The inhibitory effects of live and UV-killed Akkermansia muciniphila and its derivatives on cytotoxicity and inflammatory response induced by Clostridioides difficile RT001 in vitro

Clostridioides difficile infection (CDI) is the leading cause of healthcare-acquired infections worldwide. Probiotics are widely recommended to prevent CDI and its recurrences. Akkermansia muciniphila, as a therapeutic symbiont colonizing the intestinal mucosal layer, is considered to be a promising next-generation probiotic. In this work, we assessed the inhibitory effects of A. muciniphila MucT and its derivatives on cytotoxicity and inflammatory response induced by C. difficile RT001 in Caco-2 cells. The results obtained from SEM revealed that the morphology of UV-killed A. muciniphila remained unchanged after UV inactivation. TEM analysis showed that A. muciniphila–isolated extracellular vesicles (EVs) were spherical and ranged from 50 to 200 nm in size. Toxigenic supernatant (Tox-S) of C. difficile RT001 (500 μg/ml) significantly (P <0.01) reduced the cell viability of Caco-2 cells. Caco-2 cells treated with live (MOI 10), UV-killed (MOI 10), cell-free supernatant (CFS, 106 cfu/ml), and EVs (20 μg/ml) of A. muciniphila exhibited over 90% viability in comparison to untreated control. The neutralized CFS preparation using A. muciniphila and its derivatives could notably reduce the expression level of inflammatory markers. Additionally, A. muciniphila and its derivatives modulated the production of IL-1β, TNF-α, and IL-10 in Tox-S stimulated Caco-2 cells. We demonstrated that A. muciniphila and its derivatives can modulate changes in the gut barrier–related genes and inflammatory response caused by C. difficile Tox-S in Caco-2 cells. (AU)

Atorvastatin Effect on COVID-19 Outcomes: A Propensity Score Matched Study on Hospitalized Patients.

BACKGROUND: This study investigated the association of atorvastatin use on survival, need for intensive care unit (ICU) admission, and length of hospital stay (LOS) among COVID-19 inpatients. MATERIALS AND METHODS: A retrospective study was conducted between March 20th, 2020, and March 18th, 2021, on patients with confirmed COVID-19 admitted to three hospitals in Tehran, Iran. The unadjusted and adjusted effects of atorvastatin on COVID-19 prognosis were investigated. Propensity score matching (PSM) was used to achieve a 1:1 balanced dataset with a caliper distance less than 0.1 and the nearest neighbor method without replacement. RESULTS: Of 4322 COVID-19 patients, 2136 (49.42%) were treated with atorvastatin. After PSM, 1245 atorvastatin inpatients and 1245 controls were included with a median age of 62.0 (interquartile range [IQR]: 51.0, 76.0) and 63.0 (IQR: 51.0, 75.0) years, respectively. The standardized mean differences were less than 0.1 for all confounders, suggesting a good covariate balance. The use of atorvastatin was associated with decreased COVID-19 mortality (HR: 0.80; 95% CI: 0.68-0.95), whereas no relationship was found between atorvastatin and the need for ICU admission (HR: 1.21; 95% CI: 0.99-1.47). LOS was significantly higher in the atorvastatin cohort than controls (Atorvastatin vs. others: 7 [5, 11] vs. 6 [4, 10] days; p = 0.003). The survival rate was higher in combination therapy of atorvastatin plus enoxaparin than in those who received atorvastatin alone (p-value=0.001). CONCLUSION: Atorvastatin may reduce the risk of COVID-19 in-hospital mortality and could be a beneficial option for an add-on therapy. Randomized trials are warranted to confirm the results of the current observational studies.

The inhibitory effects of live and UV-killed Akkermansia muciniphila and its derivatives on cytotoxicity and inflammatory response induced by Clostridioides difficile RT001 in vitro.

