The Genetic Diversity and Dysfunctionality of Catalase Associated with a Worse Outcome in Crohn's Disease.

Chronic gut inflammation in Crohn's disease (CD) is associated with an increase in oxidative stress and an imbalance of antioxidant enzymes. We have previously shown that catalase (CAT) activity is permanently inhibited by CD. The purpose of the study was to determine whether there is any relationship between the single nucleotide polymorphisms (SNPs) in the CAT enzyme and the potential risk of CD associated with high levels of oxidative stress. Additionally, we used protein and regulation analyses to determine what causes long-term CAT inhibition in peripheral white mononuclear cells (PWMCs) in both active and inactive CD. We first used a retrospective cohort of 598 patients with CD and 625 age-matched healthy controls (ENEIDA registry) for the genotype analysis. A second human cohort was used to study the functional and regulatory mechanisms of CAT in CD. We isolated PWMCs from CD patients at the onset of the disease (naïve CD patients). In the genotype-association SNP analysis, the CAT SNPs rs1001179, rs475043, and rs525938 showed a significant association with CD (p < 0.001). Smoking CD patients with the CAT SNP rs475043 A/G genotype had significantly more often penetrating disease (p = 0.009). The gene expression and protein levels of CAT were permanently reduced in the active and inactive CD patients. The inhibition of CAT activity in the PWMCs of the CD patients was related to a low concentration of CAT protein caused by the downregulation of CAT-gene transcription. Our study suggests an association between CAT SNPs and the risk of CD that may explain permanent CAT inhibition in CD patients together with low CAT gene and protein expression.

Multicompartmental Lipopolyplex as Vehicle for Antigens and Genes Delivery in Vaccine Formulations.

Vector design and its characterization is an area of great interest in current vaccine research. In this article, we have formulated and characterized a multicompartmental lipopolyplex, which associates multiple liposomes and polyplexes in the same complex. These particles allow the simultaneous delivery of lipid or water-soluble antigens associated with genes to the same cell, in much higher amounts than conventional lipopolyplexes. The vector characterization and optimization were carried out using liposomes with entrapped carboxyfluorescein and adapted electrophoretic assays. Two types of lipopolyplexes (containing hydrophilic or lipophilic antigens) were employed to evaluate their interest in vaccination. The lipopolyplex loaded with an extract of water-soluble melanoma proteins proved to efficiently induce humoral response in murine melanoma model, increasing the levels of IgM and IgG. The specificity of the immune response induced by the lipopolyplex was demonstrated in mice with the lipopolyplex containing the GD3 ganglioside lipid antigen, abundant in melanoma cells. The levels of anti-GD3 IgG increased markedly without modifying the expression of humoral antibodies against other gangliosides.

Oxidative Stress in the Pathogenesis of Crohn's Disease and the Interconnection with Immunological Response, Microbiota, External Environmental Factors, and Epigenetics.

Inflammatory bowel disease (IBD) is a complex multifactorial disorder in which external and environmental factors have a large influence on its onset and development, especially in genetically susceptible individuals. Crohn's disease (CD), one of the two types of IBD, is characterized by transmural inflammation, which is most frequently located in the region of the terminal ileum. Oxidative stress, caused by an overabundance of reactive oxygen species, is present locally and systemically in patients with CD and appears to be associated with the well-described imbalanced immune response and dysbiosis in the disease. Oxidative stress could also underlie some of the environmental risk factors proposed for CD. Although the exact etiopathology of CD remains unknown, the key role of oxidative stress in the pathogenesis of CD is extensively recognized. Epigenetics can provide a link between environmental factors and genetics, and numerous epigenetic changes associated with certain environmental risk factors, microbiota, and inflammation are reported in CD. Further attention needs to be focused on whether these epigenetic changes also have a primary role in the pathogenesis of CD, along with oxidative stress.

Plasma Oncostatin M, TNF-α, IL-7, and IL-13 Network Predicts Crohn's Disease Response to Infliximab, as Assessed by Calprotectin Log Drop.

