Gram-negative enterobacteria induce tolerogenic maturation in dexamethasone conditioned dendritic cells

Dendritic cells have been investigated in clinical trials, predominantly with the aim of stimulating immune responses against tumours or infectious diseases. Thus far, however, no clinical studies have taken advantage of their specific immunosuppressive potential. Tolerogenic DCs may represent a new therapeutic strategy for human immune-based diseases, such as Crohn's disease, where the perturbations of the finely tuned balance between the immune system and the microflora result in disease. In the present report, we describe the generation of tolerogenic DCs from healthy donors and Crohn's disease patients using clinical-grade reagents in combination with dexamethasone as immunosuppressive agent and characterize their response to maturation stimuli. Interestingly, we found out that dexamethasone-conditioned DCs keep their tolerogenic properties to Gram-negative bacteria. Other findings included in this study demonstrate that the combination of dexamethasone with a specific cytokine cocktail yielded clinical-grade DCs with the following characteristics: a semi-mature phenotype, a pronounced shift towards anti-inflammatory versus inflammatory cytokine production and low T-cell stimulatory properties. Importantly, in regard to their clinical application, the tolerogenic phenotype of DCs remained stable after the elimination of dexamethasone and after a second stimulation with LPS or bacteria. All these properties make this cell product suitable to be tested in clinical trials of inflammatory conditions including Crohn's disease

Tofacitinib, an oral janus kinase inhibitor, in active ulcerative colitis

Background: ulcerative colitis is a chronic inflammatory disease of the colon for which current treatments are not universally effective. One additional treatment may be tofacitinib (CP-690,550), an oral inhibitor of Janus kinases 1, 2, and 3 with in vitro functional specificity for kinases 1 and 3 over kinase 2, which is expected to block signaling involving gamma chain-containing cytokines including interleukins 2, 4, 7, 9, 15, and 21. These cytokines are integral to lymphocyte activation, function, and proliferation. Methods: in a double-blind, placebo-controlled, phase 2 trial, we evaluated the efficacy of tofacitinib in 194 adults with moderately to severely active ulcerative colitis. Patients were randomly assigned to receive tofacitinib at a dose of 0.5 mg, 3 mg, 10 mg, or 15 mg or placebo twice daily for 8 weeks. The primary outcome was a clinical response at 8 weeks, defined as an absolute decrease from baseline in the score on the Mayo scoring system for assessment of ulcerative colitis activity (possible score, 0 to 12, with higher scores indicating more severe disease) of 3 or more and a relative decrease from baseline of 30% or more with an accompanying decrease in the rectal bleeding subscore of 1 point or more or an absolute rectal bleeding subscore of 0 or 1. Results: the primary outcome, clinical response at 8 weeks, occurred in 32%, 48%, 61%, and 78% of patients receiving tofacitinib at a dose of 0.5 mg (P=0.39), 3 mg (P=0.55), 10 mg (P=0.10), and 15 mg (P1) at 8 weeks occurred in 13%, 33%, 48%, and 41% of patients receiving tofacitinib at a dose of 0.5 mg (P=0.76), 3 mg (P=0.01), 10 mg (P<0.001), and 15 mg (P<0.001), respectively, as compared with 10% of patients receiving placebo. There was a dose-dependent increase in both low-density and high-density lipoprotein cholesterol. Three patients treated with tofacitinib had an absolute neutrophil count of less than 1500. Conclusions: patients with moderately to severely active ulcerative colitis treated with tofacitinib were more likely to have clinical response and remission than those receiving placebo. (Funded by Pfizer; ClinicalTrials.gov number, NCT00787202)

Probiotic Sonicates Selectively Induce Mucosal Immune Cells Apoptosis Through Ceramide Generation Via Neutral Sphingolyelinase