Clostridioides difficile infection (CDI) is the leading cause of healthcare-acquired infections worldwide. Probiotics are widely recommended to prevent CDI and its recurrences. Akkermansia muciniphila, as a therapeutic symbiont colonizing the intestinal mucosal layer, is considered to be a promising next-generation probiotic. In this work, we assessed the inhibitory effects of A. muciniphila MucT and its derivatives on cytotoxicity and inflammatory response induced by C. difficile RT001 in Caco-2 cells. The results obtained from SEM revealed that the morphology of UV-killed A. muciniphila remained unchanged after UV inactivation. TEM analysis showed that A. muciniphila-isolated extracellular vesicles (EVs) were spherical and ranged from 50 to 200 nm in size. Toxigenic supernatant (Tox-S) of C. difficile RT001 (500 µg/ml) significantly (P <0.01) reduced the cell viability of Caco-2 cells. Caco-2 cells treated with live (MOI 10), UV-killed (MOI 10), cell-free supernatant (CFS, 106 cfu/ml), and EVs (20 µg/ml) of A. muciniphila exhibited over 90% viability in comparison to untreated control. The neutralized CFS preparation using A. muciniphila and its derivatives could notably reduce the expression level of inflammatory markers. Additionally, A. muciniphila and its derivatives modulated the production of IL-1ß, TNF-α, and IL-10 in Tox-S stimulated Caco-2 cells. We demonstrated that A. muciniphila and its derivatives can modulate changes in the gut barrier-related genes and inflammatory response caused by C. difficile Tox-S in Caco-2 cells.

Expression Analysis of Long Noncoding RNA-MALAT1 and Interleukin-6 in Inflammatory Bowel Disease Patients.

 Inflammatory bowel disease (IBD) manifests as chronic inflammation within the gastrointestinal tract. The study focuses on a long noncoding RNA (lncRNA) known as Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). MALAT1's misregulation has been linked with various autoimmune diseases and regulates proinflammatory cytokines. The role of IL6 in immune-triggered conditions, including IBD, is another focal point. In this research, the expression of MALAT1 and IL6 in IBD patients was meticulously analyzed to uncover potential interactions. The study involved 33 IBD patients (13 with Crohn's disease and 20 with ulcerative colitis) and 20 healthy counterparts. Quantitative real-time polymerase chain reaction determined the MALAT1 and IL6 gene expression levels. The competitive endogenous RNA (ceRNA) regulatory network was constructed using several tools, including LncRRIsearch and Cytoscape. A deep dive into the Inflammatory Bowel Disease database was undertaken to understand IL6's role in IBD. Drugs potentially targeting these genes were also pinpointed using DGIdb. Results indicated a notable elevation in the expression levels of MALAT1 and IL6 in IBD patients versus healthy controls. MALAT1 and IL6 did not show a direct linear correlation, but IL6 could serve as MALAT1's target. Analyses unveiled interactions between MALAT1 and IL6, regulated by hsa-miR-202-3p, hsa-miR-1-3p, and has-miR-9-5p. IL6's pivotal role in IBD-associated inflammation, likely interacting with other cytokines, was accentuated. Moreover, potential drugs like CILOBRADINE for MALAT1 and SILTUXIMAB for IL6 were identified. This research underscored MALAT1 and IL6's potential value as targets in diagnosis and treatment for IBD patients.

Prediction of anti-TNF therapy failure in ulcerative colitis patients by ensemble machine learning: A prospective study.

Nowadays, anti-TNF therapy remarkably improves the medical management of ulcerative colitis (UC), but approximately 40 % of patients do not respond to this treatment. In this study, we used 79 anti-TNF-naive patients with moderate-to-severe UC from four cohorts to discover alternative therapeutic targets and develop a personalized medicine approach that can diagnose UC non-responders (UCN) prior to receiving anti-TNF therapy. To this end, two microarray data series were integrated to create a discovery cohort with 35 UC samples. A comprehensive gene expression and functional analysis was performed and identified 313 significantly altered genes, among which IL6 and INHBA were highlighted as overexpressed genes in the baseline mucosal biopsies of UCN, whose cooperation may lead to a decrease in the Tregs population. Besides, screening the abundances of immune cell subpopulations showed neutrophils' accumulation increasing the inflammation. Furthermore, the correlation of KRAS signaling activation with unresponsiveness to anti-TNF mAb was observed using network analysis. Using 50x repeated 10-fold cross-validation LASSO feature selection and a stack ensemble machine learning algorithm, a five-mRNA prognostic panel including IL13RA2, HCAR3, CSF3, INHBA, and MMP1 was introduced that could predict the response of UC patients to anti-TNF antibodies with an average accuracy of 95.3 %. The predictive capacity of the introduced biomarker panel was also validated in two independent cohorts (44 UC patients). Moreover, we presented a distinct immune cell landscape and gene signature for UCN to anti-TNF drugs and further studies should be considered to make this predictive biomarker panel and therapeutic targets applicable in the clinical setting.

