach on the characterization and comparability of the mucosal microbiome in gastric cancer (GC) are poorly studied. AIM: To characterize the microbiota of GC using 16S rRNA gene and its transcript and determine difference in the bacterial composition. METHODS: In this study, 316 DNA and RNA samples extracted from 105 individual stomach biopsies were included. The study cohort consisted of 29 healthy control individuals and 76 patients with GC. Gastric tissue biopsy samples were collected from damaged mucosa and healthy mucosa at least 5 cm from the tumor tissue. From the controls, healthy stomach mucosa biopsies were collected. From all biopsies RNA and DNA were extracted. RNA was reverse transcribed into cDNA. V1-V2 region of bacterial 16S rRNA gene from all samples were amplified and sequenced on an Illumina MiSeq platform. Bray-Curtis algorithm was used to construct sample-similarity matrices abundances of taxonomic ranks in each sample type. For significant differences between groups permutational multivariate analysis of variance and Mann-Whitney test followed by false-discovery rate test were used. RESULTS: Microbial analysis revealed that only a portion of phylotypes (18%-30%) overlapped between microbial profiles obtained from DNA and RNA samples. Detailed analysis revealed differences between GC and controls depending on the chosen modality, identifying 17 genera at the DNA level and 27 genera at the RNA level. Ten of those bacteria were found to be different from the control group at both levels. The key taxa showed congruent results in various tests used; however, differences in 7 bacteria taxa were found uniquely only at the DNA level, and 17 uniquely only at the RNA level. Furthermore, RNA sequencing was more sensitive for detecting differences in bacterial richness, as well as differences in the relative abundance of Reyranella and Sediminibacterium according to the type of GC. In each study group (control, tumor, and tumor adjacent) were found differences between DNA and RNA bacterial profiles. CONCLUSION: Comprehensive microbial study provides evidence for the effect of choice of sequencing modality on the microbiota profile, as well as on the identified differences between case and control.

Molecular Characterization and Seroprevalence of Hepatitis E Virus in Inflammatory Bowel Disease Patients and Solid Organ Transplant Recipients.

Seroprevalence rates and molecular characterization of hepatitis E virus (HEV) prevalent in the Lithuanian human population has not yet been evaluated. Immunosuppressed individuals have been recognized as a risk group for chronic hepatitis due to HEV genotype 3 (HEV-3) infections. The objectives of the present study were to determine prevalence rates of anti-HEV antibodies among inflammatory bowel disease (IBD) patients and solid organ transplant (SOT) recipients, to isolate and characterize HEV strain present in the Lithuanian human population, and to investigate its capacity to infect non-human primate (MARC-145 and Vero), swine (PK-15) and murine (Neuro-2a) cells in vitro. In the present study, the significant difference of anti-HEV IgG prevalence between healthy (3.0% (95% CI 0-6.3)) and immunosuppressed individuals (12.0% [95% CI 8.1-15.9]) was described. Moreover, our findings showed that anti-HEV IgG seropositivity can be significantly predicted by increasing age (OR = 1.032, p < 0.01), diagnosis of IBD (OR = 4.541, p < 0.01) and reception of SOT (OR = 4.042, <0.05). Locally isolated HEV strain clustered within genotype 3i subtype of genotype 3 and was capable of infecting MARC-145 cells. This study demonstrates higher HEV seroprevalence in the risk group compared to healthy control individuals without confidence interval overlap. The high level of genetic homology between human and animal strains in Lithuania and the capacity of locally isolated strains to infect cells of non-human origin suggests its potential for zoonotic transmission.

Environmental and Dietary Risk Factors for Colonic Diverticulosis and Diverticulitis.