Background: Probiotics appear to be beneficial in inflammatory bowel disease, but their mechanism of action is incompletely understood. We investigated whether probiotic-derived sphingomyelinase mediates this beneficial effect. Methodology/Principal Findings: Neutral sphingomyelinase (NSMase) activity was measured in sonicates of the probiotic L.brevis (LB)and S. thermophilus (ST) and the non-probiotic E. coli EC) and E. faecalis (EF). Lamina propria mononuclear cells (LPMC) were obtained from patients with Crohn"s disease (CD) and Ulcerative Colitis (UC), and peripheral blood mononuclear cells (PBMC) from healthy volunteers, analysing LPMC and PBMC apoptosis susceptibility, reactive oxygen species (ROS) generation and JNK activation. In some experiments, sonicates were preincubated with GSH or GW4869, a specific NSMase inhibitor. NSMase activity of LB and ST was 10-fold that of EC and EF sonicates. LB and ST sonicates induced significantly more apoptosis of CD and UC than control LPMC, whereas EC and EF sonicates failed to induce apoptosis. Pre-stimulation with anti-CD3/CD28 induced a significant and time-dependent increase in LB-induced apoptosis of LPMC and PBMC. Exposure to LB sonicates resulted in JNK activation and ROS production by LPMC. NSMase activity of LB sonicates was completely abrogated by GW4869, causing a dose-dependent reduction of LB -induced poptosis. LB and ST selectively induced immune cell apoptosis, an effect dependent on the degree of cell activation and mediated by bacterial NSMase. Conclusions: These results suggest that induction of immune cell apoptosis is a mechanism of action of some probiotics and that NSMase-mediated ceramide generation contributes to the therapeutic effects of probiotics

El registro ENEIDA (Estudio Nacional en Enfermedad Inflamatoria intestinal sobre Determinantes geneticos y Ambientales) de GETECCU: diseno, monitorizacion y funciones

El registro ENEIDA, promovido por el Grupo Espa ̃nol de Trabajo en Enfermedad deCrohn y Colitis Ulcerosa (GETECCU), fue creado en 2005 por un grupo de gastroenterólogosinteresados en mejorar el manejo de los pacientes con enfermedad inflamatoria intestinal. Losobjetivos principales del registro fueron facilitar la recogida de datos clínicos de interés parala práctica clínica asistencial, así como la elaboración de estudios colaborativos a partir dedatos clínicos y muestras biológicas. En sus 15 a ̃nos de existencia, ENEIDA ha evolucionado enmúltiples aspectos, desde su contenido o su soporte tecnológico hasta el número de centrosparticipantes, para convertirse en uno de los registros de referencia para el estudio y cuidadode los pacientes con enfermedad inflamatoria intestinal, con una producción científica continua y de alta calidad que lo ha situado como ejemplo de explotación científica colaborativa en elámbito internacional. En este artículo se revisan los objetivos, el dise ̃no, las característicasestructurales, la monitorización y la explotación científica del registro ENEIDA

Differentially Deregulated MicroRNAs as Novel Biomarkers for Neoplastic Progression in Ulcerative Colitis

Introduction: Colorectal cancer (CRC) is a potentially life-threatening complication of long-standing ulcerative colitis (UC). MicroRNAs (miRNA) are epigenetic regulators that have been involved in the development of UC-associated CRC. However, their role as potential mucosal biomarkers of neoplastic progression has not been adequately studied. Methods: In this study, we analyzed the expression of 96 preselected miRNAs in human formalin-fixed and paraffin-embedded tissue of 52 case biopsies (20 normal mucosa, 20 dysplasia, and 12 UC-associated CRCs) and 50 control biopsies (10 normal mucosa, 21 sporadic adenomas, and 19 sporadic CRCs) by using Custom TaqMan Array Cards. For validation of deregulated miRNAs, we performed individual quantitative real-time polymerase chain reaction in an independent cohort of 50 cases (13 normal mucosa, 25 dysplasia, and 12 UC-associated CRCs) and 46 controls (7 normal mucosa, 19 sporadic adenomas, and 20 sporadic CRCs). Results: Sixty-four miRNAs were found to be differentially deregulated in the UC-associated CRC sequence. Eight of these miRNAs were chosen for further validation. We confirmed miR-31, -106a, and -135b to be significantly deregulated between normal mucosa and dysplasia, as well as across the UC-associated CRC sequence (all P < 0.01). Notably, these miRNAs also confirmed to have a significant differential expression compared with sporadic CRC (all P < 0.05). Discussion: UC-associated and sporadic CRCs have distinct miRNA expression patterns, and some miRNAs indicate early neoplastic progression

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

Cardiovascular disease in immune-mediated inflammatory diseases: A cross-sectional analysis of 6 cohorts