Plasma Extracellular Vesicle LncRNA H19 as a Potential Diagnostic Biomarker for Inflammatory Bowel Diseases.

BACKGROUND: Inflammatory bowel disease (IBD) is a complex gastrointestinal disease with 2 main subtypes of Crohn's disease (CD) and ulcerative colitis (UC), whose diagnosis mainly depends on the medical history, clinical symptoms, endoscopic, histologic, radiological, and serological findings. Extracellular vesicles (EVs) are now considered an additional mechanism for intercellular communication, allowing cells to exchange biomolecules. Long noncoding RNAs (lncRNAs) that are enriched in EVs have been defined as an ideal diagnostic biomarker for diseases. In this study, we investigated the expression differences of 5 lncRNAs in tissue and plasma EVs of active IBD patients compared with patients in the remission phase and healthy controls to introduce an EV-lncRNA as a noninvasive IBD diagnostic biomarker. METHODS: Twenty-two active IBD patients, 14 patients in the remission phase, 10 active rheumatoid arthritis (RA) patients, 14 irritable bowel syndrome (IBS) patients, and 22 healthy individuals were recruited in the discovery cohort. In addition, 16 patients with active IBD, 16 healthy controls, 10 inactive IBD patients, 12 active RA patients, and 14 IBS patients were also included in the validation cohort. The expression levels of 5 lncRNAs in tissue and EV-plasma were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) . Machine learning and receiver operating characteristic (ROC) curve analysis were performed to investigate the distinguishing ability of the candidate biomarkers. RESULTS: While the expression levels of lncRNAs CDKN2B-AS1, GAS5, and TUG1 were significantly downregulated, lncRNAs H19 and CRNDE were overexpressed in active IBD lesions. Expression of H19 was detected in plasma EVs whose isolation had been confirmed via dynamic light scattering, microscopy images, and western blotting. The classification results demonstrated the excellent ability of H19 in distinguishing IBD/active from IBD/remission, healthy control, RA, and IBS (area under the ROC curve = 0.95, 0.97,1, and 0.97 respectively). CONCLUSIONS: Our study suggests that circulating EV-lncRNA H19 exhibited promising potential for the diagnosis of active IBD.

The upregulation of lncRNA H19 in active IBD tissues and plasma extracellular vesicles indicated the possible association of H19 with the disease activity. In addition, the high sensitivity and specificity of this marker proposed it as a potential biomarker for the diagnosis of IBD patients.

A novel stop codon mutation in STK11 gene is associated with Peutz-Jeghers Syndrome and elevated cancer risk: a case study.

Based on the analysis of patients with Peutz-Jeghers syndrome (PJS), Serine threonine kinase11 (STK11) is known as a tumor suppressor gene, which is involved in cell polarization, regulation of apoptosis, and DNA damage response. In this case report study, we examined STK11 gene sequencing in a 42-year-old woman with mucocuta neous pigmentation and positive family history. Endoscopy and colonoscopy showed >1000 polyps throughout the stomach/colon (PJ-type hamartomas). The larger polyp in the stomach was resected and the small bowel imaging detected multiple jejunum/ileum small polyps. The data released from the sequencing results revealed five alterations in exons 1 to 5. The major mutation in stop codon was reported as converted to the amino acid tryptophan (TRP) to tyrosine (TER). The TGG codon was converted to TAG by mutation. Finally, another novel mutation in STK11 stop codon as a 'de novo' variant was seen. It is predicted that stop codon mutations make the affected person susceptible to developing colorectal cancer.

Long non-coding RNAs ANRIL, THRIL, and NEAT1 as potential circulating biomarkers of SARS-CoV-2 infection and disease severity.

