Estudi transcripcional de la Colitis Ulcerosa. Caracterització i cerca de biomarcadors

[cat] La colitis ulcerosa (CU) és una malaltia inflamatòria intestinal (MII) idiopàtica, de caràcter crònic i que afecta el còlon. Tot i que se’n desconeix l’etiologia, actualment existeixen diferents tractament pal·liatius, principalment fàrmacs antiinflamatoris, immunosupressors i biològics, que permeten induir i mantenir la remissió de la malaltia en un elevat nombre de casos. Per la vigilància i el seguiment de la patologia s’han descrit varis marcadors d’inflamació, tot i així, l’avaluació endoscòpica és el procediment de referència per determinar l’activitat de la malaltia. Mitjançant l’anàlisi transcripcional de mostres de pacients amb CU, el projecte que es presenta té per objectiu descriure el perfil transcripcional de la mucosa intestinal en remissió i identificar biomarcadors d’inflamació intestinal en sang perifèrica. L’anàlisi transcripcional de la mucosa intestinal comparant entre pacients amb CU activa, CU en remissió i controls sans, mitjançant microarrays, ens ha permès la identificació d’un perfil transcripcional característic de la mucosa en remissió. Les troballes senyalen l’existència d’alteracions permanents en cèl·lules epitelials en la mucosa endoscòpica i histològicament curada dels pacients amb CU. Alguns dels gens identificats són REG4, S100P, SERPINB5, AQP3 i TFF1; veient-se també alterada l’expressió proteica de REG4, S100P i SERPINB5 mitjançant tècniques d’immunohistoquímica. Els resultats d’aquest primer estudi van publicar-se a la revista “GUT” amb el títol: “Transcriptional analysis of the intestinal mucosa of patients with ulcerative colitis in remission reveals lasting epithelial cell alterations”. Per altra banda, s’han explorat les alteracions en el perfil transcripcional de sang perifèrica de pacients amb CU associades a l’activitat endoscòpica de la malaltia. L’anàlisi transcripcional en sang perifèrica ens ha permès identificar un subconjunt de gens associats a la presència de lesions intestinals, incloent els gens CD177, HP, S100A12, GPR84 i ARG1, entre altres. Els gens més rellevants s’han validat mitjançant PCR quantitativa en una cohort independent i s’ha identificat l’expressió d’haptoglobina (HP) com un bon gen predictor de l’activitat endoscòpica, mostrant major precisió que els marcadors utilitzats actualment en la pràctica clínica (PCR, VSG i recompte de plaquetes). També hem vist com els canvis en l’expressió d’HP, CD177, GPR84 i S100A12 es correlacionen bé amb la resposta endoscòpica a l’anti-TNF. Els resultats d’aquest segon estudi van publicar-se a la revista “Journal of Crohn’s and Colitis” amb el títol: “Usefulness of transcriptional blood biomarkers as a non-invasive surrogate marker of mucosal healing and endoscopic response in ulcerative colitis”.[eng] Ulcerative Colitis (UC) is an inflammatory bowel disease (IBD); an idiopathic and chronic condition confined to the colon. Although the unknown etiology, nowadays there are different treatments to control this disease, mainly anti-inflammatory, immunosuppressors and biologic drugs, allowing the induction and maintenance of disease remission in a large number of disease patients. For the disease vigilance and follow-up there are several inflammatory markers described, however, the endoscopic evaluation still being the gold standard for the disease activity. Through the transcriptional analysis of samples from UC patients, the project aims to describe the transcriptional profile of intestinal mucosa in remission and identify biomarkers of intestinal inflammation in peripheral blood. Transcriptional analysis of intestinal mucosa between active UC, UC in remission and healthy individuals, using microarrays, allowed the identification of a specific transcriptional profile for mucosa in remission. The findings points to permanent alterations in epithelial cells; within the genes identified there are REG4, S100P, SERPINB5, AQP3 and TFF1, confirming the alterations at protein level (immunohistochemistry) for REG4, S100P and SERPINB5. The results of this first study have been published in the “GUT” journal with the title: “Transcriptional analysis of the intestinal mucosa of patients with ulcerative colitis in remission reveals lasting epithelial cell alterations”. By other way, transcriptional alterations in peripheral blood associated with disease endoscopic activity had been explored. The transcriptional analysis of blood highlights a subset of genes related with the presence of intestinal lesions, including CD177, HP, S100A12, GPR84 and ARG1. Most relevant genes have been validated through quantitative PCR in an independent cohort and the expression of haptoglobin (HP) has been identified as a good predictor of endoscopic activity, showing higher precision than other biomarkers commonly used in clinics (CRP, ESR and platelets counts). We also see that changes in gene expression of HP, CD177, GPR84 and S100A12 show good correlation with endoscopic response to anti-TNF therapy. The results of this second study have been published in the “Journal of Crohn’s and Colitis” with the title: “Usefulness of transcriptional blood biomarkers as a non-invasive surrogate marker of mucosal healing and endoscopic response in ulcerative colitis”