BACKGROUND AND AIMS: Colonic diverticulosis (CD) is among the most common conditions of the large bowel. Several factors have been associated with an increased risk of CD and its complications, including advanced age, obesity, physical inactivity, and a low-fiber diet. Available data is conflicting and a comprehensive analysis of different bowel, dietary and environmental habits linked with CD is lacking. We aimed to investigate the relationship between potential risk factors and CD prevalence using full data from a colonoscopy-based cross-sectional study in Europe. METHODS: The study was conducted at three tertiary referral centers in Germany and Lithuania. It included consecutive adult patients referred for routine colonoscopy who completed a detailed questionnaire on our considered multiple risk factors for diverticulosis and diverticulitis, including dietary and environmental factors, and bowel habits. RESULTS: The study included 1,333 patients, 696 women and 635 men. Colonic diverticulosis was diagnosed in 858 (64%) of patients. Multivariate analysis revealed that age (OR: 1.08, 95%CI: 1.06-1.10, p<0.001) and obesity (OR: 1.05, 95%CI: 1.02-1.09, p=0.004) were associated with CD. We also revealed new risk factors for CD: increased frequency of bowel movements (OR: 0.10, 95%CI: 0.03-0.33, p<0.001) and feeling of incomplete bowel emptying (OR: 2.05, 95%CI: 1.47-2.87, p<0.001). Older participants had reduced odds (OR: 0.921, 95 CI: 0.89-0.95, p<0.05) of diverticulitis compared to younger subjects. Feeling of incomplete bowel emptying after defecation was associated with increased odds (OR: 2.769, 95% CI 1.35-5.7, p<0.006) for diverticulitis. Moreover, participants with a higher educational status had increased odds (OR: 2.453, 95%CI: 1.31-4.59, p=0.005) for diverticulitis compared to the lower education group. CONCLUSIONS:  Study shows that older age, obesity, frequency of bowel movements, and feeling of incomplete bowel emptying are associated with the risk of CD. Furthermore, older age, feeling of incomplete bowel emptying, and higher education were associated with the risk of diverticulitis among CD patients.

Drug-Induced Intersticial Nephritis. Clinical Case.

5-Aminosalicylic acid (5-ASA) preparations are widely used in the treatment of inflammatory bowel diseases. The most commonly used medicine is mesalamine. Overall, it is a very safe drug with few side effects. A rare side effect of this medicine is interstitial nephritis (IN). With discontinuation of the drug the renal function usually restores. However, if damage has not been detected for a long time, irreversible changes may occur. In this article, we present a clinical case of mesalamine induced IN. A 56-year-old man who has had ulcerative colitis for 20 years, was admitted due to mesalamine induced acute renal failure. A year before, the patient had been diagnosed with mesalamine-induced interstitial nephritis and the treatment with mesalamine was discontinued. The symptoms of ulcerative colitis worsened, and the patient independently decided to start taking mesalamine, which resulted in worsening of his health condition and impaired renal function. Mesalamine has been discontinued, additional treatment for acute kidney failure has been administered including hemodialysis. Renal function recovered and the patient was released for further treatment of ulcerative colitis and monitoring of renal function.

Evaluation of a novel 14C-urea breath test "Heliprobe" in diagnosis of Helicobacter pylori infection.

BACKGROUND: At present, (14)C-urea breath test is considered a gold standard for diagnosis of Helicobacter pylori infection, but they are time-consuming, comparably expensive, and usually not portable tests. The aim of our study was to establish the diagnostic value of the novel, inexpensive, quick, and convenient to use (14)C-urea breath test "Heliprobe", Noster AB, Sweden. MATERIAL AND METHODS: Helicobacter pylori testing using "Heliprobe" was performed in 108 consecutive patients. Helicobacter pylori was also investigated using rapid urease test and Giemsa stained histological specimens according to Sydney system. RESULTS: The diagnostic values of "Heliprobe" assuming the Helicobacter pylori positivity, if the results of two tests (rapid urease test and histology) are positive, were: sensitivity--97%, specificity--87%, positive predictive value--93%, negative predictive value--95%, accuracy--94%. The diagnostic values of "Heliprobe" assuming the Helicobacter pylori positivity, if at least the results of one test are positive: sensitivity--92%, specificity--100%, positive predictive value--100%, negative predictive value--84%, accuracy--94%. CONCLUSIONS. The novel, quick, convenient to use (14)C-urea breath test "Heliprobe" is accurate, reliable, and useful for the diagnosis of Helicobacter pylori infection in routine clinical practice.