The current outbreak of coronavirus disease 2019 (COVID-19) is a global emergency, as its rapid spread and high mortality rate, which poses a significant threat to public health. Innate immunity plays a crucial role in the primary defense against infections, and recent studies have highlighted the pivotal regulatory function of long non-coding RNAs (lncRNAs) in innate immune responses. This study aims to assess the circulating levels of lncRNAs namely ANRIL, THRIL, NEAT1, and MALAT1 in the blood of moderate and severe SARS-CoV-2 infected patients, in comparison to healthy individuals. Additionally, it aims to explore the potential of these lncRNAs as biomarkers for determining the severity of the disease. The blood samples were collected from a total of 38 moderate and 25 severe COVID-19 patients, along with 30 healthy controls. The total RNA was extracted and qPCR was performed to evaluate the blood levels of the lncRNAs. The results indicate significantly higher expression levels of lncRNAs ANRIL and THRIL in severe patients when compared to moderate patients (P value = 0.0307, P value = 0.0059, respectively). Moreover, the expression levels of lncRNAs ANRIL and THRIL were significantly up-regulated in both moderate and severe patients in comparison to the control group (P value < 0.001, P value < 0.001, P value = 0.001, P value < 0.001, respectively). The expression levels of lncRNA NEAT1 were found to be significantly higher in both moderate and severe COVID-19 patients compared to the healthy group (P value < 0.001, P value < 0.001, respectively), and there was no significant difference in the expression levels of NEAT1 between moderate and severe patients (P value = 0.6979). The expression levels of MALAT1 in moderate and severe patients did not exhibit a significant difference compared to the control group (P value = 0.677, P value = 0.764, respectively). Furthermore, the discriminative power of ANRIL and THRIL was significantly higher in the severe patient group than the moderate group (Area under curve (AUC) = 0.6879; P-value = 0.0122, AUC = 0.6947; P-value = 0.0093, respectively). In conclusion, the expression levels of the lncRNAs ANRIL and THRIL are correlated with the severity of COVID-19 and can be regarded as circulating biomarkers for disease progression.

Characterisation of extracellular vesicles isolated from hydatid cyst fluid and evaluation of immunomodulatory effects on human monocytes.

Hydatidosis is a disease caused by the larval stage of Echinococcus granulosus, which involves several organs of intermediate hosts. Evidence suggests a communication between hydatid cyst (HC) and hosts via extracellular vesicles. However, a little is known about the communication between EVs derived from HC fluid (HCF) and host cells. In the current study, EVs were isolated using differential centrifugation from sheep HCF and characterized by western blot, electron microscope and size distribution analysis. The uptake of EVs by human monocyte cell line (THP-1) was evaluated. The effects of EVs on the expression levels of pro- and anti-inflammatory cytokines were investigated using quantitative real-time PCR (RT-PCR), 3 and 24 h after incubation. Moreover, the cytokine level of IL-10 was evaluated in supernatant of THP-1 cell line at 3 and 24 h. EVs were successfully isolated and showed spherical shape with size distribution at 130.6 nm. After 3 h, the expression levels of pro-inflammatory cytokine genes (IL1Β, IL15 and IL8) were upregulated, while after 24 h, the expression levels of pro-inflammatory cytokines were decreased and IL13 gene expression showed upregulation. A statistically significant increase was seen in the levels of IL-10 after 24 h. The main mechanism of the communication between EVs derived from HCF and their host remains unclear; however, time-dependent anti-inflammatory effects in our study suggest that HC may modulate the immune responses via EVs.

Iranian Consensus Guideline for Pharmacotherapy with Biologics and Small Molecules Drugs in Adults with Inflammatory Bowel Diseases.

Background: Pharmacotherapy with biologics and small molecules, as the more effective therapies for moderate to severe ulcerative colitis (UC) and Crohn's disease (CD), is complex. Choosing the best methods for their utilization in order to induce and maintain remission are critical for practicing gastroenterologists. We aimed to develop an Iranian consensus on the management of inflammatory bowel disease (IBD) patients with biologics and small molecules. Methods: A Delphi consensus was undertaken by experts who performed a literature summary and voting process. Quality of evidence was assessed using the Grading and Recommendations Assessment, Development, and Evaluation; and an additional risk of bias-protocol. Results: Following an extensive search of the literature, 219 studies were used to determine the quality of the evidence. After three rounds of voting, consensus (defined as≥80% agreement) was reached for 87 statements. Conclusion: We considered different aspects of pharmacotherapy in this consensus. This guideline, along with clinical judgment, can be used to optimize management of IBD patients.

Translation and cross-cultural adaptation of the Persian version of inflammatory bowel disease-fatigue (IBD-F) self-assessment questionnaire.