To analyze in several immune-mediated inflammatory diseases (IMIDs) the influence of demographic and clinical-related variables on the prevalence of cardiovascular disease (CVD), and compare their standardized prevalences.Cross-sectional study, including consecutive patients diagnosed with rheumatoid arthritis, psoriatic arthritis, psoriasis, systemic lupus erythematosus, Crohn disease, or ulcerative colitis, from rheumatology, gastroenterology, and dermatology tertiary care outpatient clinics located throughout Spain, between 2007 and 2010. Our main outcome was defined as previous diagnosis of angina, myocardial infarction, peripheral vascular disease, and/or stroke. Bivariate and multivariate logistic and mixed-effects logistic regression models were performed for each condition and the overall cohort, respectively. Standardized prevalences (in subjects per 100 patients, with 95% confidence intervals) were calculated using marginal analysis.We included 9951 patients. For each IMID, traditional cardiovascular risk factors had a different contribution to CVD. Overall, older age, longer disease duration, presence of traditional cardiovascular risk factors, and male sex were independently associated with a higher CVD prevalence. After adjusting for demographic and traditional cardiovascular risk factors, systemic lupus erythematosus exhibited the highest CVD standardized prevalence, followed by rheumatoid arthritis, psoriasis, Crohn disease, psoriatic arthritis, and ulcerative colitis (4.5 [95% confidence interval (CI): 2.2, 6.8], 1.3 [95% CI: 0.8, 1.8], 0.9 [95% CI: 0.5, 1.2], 0.8 [95% CI: 0.2, 1.3], 0.6 [95% CI: 0.2, 1.0], and 0.5 [95% CI: 0.1, 0.8], respectively).Systemic lupus erythematosus, rheumatoid arthritis, and psoriasis are associated with higher prevalence of CVD compared with other IMIDs. Specific prevention programs should be established in subjects affected with these conditions to prevent CVD

Tofacitinib as induction and maintenance therapy for ulcerative colitis

Background: Tofacitinib, an oral, small-molecule Janus kinase inhibitor, was shown to have potential efficacy as induction therapy for ulcerative colitis in a phase 2 trial. We further evaluated the efficacy of tofacitinib as induction and maintenance therapy. METHODS: We conducted three phase 3, randomized, double-blind, placebo-controlled trials of tofacitinib therapy in adults with ulcerative colitis. In the OCTAVE Induction 1 and 2 trials, 598 and 541 patients, respectively, who had moderately to severely active ulcerative colitis despite previous conventional therapy or therapy with a tumor necrosis factor antagonist were randomly assigned to receive induction therapy with tofacitinib (10 mg twice daily) or placebo for 8 weeks. The primary end point was remission at 8 weeks. In the OCTAVE Sustain trial, 593 patients who had a clinical response to induction therapy were randomly assigned to receive maintenance therapy with tofacitinib (either 5 mg or 10 mg twice daily) or placebo for 52 weeks. The primary end point was remission at 52 weeks. RESULTS: In the OCTAVE Induction 1 trial, remission at 8 weeks occurred in 18.5% of the patients in the tofacitinib group versus 8.2% in the placebo group (P=0.007); in the OCTAVE Induction 2 trial, remission occurred in 16.6% versus 3.6% (P<0.001). In the OCTAVE Sustain trial, remission at 52 weeks occurred in 34.3% of the patients in the 5-mg tofacitinib group and 40.6% in the 10-mg tofacitinib group versus 11.1% in the placebo group (P<0.001 for both comparisons with placebo). In the OCTAVE Induction 1 and 2 trials, the rates of overall infection and serious infection were higher with tofacitinib than with placebo. In the OCTAVE Sustain trial, the rate of serious infection was similar across the three treatment groups, and the rates of overall infection and herpes zoster infection were higher with tofacitinib than with placebo. Across all three trials, adjudicated nonmelanoma skin cancer occurred in five patients who received tofacitinib and in one who received placebo, and adjudicated cardiovascular events occurred in five who received tofacitinib and in none who received placebo; as compared with placebo, tofacitinib was associated with increased lipid levels. CONCLUSIONS: In patients with moderately to severely active ulcerative colitis, tofacitinib was more effective as induction and maintenance therapy than placebo. (Funded by Pfizer; OCTAVE Induction 1, OCTAVE Induction 2, and OCTAVE Sustain ClinicalTrials.gov numbers, NCT01465763 , NCT01458951 , and NCT01458574 , respectively.

