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Background Bowel preparation prior to colonoscopy is a routine procedure and supposed to be safe. However, the exacerbation of the complaints could be observed in numerous cases after colonoscopy in patients with inflammatory bowel disease (IBD, Crohn’s disease CD, ulcerative colitis UC). Therefore, our study aimed to investigate the short- and long-term changes of the fecal microbiota composition influenced by the bowel preparation with sodium-picosulphate and magnesium-oxide. Methods IBD patients (inactive or mild) preparing for colonoscopy with bowel preparation were enrolled in the study. Control group (HC) consisted of non-IBD patients who were recommended to undergo colonoscopy due to abdominal complaints. Clinical data, blood and stool samples were collected before colonoscopy (point A), 3 days later (point B) and 4 weeks later (point C) to assess disease activity and the changes of the gut microbiome. Fecal microbiota structure was determined by sequencing V4 region of the 16S rRNA gene. Microbiome structure was determined at family level. Statistical analysis included differential abundance analysis and Mann-Whitney test, values of p Results 41 patients (9 CD, 13 UC, 19 HC) were enrolled in our study. After the bowel preparation, alpha diversity in the CD group was lower compared to UC(p=0.01) and control(p=0.02) patients at point B. Alpha diversity of UC patients was significantly higher than CD (p‹0.01) and control (p=0.03) groups at point C. Alpha diversity of UC patients increased from point A to C (p=0.04). However, a decreasing tendency in alpha diversity was observed in patients with CD at A to C period (p=0.26). These changes couldn’t be observed in the control group. Beta diversity showed slight differences between the IBD groups and the control group. After bowel preparation, beta diversity of CD patients immediately (from A to B) changed p=0.03), while beta diversity of UC patients seems to continually change from point A to C(p=0.03). Based on the differential abundance analysis, Clostridiales and Pseudomonadaceae families increased, while Bifidobacteriaceae, Carnobacteriaceae, Veillonellaceae and Pasteurellaceae families decreased in CD patients compared to the HC at point B. The most growing families in HC group were Brucellaceae, Moraxellaceae and Alcaligenaceae at A to B period. Conclusion Our findings may suggest that bowel preparation can result in a significant change in faecal microbial composition in IBD. Microbial alterations recovering earlier in UC patients due to changed mucosa. Reduced alpha diversity and altered abundance in CD may have potential role in disease exacerbation. Further examinations are needed to clarify our pilot results.

Background Changes in microbiome of the gut and decreased diversity in inflammatory bowel disease(IBD) is the subject of intense researches. The exact pathophysiological connection is lacking, but the systemic inflammation due to the microbial alterations are assumed. This study aimed to perform analysis of faecal microbiota composition simultaneously with body composition by bioelectrical impedance and nutrition habits in Crohn’s disease(CD) patients to find specific microbiota profiles connected with obesity, mesenterial fat, signs of systemic inflammation, nutritional and body composition characteristics. Methods In our prospective cohort study, CD patients were enrolled. At the inclusion, demographic and clinical data, blood, and faecal samples were obtained. Clinical disease activity was assessed by CDAI, and SES-CD. Laboratory tests were made including C-reactive protein(CRP), albumin and lipids (triglyceride, cholesterol). Faecal bacterial composition was assessed using shotgun metagenomics sequencing technique. Each patient underwent a body composition analysis via bioelectrical impedance analysis. A nutritional questionnaire was filled by each subject. Results Data of 27 CD patients with different localisation was analysed in this study (mean age was 35±11 years). According to the gut inflammation, some Blautia species correlated positively with SES-CD (p=0,004), and correlated with faecal calprotectin negatively (p=0,02), while Roseburia hominis negatively with CDAI scores(p=0,01). High CDAI scores were also associated with dysbiosis. Adlercreutzia equolifaciens and Blautia hansenii associated with triglyceride(negatively p=0,04, positively p=0,05). Firmicutes has a lower, while Bacteroidetes has a higher abundance at high cholesterol level(p=0,001, p=0,023). Lower Adlercreutzia and Lactobacillus abundance were seen in patients with high visceral fat area(p=0,05, p=0,01), while Clostridia correlated with percent body fat(p=0,025).Bifidobacterium bifidum were associated in people with low body weight or low skeletal muscle mass(p=0,01, p=0,03). Beside Clostridia, Eubacteriales also correlated with waist-hip ratio(p=0,04, p=0,04). Conclusion To our knowledge this is the first study to analyse microbiota profiles by shotgun sequencing technique in CD patients depending on body composition via bioelectrical impedance and nutrition. Changes in Blautia, Lactobacillus, Adlercreutzia, Roseburia and Bifidobacterium abundances in patients with CD and obesity highlight the importance of correlations between lipid metabolism, adipose tissue mass, and chronic inflammation. Through this, it helps to develop new therapeutic approaches, such as personalized pre- or probiotics or faecal microbiota transplantation.