BACKGROUND: This study appraises the psychometrics properties of the Inflammatory bowel disease-fatigue (IBD-F) Persian version questionnaire. METHODS: The original IBD-F questionnaire was translated into the Persian version in a standard forward-back manner. The validation was performed through the face, content, and construct validity. Fifteen experts scored each item's necessity on an ordinal Likert scale of three; then, the content validity ratio was calculated using the Lawshe formula. Eight judges from pre-defined panel rated each item on an ordinal 4-point Likert scale concerning its relevancy, clarity, and simplicity for individual-CVI calculation. The mean individual-CVI was considered as the Scale-CVI for each domain. Twenty lay experts (selected from the target population) were asked to express their opinion on each item's importance by scoring on a 5-point Likert scale; subsequently, face validity was determined by the impact score formula. The questions that had minimum values of CVR, CVI, and impact score were retained in the final version of the questionnaire for reliability evaluation. Construct validity was evaluated via Confirmatory Factor Analysis. Internal consistency and test-retest reliability were checked using Cronbach's α and intraclass correlation coefficients (ICC). Fifty-four patients recruited based on inclusion and exclusion criteria to participate in the reliability analysis. RESULTS: All the questions received the qualified values of CVR (exceeding 0.49 points) and impact score (more than 1.5 points) and were retained in the questionnaire; however, revisions were made for questions with a CVI 0.7-0.9 for clarity and simplicity. The result demonstrated relative goodness CFA and proper internal consistency, as Cronbach's α coefficient was 0.964 for the test (0.845 and 0.963 for the first and second part of the questionnaire, respectively (and 0.888 for the re-test (0.793 and 0.876 for the first and second section of the questionnaire, respectively). The ICC values between test and re-test for the first and second part and the whole questionnaire were obtained as 0.904, 0.922, and 0.921, respectively. CONCLUSION: The Persian version of the IBD-F questionnaire was valid and reliable; thus, an appropriate scale was deemed to measure fatigue (severity, frequency, and impact on daily activities).

Toll-Like Receptor 7 a Novel Non-Invasive Inflammatory Genetic Sensor for Ulcerative Colitis Remission Monitoring.

Background: Ulcerative colitis (UC) and Crohn's disease (CD) are two major types of inflammatory bowel diseases (IBDs). Toll-like receptors (TLRs) are expressed in the innate immune system compartments, in charge of identifying a wide range of microorganisms. The aim of the present study was to evaluate the expression of TLR-2, -7, and -8 in peripheral blood mononuclear cells (PBMC) of UC patients as a novel non-invasive primary inflammation sensor for monitoring the clinical course of UC candidates. Materials and Methods: In this cross-sectional study, total RNA was extracted from the PBMC of 42 UC patients along with 20 healthy donors. The mRNA levels of TLR-2, -7, and -8 were assessed using the quantitative real-time polymerase chain (qRT-PCR) reaction. Results: The present research study demonstrated no significant changes in TLR-2 mRNA expression in UC patients in comparison with the control group (P = 0.1264), whereas significant elevation (P = 0.0008) was distinguished in the TLR-7 expression of UC participants specifically during the remission course compared with healthy donors and flareup patients (P = 0.0004 and P = 0.0063, respectively). The last selected TLR, TLR-8 was not shown remarkable changes either between UC patients and the control group or between clinical courses of the disease. Conclusion: Here, among three nominated TLRs for predicting UC patients, TLR-7 was potentially selected according to the significant difference in mRNA expression in flareup UC patients and control donors. TLR-7 could be used as a novel non-invasive biomarker for monitoring UC patients in the active course of the disease.

The effects of flaxseed supplementation on concentration of circulating adipokines in patients with ulcerative colitis.