Kernel PCA per a l’anàlisi de dades òmiques

Les funcions kernel, per les seves propietats, permeten combinar diferents tipus de dades, essent una possible via per a la integració de dades òmiques. Amb l'objectiu d'explorar l'ús d'aquestes, s'estudien els fonaments estadístics de l'anàlisi de components principals basat en funcions kernel (kernel PCA), es proporcionen eines que permeten integrar conjunts de dades, representar variables originals i cercar variables d'interès en el kernel PCA i, finalment, s'inspecciona un conjunt de dades transcripcionals de pacients amb colitis ulcerosa utilitzant les eines desenvolupades

Kernel PCA per a l’anàlisi de dades òmiques

Les funcions kernel, per les seves propietats, permeten combinar diferents tipus de dades, essent una possible via per a la integració de dades òmiques. Amb l'objectiu d'explorar l'ús d'aquestes, s'estudien els fonaments estadístics de l'anàlisi de components principals basat en funcions kernel (kernel PCA), es proporcionen eines que permeten integrar conjunts de dades, representar variables originals i cercar variables d'interès en el kernel PCA i, finalment, s'inspecciona un conjunt de dades transcripcionals de pacients amb colitis ulcerosa utilitzant les eines desenvolupades

Methanol opportunities for electricity and hydrogen production in bioelectrochemical systems

An anodic syntrophic consortium (exoelectrogenic plus fermentative bacteria) able to use methanol as sole carbon source was developed for the first time in a bioelectrochemical system. In this frame, promising results were obtained in single chamber MFC, comparable to those obtained with readily biodegradable substrates. Regarding MEC operation, the presence of homoacetogenic bacteria led to electron recycling, avoiding net hydrogen production in single chamber MEC. In a double chamber MEC, satisfying results (in terms of coulombic efficiency and cathodic gas recovery) were obtained even though energy recovery still restrained the feasibility of the process. The approach used in this work with methanol opens a new range of possibilities for other complex substrates as electron donors for bioelectrosynthesis

Defective Apoptosis In Intestinal And Mesenteric Adipose Tissue Of Crohn's Disease Patients.

Crohn's disease (CD) is associated with complex pathogenic pathways involving defects in apoptosis mechanisms. Recently, mesenteric adipose tissue (MAT) has been associated with CD ethiopathology, since adipose thickening is detected close to the affected intestinal area. However, the potential role of altered apoptosis in MAT of CD has not been addressed. To evaluate apoptosis in the intestinal mucosa and MAT of patients with CD. Samples of intestinal mucosa and MAT from patients with ileocecal CD and from non-inflammatory bowel diseases patients (controls) were studied. Apoptosis was assessed by TUNEL assay and correlated with the adipocytes histological morphometric analysis. The transcriptional and protein analysis of selected genes and proteins related to apoptosis were determined. TUNEL assay showed fewer apoptotic cells in CD, when compared to the control groups, both in the intestinal mucosa and in MAT. In addition, the number of apoptotic cells (TUNEL) correlated significantly with the area and perimeter of the adipose cells in MAT. Transcriptomic and proteomic analysis reveal a significantly lower transcript and protein levels of Bax in the intestinal mucosa of CD, compared to the controls; low protein levels of Bax were found localized in the lamina propria and not in the epithelium of this tissue. Furthermore, higher level of Bcl-2 and low level of Caspase 3 were seen in the MAT of CD patients. The defective apoptosis in MAT may explain the singular morphological characteristics of this tissue in CD, which may be implicated in the pathophysiology of the disease.9e9854

Serum methylation of GALNT9, UPF3A, WARS, and LDB2 as noninvasive biomarkers for the early detection of colorectal cancer and advanced adenomas

Background Early detection has proven to be the most effective strategy to reduce the incidence and mortality of colorectal cancer (CRC). Nevertheless, most current screening programs suffer from low participation rates. A blood test may improve both the adherence to screening and the selection to colonoscopy. In this study, we conducted a serum-based discovery and validation of cfDNA methylation biomarkers for CRC screening in a multicenter cohort of 433 serum samples including healthy controls, benign pathologies, advanced adenomas (AA), and CRC.Results First, we performed an epigenome-wide methylation analysis with the MethylationEPIC array using a sample pooling approach, followed by a robust prioritization of candidate biomarkers for the detection of advanced neoplasia (AN: AA and CRC). Then, candidate biomarkers were validated by pyrosequencing in independent individual cfDNA samples. We report GALNT9, UPF3A, WARS, and LDB2 as new noninvasive biomarkers for the early detection of AN. The combination of GALNT9/UPF3A by logistic regression discriminated AN with 78.8% sensitivity and 100% specificity, outperforming the commonly used fecal immunochemical test and the methylated SEPT9 blood test.Conclusions Overall, this study highlights the utility of cfDNA methylation for CRC screening. Our results suggest that the combination methylated GALNT9/UPF3A has the potential to serve as a highly specific and sensitive blood-based test for screening and early detection of CRC