Experimental and genetic evidence for the impact of CD5 and CD6 expression and variation in inflammatory bowel disease

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) resulting from the interaction of multiple environmental, genetic and immunological factors. CD5 and CD6 are paralogs encoding lymphocyte co-receptors involved in fine-tuning intracellular signals delivered upon antigen-specific recognition, microbial pattern recognition and cell adhesion. While CD5 and CD6 expression and variation is known to influence some immune-mediated inflammatory disorders, their role in IBD remains unclear. To this end, Cd5- and Cd6-deficient mice were subjected to dextran sulfate sodium (DSS)-induced colitis, the most widely used experimental animal model of IBD. The two mouse lines showed opposite results regarding body weight loss and disease activity index (DAI) changes following DSS-induced colitis, thus supporting Cd5 and Cd6 expression involvement in the pathophysiology of this experimental IBD model. Furthermore, DNA samples from IBD patients of the ENEIDA registry were used to test association of CD5 (rs2241002 and rs2229177) and CD6 (rs17824933, rs11230563, and rs12360861) single nucleotide polymorphisms with susceptibility and clinical parameters of CD (n=1352) and UC (n=1013). Generalized linear regression analyses showed association of CD5 variation with CD ileal location (rs2241002CC) and requirement of biological therapies (rs2241002C-rs2229177T haplotype), and with poor UC prognosis (rs2241002T-rs2229177T haplotype). Regarding CD6, association was observed with CD ileal location (rs17824933G) and poor prognosis (rs12360861G), and with left-sided or extensive UC, and absence of ankylosing spondylitis in IBD (rs17824933G). The present experimental and genetic evidence support a role for CD5 and CD6 expression and variation in IBD's clinical manifestations and therapeutic requirements, providing insight into its pathophysiology and broadening the relevance of both immunomodulatory receptors in immune-mediated disorders

Influencia de la lesión hepática y de la actividad alcohol deshidrogenasa y aldehido deshidrogenasa hepáticas en el metabolismo del etanol