Aim: This study aimed to evaluate the effect of supplementation with ground flaxseed (GF) on the concentrations of adiponectin, resistin, and visfatin in patients with ulcerative colitis (UC). Background: Inflammatory bowel disease (IBD) is one of the most common gastrointestinal diseases affecting people of all ages. Adipokines secreted from adipose tissue have been shown to play an essential role in the pathogenesis of UC. Methods: This trial is an open-labeled randomized controlled trial conducted on 70 patients with UC. The patients were randomly divided into two groups: flaxseed and control. The patients in the intervention received 30 g/day flaxseed powder for 12 weeks. Patients' anthropometric, nutritional, and biochemical factors were evaluated at the beginning and end of the intervention period. Results: Totally, 64 patients (36 men and 28 women) with a mean age of 31.12±9.67 were included in the final analysis. There was no significant difference between the two groups regarding baseline weight and height (P>0.05). After the 12-week intervention, flaxseed supplementation led to a significant reduction in the resistin (-4.85±1.89 vs. -1.10±2.25, P<0.001) and visfatin concentration (-1.33±1.14 vs. -0.53±1.63, P=0.018). Further, we found a significant increase in the adiponectin levels after the GF supplementation (3.49±1.29 vs. -0.35±0.96, P<0.001). Conclusion: Flaxseed supplementation could exert beneficial effects on adipokine levels in patients with UC.

Toxoplasma gondii profilin induces NLRP3 activation and IL-1ß production/secretion in THP-1 cells.

Toxoplasma gondii is a highly prevalent protozoan that infects a broad spectrum of warm-blooded animals. Profilin is a critical protein that plays a role in the movement and invasion of T. gondii. In the current study, we assessed how profilin stimulates inflammasomes and how it induces transcription and secretion of IL-1ß. For this purpose, we assessed the level of TLR 2, 4, 5, and 9 expressions in a THP-1 cell line treated with profilin from T. gondii (TgP). In addition, we analyzed the expression levels of various inflammasomes, as well as IL-1ß, and IL-18 in THP-1 cells treated with the NLRP3 inhibitor MCC950. TgP significantly increased the expression of TLR5 but the expression of TLR2, 4, and 9 was not significantly increased. In addition, TgP did not significantly increase the level of inflammasomes after 5 h. Treatment with MCC950 significantly reduced NLRP3 and IL-1ß on both transcription and protein levels. Although the transcription level of NLRP3 was reduced 5 h after treatment with TgP, western blot analysis showed an increase in NLRP3. The western blot and ELISA analysis also showed that TgP increased both pro- and mature IL-1ß. In summary, our study showed that NLRP3 most probably plays a pivotal role in the expression and production levels of IL-1ß during the interaction between TgP and macrophages.

Intestinal microbiota changes pre- and post-fecal microbiota transplantation for treatment of recurrent Clostridioides difficile infection among Iranian patients with concurrent inflammatory bowel disease.

Introduction: Patients with inflammatory bowel disease (IBD) are at a greater risk for the recurrence of Clostridioides difficile infection (rCDI) that is triggered by intestinal microbiota dysbiosis. Fecal microbiota transplantation (FMT) has emerged as a highly effective therapeutic option for this complication. However, little is known about the impact of FMT on intestinal microbiota alterations in rCDI patients suffering from IBD. In this study, we aimed to investigate post-FMT intestinal microbiota alterations in Iranian rCDI patients with underlying IBD. Methods: A total of 21 fecal samples were collected including 14 samples pre- and post-FMT and 7 samples from healthy donors. Microbial analysis was performed by quantitative real-time PCR (RT-qPCR) assay targeting the 16S rRNA gene. The pre-FMT profile and composition of the fecal microbiota were compared to the microbial changes of samples collected 28 days after FMT. Results and discussion: Overall, the fecal microbiota profile of recipients was more similar to donor samples after the transplantation. We observed a significant increase in the relative abundance of Bacteroidetes post-FMT, compared to the pre-FMT microbial profile. Furthermore, there were remarkable differences between the microbial profile of pre-FMT, post-FMT, and healthy donor samples by PCoA analysis based on the ordination distance. This study demonstrates FMT as a safe and effective approach to restore the indigenous composition of the intestinal microbiota in rCDI patients and ultimately results in the treatment of concurrent IBD.

Evaluation of the mTORC activity in the presence of Toxoplasma gondii and azathioprine in human monocyte cell line.