Background Inflammatory bowel disease (IBD) is characterized by an unregulated immune response generating unbalance cytokine homeostasis. Analysis of patient-specific cytokine profiles may open new therapeutic targets or identify biomarkers that distinguish responders from non-responders before initiating therapy. Therefore, the aim of the present study was to determine cytokine profile of IBD patients and identify cytokines with predictive potential. Methods Blood and biopsy samples were obtained from 79 clinically active IBD patients and 28 controls without IBD. Inclusion criteria included change of therapy or initiation of new treatment for IBD patients. Organoid culture (OC) was generated from 5 clinically active IBD patients and 5 controls. Cytokine Array was used to analyse cytokine expression patterns. Total protein and mRNA were isolated from biopsies and from OCs. Protein levels were measured by ELISA and gene expression by qRT-PCR in serum, mucosa and in OCs. Results Pro-inflammatory cytokines were not detected in control samples, whereas in IBD biopsies the cytokine profiles enabled the discrimination between inflamed or non-inflamed areas. The mucosal expression of Serpin E1 was detected in all inflamed biopsy samples, whereas it was below the detection limit in healthy subjects. In responders Serpin E1 gene expressions were significantly (p Conclusion Our results suggest that serum and mucosal Serpin E1 expression reflects endoscopic activity in IBD, which could be used to follow-up disease activity and possibly therapeutic response. Organoids mimicked the expression of Serpin E1 in the original biopsies suggesting that OCs can be used to study the effect of Serpin E1 inhibition.

Background Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastrointestinal tract which is associated with imbalanced pro- and anti-inflammatory cytokines. Anti-TNF-α therapy is widely used, but 10–30% of the patients are primarily not responding to the treatment or lose response over time. In vitro organoid cultures (OCs) generated from tissue specific adult stem cells can mimic cellular diversity and function of the organ of origin and might be used as a predictive tool for patient specific therapeutic response. However, the reductionist nature of the OCs and lack of knowledge about the proper representation of the disease features currently limit the utilization of the OCs as ex vivo IBD models. Methods Therefore our aims were to determine and compare cytokine profiles of colonic biopsies collected from IBD patients and OCs generated from the same biopsies, and to follow the changes of cytokine expression over time. In this study samples from 20 IBD and 8 non-IBD patients were used. Biopsies were taken during colonoscopy from inflamed part of the colon of IBD patients. Crypts were isolated from biopsy samples to establish colon OCs. Total protein and RNA were isolated from biopsies and OCs. Cytokine Array was used to determine cytokine patterns in our samples. Gene expressions of control and IBD OCs after first passage was compared by qPCR. TNF-α concentrations and cellular expression was determined by EILSA and immunostainings, respectively in control and IBD OCs. To determine therapeutic response OCs were treated with anti-TNF-α therapy. Results Cytokine patterns of colon biopsies and OCs were remarkably similar until first passage. The major pro-inflammatory cytokines, IL-1β, IL-6, IL-8, were detected both in biopsies and OCs. After second passage the cytokine expression decreased or disappeared in OCs. TNF-α was also measurable in the OCs, however a similar decrease was observed, which was also confirmed by immunostaining. Gene expression analysis in IBD OCs showed an increased TNF-α, IL-1β and IL-6 levels compared to healthy control OCs after first passage. ELISA also showed higher TNF-α concentration in organoids from inflamed origin compared to controls. After anti-TNF-α treatment we detected a decrease in the IL-6 gene expression in treated OCs. Conclusion Our results suggest that colon OCs maintain the cytokine expression ex vivo until the 2nd passage and show inflammatory characteristics. Moreover, in vitro treatment induces changes in the cytokine expression. Based on these results the utility of patient-derived organoids to predict the therapeutic response can be investigated.