Obtaining microbial communities with exoelectrogenic activity from anaerobic sludge using a simplified procedure

BACKGROUND: The microbial fuel cell (MFC) technology transforms the chemical energy present in substrates into electricity. Starting-up these systems, i.e. enriching the anodic community in exoelectrogenic bacteria, is a lengthy process or requires expensive equipment. - RESULTS: An easy and low-cost procedure based on a sediment MFC was developed to select microbial communities with exoelectrogenic activity from the anaerobic sludge of a waste water treatment plant (WWTP). The configuration was based on a simple vessel working as a single chamber MFC with a cathode of stainless steel wool in the liquid surface and a submerged graphite fibre brush as anode. In 30 days of operation, a biofilm with remarkable exoelectrogenic activity was grown on the anode of the MFC. This graphite fibre brush anode was able to supply 0.9Wm-2 when working in an air-cathode MFC (AC-MFC) for 45 days. - CONCLUSION:The procedure presented was demonstrated to be a successful, low-costandlow-maintenance procedure to obtain exoelectrogenic activity and had performances comparable with other more costly and complex inoculation procedures. The Sed-MFC does not require a potentiostat, external aeration, stirring, membranes or an enriched inoculum in the exoelectrogenic biomass

RIPK1 Mediates TNF-Induced Intestinal Crypt Apoptosis During Chronic NF-κB Activation

Tumor necrosis factor (TNF) is a major pathogenic effector and a therapeutic target in inflammatory bowel disease (IBD), yet the basis for TNF-induced intestinal epithelial cell (IEC) death is unknown, because TNF does not kill normal IECs. Here, we investigated how chronic nuclear factor (NF)- κB activation, which occurs in human IBD, promotes TNF-dependent IEC death in mice. Human IBD specimens were stained for p65 and cleaved caspase-3. C57BL/6 mice with constitutively active IKKβ in IEC (Ikkβ(EE) IEC ), Ripk1 D138N/D138N knockin mice, and Ripk3 -/- mice were injected with TNF or lipopolysaccharide. Enteroids were also isolated from these mice and challenged with TNF with or without RIPK1 and RIPK3 inhibitors or butylated hydroxyanisole. Ripoptosome-mediated caspase-8 activation was assessed by immunoprecipitation. NF-κB activation in human IBD correlated with appearance of cleaved caspase-3. Congruently, unlike normal mouse IECs that are TNF-resistant, IECs in Ikkβ(EE) IEC mice and enteroids were susceptible to TNF-dependent apoptosis, which depended on the protein kinase function of RIPK1. Constitutively active IKKβ facilitated ripoptosome formation, a RIPK1 signaling complex that mediates caspase-8 activation by TNF. Butylated hydroxyanisole treatment and RIPK1 inhibitors attenuated TNF-induced and ripoptosome-mediated caspase-8 activation and IEC death in vitro and in vivo. Contrary to common expectations, chronic NF-κB activation induced intestinal crypt apoptosis after TNF stimulation, resulting in severe mucosal erosion. RIPK1 kinase inhibitors selectively inhibited TNF destructive properties while preserving its survival and proliferative properties, which do not require RIPK1 kinase activity. RIPK1 kinase inhibition could be a potential treatment for IBD

Differences in peripheral and tissue immune cell populations following haematopoietic stem cell transplantation in Crohn's disease patients

Background and aims: recent studies have shown the efficacy of autologous haematopoietic stem cell transplantation [HSCT] in severely refractory Crohn's disease [CD] patients. HSCT is thought to eliminate auto-reactive cells; however, no specific studies of immune reconstitution in CD patients are available. Methods: we followed a group of CD patients [n = 18] receiving autologous HSCT, with 50% of them achieving endoscopic drug-free remission. To elucidate the mechanisms driving efficacy, we monitored changes after HSCT in blood and intestine immune-cell composition. CD patients [n = 22] receiving anti-tumour necrosis factor [TNF]-α were included for comparison. Results: severe immune ablation followed by HSCT induced dramatic changes in both peripheral blood T and B cells in all patients regardless of the efficacy of the treatment. Endoscopic remission at week 52 following HSCT was associated with significant intestinal transcriptional changes. A comparison of the remission signature with that of anti-TNFα identified both common and unique genes in the HSCT-induced response. Based on deconvolution analysis of intestinal biopsy transcriptome data, we show that response to HSCT, but not to anti-TNFα, is associated with an expansion of naïve B-cells, as seen in blood, and a decrease in the memory resting T-cell content. As expected, endoscopic remission, in response to both HSCT and anti-TNFα, led to a significant reduction in intestinal neutrophil and M1 macrophage content. Conclusions: peripheral blood immune remodelling after HSCT does not predict efficacy. In contrast, a profound intestinal T-cell depletion that is maintained long after transplant is associated with mucosal healing following HSCT, but not anti-TNFα