BACKGROUND: Autophagy is an important part of pathogenesis of IBD. Thiopurines such as azathioprine (AZA) are approved drugs for clinical practices in IBD patients. Besides, as an escape strategy, Toxoplasma gondii can use the mTORC1 complex to inactivate autophagy. METHODS: In this study, we investigated whether T. gondii tachyzoites may modulate autophagy and interfere the effects of azathioprine in IBD treatment. PMA-activated human monocyte cell line (THP-1) was infected with fresh T. gondii RH tachyzoites. After 5 h of infection, the cells were treated with AZA for 6 h. The expression of atg5, atg7, atg12, lc3b, and ß-actin (BACT) genes was evaluated using quantitative real-time PCR. To analyze the phosphorylation of ribosomal protein S6 (rpS6), western blot using specific primary antibodies was performed. RESULTS: The results of real-time PCR revealed that AZA, T. gondii tachyzoites, and a combination of AZA and T. gondii tachyzoites upregulated atg5 gene for 4.297-fold (P-value = 0.014), 2.49-fold (P-value = 0.006), and 4.76-fold (P-value = 0.001), respectively. The atg7 gene showed significant upregulation (2.272-fold; P-value = 0.014) and (1.51-fold; P-value = 0.020) in AZA and AZA / T. gondii, respectively. The expression of atg12 gene was significantly downregulated in AZA and T. gondii tachyzoites for (8.85-fold; P-value = 0.004) and (2.005-fold; P-value = 0.038), respectively, but upregulated in T. gondii/AZA (1.52-fold; P-value = 0.037). In addition, the lc3b gene was only significantly changed in AZA / T. gondii (3.028-fold; P-value = 0.001). Western blot analysis showed that T. gondii tachyzoites significantly phosphorylated rpS6, and tachyzoites did not interfere the effects of AZA to phosphorylate the rpS6. CONCLUSION: Taken together, although AZA and T. gondii similarly affects the expression levels of atg5, atg7, and atg12, but T. gondii does not seem to modulate the effects of AZA via mTORC functions.

TGF-ß receptor I inhibitor may restrict the induction of EMT in inflamed intestinal epithelial cells.

Despite the extensive body of research, understanding the exact molecular mechanisms governing inflammatory bowel diseases (IBDs) still demands further investigation. Transforming growth factor-ß1 (TGF-ß1) signaling possesses a multifacial effect on a broad range of context-dependent cellular responses. However, long-term TGF-ß1 activity may trigger epithelial-mesenchymal transition (EMT), followed by fibrosis. This study aimed to determine the role of epithelial TGF-ß1 signaling in inflammatory bowel disease (IBD) pathogenesis. The expression of TGF-ß1 signaling components and EMT-related and epithelial tight junction markers was examined in IBD patients (n = 60) as well as LPS-induced Caco-2/RAW264.7 co-culture model using quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorescence staining. Furthermore, the effect of A83-01, as a TGF-ß receptor I (TßRI) inhibitor, on the inflamed epithelial cells was evaluated in vitro. To evaluate the cytotoxic effects of the TßRI inhibitor, a cell viability assay was performed by the MTS method. Considering the activation of canonical and non-canonical TGF-ß1 signaling pathways in IBD patients, expression results indicated that administering A83-01 in inflamed Caco-2 cells substantially blocked the expression level of TGF-ß1, SMAD4, and PI3K and the phosphorylation of p-SMAD2/3, p-AKT, and p-RPS6 as well as prevented downregulation of LncGAS5 and LncCDKN2B. Further analysis revealed that the inhibition of TGF-ß1 signaling in inflamed epithelial cells by the small molecule could suppress the EMT-related markers as well as improve the expression of epithelial adherens and tight junctions. Collectively, these findings indicated that the inhibition of the TGF-ß1 signaling could suppress the induction of EMT in inflamed epithelial cells as well as exert a protective effect on preserving tight junction integrity. There is a pressing need to determine the exact cellular mechanisms by which TGF-ß1 exerts its effect on IBD pathogenesis.

Characterization of the mucosal microbiota in patients with nodular lymphoid hyperplasia with concurrent irritable bowel syndrome compared to healthy controls.