Background Inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastrointestinal tract which is associated with the imbalanced pro- and anti-inflammatory cytokines. Anti-TNF-α is widely used as a therapeutic agent, but 10–30% of the patients are not responding to the treatment or in time it become less affective. In vitro organoid cultures generated from stem cells can mimic the cellular diversity and function of the organ of origin and might be used as a predictive tool for patient specific response. However, the in vitro cytokine expression pattern of the IBD colon organoid cultures have never been determined or compared with the cytokine expression of the colon mucosa. Methods Our aim was to determine and compare the cytokine profiles of the colon biopsy samples in IBD patients and organoid cultures generated from these biopsies. In this study samples from 5 IBD patients were used. Biopsies were taken during colonoscopy from inflamed part of the colon. Crypts were isolated from the biopsy samples to start colon organoid culture. Total protein was isolated from the biopsies and the organoids. Cytokine Array was used to determine the cytokine patterns in both samples. ELISA was used to measure the TNF-α concentrations and immunofluorescence staining to localize it in the organoids. Results We determined the cytokine profile of the biopsy samples and the colon organoid cultures. The cytokine patterns were remarkably similar until the first passage. The major pro-inflammatory cytokines, IL-1β, IL-6, IL-8, were detected in the organoids after the first passage. After the second passage the cytokine expression decreased or disappeared. ELISA revealed that TNF-α was also expressed in the organoid cultures, however a similar decrease in the expression was observed, which was also confirmed by immunofluorescence staining. TNF-α positive cells were present in the organoids, but it decreased after every passage. Conclusion Our results suggest that the colon organoid cultures maintain the cytokine profile of the tissue of origin until a limited time (until the first passage). Therefore, organoids might be used to determine patient specific therapy for drug tests.

Background Inflammatory Bowel Disease (IBD) occurs as a consequence of abnormal immune response generating unbalance between pro- and anti-inflammatory signalling. Analysis of cytokine profiles in view of different cytokine targeting or immunosuppressive therapy may open up new therapeutic targets and may reveal biological profiles that distinguish responders from non-responders before initiating therapy. The aim of present study was to determine cytokine profile of IBD patients and identify cytokines with predictive potential. Methods IBD patients with clinically active disease were enrolled in study. Blood and biopsy samples were obtained from 22 IBD patients and 5 healthy controls. Biopsies were taken from inflamed and non-inflamed part of colon of IBD patients. Total protein and mRNA were isolated from biopsy samples. Cytokine Array was used to analyse cytokine expression patterns. Serum and mucosal SerpinE1 levels were measured by ELISA and qRT-PCR. Results In samples from IBD patients, remarkable discrimination between inflamed, or non-inflamed areas was observed, whereas no pro-inflammatory cytokines were detected in control samples. SerpinE1 was presented in every inflamed biopsy samples, which was analyzed in more details. Mucosal expression of SerpinE1 differed significantly in healthy subjects compared to IBD patients with active disease (0 vs 24.06 pg/mg, p=0.02). After therapy induction a remarkable decrease was observed in the mucosal SerpinE1 concentration in responders (45.5 vs 9.7 pg/mg, p=0.02) versus non-responders (45 vs 61.2 pg/mg, p=0.3). Moreover, mean value of mucosal SerpinE1 did not differ significantly in healthy subjects compared to responders (5.7 vs. 0 pg/mg, p=0.12). In non-responders the fold changes of SerpinE1 gene expressions were significantly (p=0.001) higher than in responders. Lowest expression of SerpinE1 gene was measured in control samples, whereas the highest in untreated, inflamed biopsy samples. Serum and mucosal SerpinE1 concentrations were significantly higher in patients with active disease compared to inactive (tissue: 5 vs 47.4 pg/mg, p=0.00003; serum: 22.4 vs 25.94 mg/ml, p=0.022). Correlation analysis revealed that serum SerpinE1 correlates with disease activity (p0.1, sensitivity=72%, specificity=77.8%, accuracy=73.5%). Conclusion These results suggest that serum and mucosal SeprinE1 expression reflects endoscopic activity of IBD. Correlation of SerpinE1expression between the blood and the bowel mucosa would open up new possibilities in non-invasive disease monitoring of IBD.

The pathways of the conformational transitions in flexible molecular systems were studied by the DDRP (Dynamically Determined Reaction Path) method implemented in TINKER molecular modelling package. Our first model systems were conformational transitions in small organic molecules (determination of rotational transitions, boat-chair transitions, etc.). The method was found to be very effective in finding such transitions and the location of the transition state. In comparison with other methods (modified Elber and Czerminski (EC) method implemented also in TINKER), similar results were obtained. For the conformational change of di- and small peptide fragments also acceptable paths and barriers were obtained. To determine the conformational transition in large peptides (i.e. folding), the effectivity of the procedure demands further studies.