[spa] A) OBJETIVOSLos principales objetivos de la tesis han sido: 1) analizar el metabolismo hepático del etanol en los alcohólicos crónicos, 2) la producción de acetaldehido, 3) la actividad e isoenzimas de la alcohol deshidrogenasa (ADH) y aldehído deshidrogenasa (ALDH) hepáticas, y 4) la relación de cada uno de estos factores con el alcoholismo y el grado de lesión hepática.B) PACIENTESLa ADH y ALDH hepáticas se han estudiado en 84 pacientes, 60 eran alcohólicos (5 con hígado normal, 9 esteatosis, 9 fibrosis, 11 hepatitis alcohólica, 19 cirrosis alcohólica y 7 cirrosis y hepatitis alcohólica) y 24 eran hepatópatas no alcohólicos (14 hepatitis crónica y 10 cirrosis). Además, en 25 de los alcohólicos (4 hígado normal, 4 esteatosis, 4 fibrosis, 7 hepatitis alcohólica y 7 cirrosis no alcohólica) y en 6 de los cirróticos no alcohólicos se ha estudiado el metabolismo hepático del etanol.C) ESTUDIO DE LA CAPACIDAD OXIDATIVA DEL ETANOLLa capacidad de oxidación del etanol resultó superior en los pacientes alcohólicos que en los no alcohólicos de manera que en los alcohólicos la velocidad de metabolización del etanol (VME) fue de 113+/-16.9 mg/Kg/h y en los no alcohólicos de 90.5+/-18,9 mg/Kg/h (P pH 10.5). La prevalencia de la ADH "atípica" fue similar en pacientes alcohólicos (4/60, 6.7%) y no alcohólicos (2/24, 8.31%).No se halló el fenotipo "Indianápolis" en ninguna de las muestras estudiadas. El fenotipo del locus ADH3 pudo ser estudiado en 60 pacientes, 38 alcohólicos y 22 no alcohólicos. El fenotipo T1T1 se observó en 16 pacientes (27%), 39 casos (65%) presentaron el fenotipo T1T2 y 5 casos (81%) el fenotipo T2T2. La frecuencia fenotípica observada fue significativamente distinta a la calculada a partir de la frecuencia génica según la ley de la distribución de equilibrio de los genotipos. Entre los pacientes alcohólicos la frecuencia fenotípica resultó significativamente distinta a la calculada según esta ley. En cambio, entre los individuos no alcohólicos no se hallaron diferencias entre las frecuencias fenotípicas esperadas y las observadas.Este desequilibrio puede venir dado por una mayor predisposición al desarrollo de lesiones hepáticas en los pacientes alcohólicos con fenotipo T1T2 ya que en los alcohólicos con hígado normal o esteatosis la frecuencia fenotípica del locus ADH3 siguió el principio de equilibrio de los genotipos, mientras que en los pacientes con lesiones hepáticas severas como fibrosis, hepatitis alcohólica ó cirrosis la frecuencia fenotípica fue significativamente distinta a la calculada según la ley de Hardy-Weinberg, fundamentalmente por un aumento de la frecuencia observada del fenotipo T1T2 respecto a la esperada.El estudio de los isoenzimas de la ALDH reveló la banda correspondiente a la ALDH1, en todas las muestras estudiadas, tanto de los pacientes alcohólicos como de los no alcohólicos.La banda del isoenzima ALDH2 fue indetectable en 17 alcohólicos (39.51%) y en dos pacientes no alcohólicos (9.5%), existiendo entre ambas proporciones una diferencia estadísticamente significativa (p<0.02). Entre los individuos alcohólicos, la proporción de pacientes con ausencia de la banda de ALDH2 fue mayor a medida que aumentó la severidad de la lesión hepática, lo que sugiere que la falta de detección de la ALDH2 es probablemente una consecuencia de la disminución de actividad de este isoenzima, que resulta más marcada en pacientes alcohólicos con lesiones hepáticas severas como hepatitis alcohólica ó cirrosis.[eng] The influence of liver injury on the hepatic alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities (total, high-Km and low-Km ALDH) has been investigated in 60 alcoholics -5 normal liver (N), 9 steatosis (S), 9 fibrosis (F), 11 alcoholic hepatitis (AH), 19 cirrhosis (AC) and 7 cirrhosis with alcoholic hepatitis (ACAH)- and 24 non-alcoholics -14 chronic hepatitis (CH) and 10 cirrhosis (NAC)-. ADH and ALDH activities decreased proportionally with the progression of liver disease in alcoholics (ADH: N: 39.9 +/- 9,6; S: 32.6 +/- 6; F: 29.9 +/-4.9; AH: 20.8+/-3.8; AC:15.3+/-2.9; ACAH: 10+/- 2.8. Low-Km ALDH: N: 4.4 +/- 0.9; S: 4.1+/- 0.5; F: 2.3+/- 0.5; AH: 2.4+/- 0.5; AC:1.9 +/- 0.5; ACAH:1.2+/-0.5 mU/mg protein). By contrast, in non-alcoholics, there was a reduction of low-Km ALDH related to the severity of liver injury (CH: 5.3 +/- 0.8: NAC: 2.4 +/- 0.7 mU/mg protein), but not of ADH (CH:36.1+/-14.2; NAC: 36.0 +/- 8.7 mU/mg protein), Atypical ADH was present in 6.6% alcoholics and in 8.3% non-alcoholics. AH patients exhibited the isozyme ALDH II, but isozyme ALDH I was not detected in 39.5% alcoholic patients and in 9.5% of those with non-alcoholic liver disease. These results suggests that the decrease of ADH and ALDH activities in alcoholics are a consequence of liver damage. The diminution of ADH found particularly in alcoholics could be due to the leakage of the enzyme secondary to centrilobular cell necrosis.In 31 of the patients, 25 alcoholics (4 N, 4 S, 4 F, 7 AH, 7 AC) and 6 non-alcoholics (NAC), the blood ethanol and acetaldehyde concentrations after an intravenous infusion of ethanol were analyzed. In the alcoholics the ethanol metabolic rate (EHR) was significantly higher than in non-alcoholics. A significant positive correlation was observed between EMR and liver function estimated by the aminopyrine breath test (r=0.70, P<0.001). In non-alcoholics acetaldehyde levels were below the detection limits (<0.5 milimicres), in contrast, an elevated blood acetaldehyde was found in 14/15 alcoholics. The clinical characteristics and hepatic ADH and ALDH activities were similar in both groups of alcoholics. The only difference between alcoholics with elevated blood acetaldehyde and those without was EMR (120+/-17.4 vs 104+/-11.7 mg/Kg/h). Furthermore, the peak blood acetaldehyde level correlated positively with EMR. These results suggest that the main reason for blood acetaldehyde elevation in chronic alcoholics is their higher capacity to metabolize ethanol