BACKGROUND: Nodular lymphoid hyperplasia (NLH) is known as a lymphoproliferative lesion in which multiple small nodules appear on the intestinal wall. It has been documented that patients who struggle with irritable bowel syndrome (IBS) are at greater risk of developing NLH. Here, we aimed to investigate the previously reported pathogens and the abundance of a selection of mucosal microbiota in IBS + NLH patients compared to IBS, and healthy controls. METHODS AND RESULTS: Terminal ileum biopsies were collected from 37 IBS + NLH, 37 IBS, and 29 healthy controls. Bacterial culture and PCR was performed to detect the presence of pathogens in biopsies. A qPCR assay was applied to assess the abundance of a selection of bacterial taxa. Totally, five bacterial isolates including two enteropathogenic and one enteroaggregative Escherichia coli (EPEC, EAEC), one enterotoxigenic Staphylococcus aureus (SEA), and one Yersinia enterocolitica strains were detected among the IBS + NLH cases. The relative abundance of Bacteroidetes and Streptococcus spp. in IBS + NLH patients was significantly less than IBS and healthy controls. Firmicutes, Pseudomonas spp., Haemophilus spp., and Campylobacter spp. were notably more abundant in IBS + NLH than in IBS patients. The abundance of Verrucomicrobia was higher in NLH + IBS than in healthy controls. Actinobacteria was also significantly more abundant among NLH + IBS patients than the controls. CONCLUSION: Our results demonstrated that mucosal microbiota composition in NLH + IBS patients slightly differs from that of IBS patients and healthy controls. Further research using large-scale cohorts are needed to enhance current understanding of the contribution of the mucosal microbiota to NLH pathogenesis with concurrent IBS.

The role of mir-197-3p in regulating the tight junction permeability of celiac disease patients under gluten free diet.

BACKGROUND: Celiac disease (CD) is a hereditary immune-mediated disorder, which is along with the enormous production of pro-inflammatory cytokines and the reduced level of tight junction proteins. The aim of this study was to determine the expression of TNF-α, IFN-γ, IL-18, Occludin, miR-122-5p and miR-197-3p genes in duodenal biopsies of treated CD patients in comparison to the controls. METHODS AND RESULTS: Biopsy specimens were taken from the duodenum of 50 treated CD patients (36 (72%) females and 14 (28%) males with mean age of 37.06 ± 7.02 years) and 50 healthy controls (17 (34%) females and 33 (66%) males with mean age of 34.12 ± 4.9). Total RNA was isolated, cDNA was synthesized and mRNA expression of TNF-α, IFN-γ, IL-18, Occludin, miR-122-5p and miR-197-3p were quantified by relative qPCR using B2M and U6 as internal control genes. All data were evaluated using SPSS (V.21) and GraphPad Prism (V.5). Our results showed that there was no significant difference between patients and controls for intestinal mRNA expression of TNF-α, IFN-γ, IL-18, Occludin, and miR-122-5p (p > 0.05) and the expression of miR-197-3p was significantly increased in CD patients relative to control subjects (p = 0.049). CONCLUSION: This study suggests that adherence to GFD may have a positive effect on the tight junction (TJ) permeability and in this process, miR-197-3p plays an important role. Increased expression of miR-197-3p with a final protective effect on Occludin expression can be further studied as a complement therapeutic target for Celiac disease.

Selective serotonin reuptake inhibitors and inflammatory bowel disease; Beneficial or malpractice.

IBD, a chronic inflammatory disease, has been manifested as a growing health problem. No Crohn's and Colitis councils have officially ratified anti-depressants as a routine regimen for IBD patients. However, some physicians empirically prescribe them to rectify functional bowel consequences such as pain and alleviate psychiatric comorbidities. On the other side, SSRIs' prescription is accompanied by adverse effects such as sleep disturbances. Prolonged intermittent hypoxia throughout sleep disturbance such as sleep apnea provokes periodic reductions in the partial oxygen pressure gradient in the gut lumen. It promotes gut microbiota to dysbiosis, which induces intestinal inflammation. This phenomenon and evidence representing the higher amount of serotonin associated with Crohn's disease challenged our previous knowledge. Can SSRIs worsen the IBD course? Evidence answered the question with the claim on anti-inflammatory properties (central and peripheral) of SSRIs and illuminated the other substantial elements (compared to serotonin elevation) responsible for IBD pathogenesis. However, later clinical evidence was not all in favor of the benefits of SSRIs. Hence, in this review, the molecular mechanisms and clinical evidence are scrutinized and integrated to clarify the interfering molecular mechanism justifying both supporting and disproving clinical evidence. Biphasic dose-dependent serotonin behavior accompanying SSRI shifting function when used up for the long-term can be assumed as the parameters leading to IBD patients' adverse outcomes. Despite more research being needed to elucidate the effect of SSRI consumption in IBD patients, periodic prescriptions of SSRIs at monthly intervals can be recommended.