BACKGROUND: Cytokines emerge as possible biomarkers of response in Crohn's disease (CD). We aimed to determine the plasmatic cytokine profiles of active CD patients who started infliximab (IFX) treatment and their capacity to predict the response to IFX. METHODS: A total of 30 active CD patients receiving an induction therapy of IFX were enrolled in the study. Peripheral blood samples pretreatment were collected. Concentrations of 15 cytokines were measured by Luminex technology. Responses to IFX were evaluated by the drop in fecal calprotectin based on its logarithm-transformed values. A random forest (RF) predictive model was used for data analyses. RESULTS: Samples of 22 patients were analyzed. The RF model ranked the following cytokines as the top predictors of the response: tumor necrosis factor alpha (TNFα), interleukin (IL)-13, oncostatin M (OSM), and IL-7 (p < 0.005). Partial dependency plots showed that high levels of IL-13 pretreatment, low TNFα levels, and low IL-7 levels were associated with a favorable IFX response. Increased levels of OSM and TNFα predicted unfavorable responses to IFX. CONCLUSIONS: We here show that a log drop in calprotectin strongly correlates with clinical parameters and it can be proposed as a useful objective clinical response predictor. Plasma TNFα, IL-13, Il-7, and OSM network could predict CD response to IFX before induction therapy, as assessed by calprotectin log drop.

Bases for the Adequate Development of Nutritional Recommendations for Patients with Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory condition of the gastrointestinal tract; it is a heterogeneous and multifactorial disorder resulting from a complex interplay between genetic variation, intestinal microbiota, the host immune system and environmental factors such as diet, drugs, breastfeeding and smoking. The interactions between dietary nutrients and intestinal immunity are complex. There is a compelling argument for environmental factors such as diet playing a role in the cause and course of IBD, given that three important factors in the pathogenesis of IBD can be modulated and controlled by diet: intestinal microbiota, the immune system and epithelial barrier function. The aim of this review is to summarize the epidemiological findings regarding diet and to focus on the effects that nutrients exert on the intestinal mucosa-microbiota-permeability interaction. The nature of these interactions in IBD is influenced by alterations in the nutritional metabolism of the gut microbiota and host cells that can influence the outcome of nutritional intervention. A better understanding of diet-host-microbiota interactions is essential for unravelling the complex molecular basis of epigenetic, genetic and environmental interactions underlying IBD pathogenesis as well as for offering new therapeutic approaches for the treatment of IBD.

A Nomogram Combining Fecal Calprotectin Levels and Plasma Cytokine Profiles for Individual Prediction of Postoperative Crohn's Disease Recurrence.

BACKGROUND: The aims of this study were to characterize the immune response profile in patients with Crohn's disease (CD) and early postoperative recurrence (POR), to identify predictive biomarkers, and to develop a noninvasive predictive tool for individual estimation of POR risk. METHODS: Sixty-one patients who had undergone ileocolonic resection for CD were prospectively included and followed up for 24 months. Fecal calprotectin (FC), analytical parameters, and plasma cytokines were obtained before surgery and at various time points during postoperative follow-up. Morphological recurrence was assessed by ileocolonoscopy or magnetic resonance enterography within 6-12 months after surgery. Clinical activity was scored using the Harvey-Bradshaw Index. RESULTS: Twenty-seven patients (44.3%) had morphological recurrence during follow-up. Fecal calprotectin values were significantly associated with POR risk over time. The receiver operating characteristic curve for FC provided an area under the curve (AUC) of 0.88 (95% confidence interval, 0.75-0.96), and morphological recurrence was best predicted by FC ≥160 µg/g at 6 months after surgery (85% sensitivity, 70% specificity, 26% predictive positive value, 98% negative predictive value [NPV]). The plasma cytokine profile showed higher presurgery interleukin (IL)-13 plasma levels and higher IL-6 and interferon (IFN)-γ levels at 6 months after surgery in patients with POR compared with patients without recurrence. The combination of FC, IL-6, and IFN-γ values at 6 months gave an AUC of 0.90 for predicting an early recurrence. CONCLUSIONS: FC values <160 µg/g at 6 months have a high NPV to rule out early lesions. Combined values of FC, IL-6, and IFN-γ levels at 6 months postsurgery constitute a prognostic index with a high predictive capacity to assess the risk of early POR.

Fecal Calprotectin Pretreatment and Induction Infliximab Levels for Prediction of Primary Nonresponse to Infliximab Therapy in Crohn's Disease.

INTRODUCTION: The association between infliximab (IFX) and fecal calprotectin (FC) levels on one hand, and the clinical and endoscopic response of patients with inflammatory bowel disease on the other, is well established. OBJECTIVE AND METHODS: To investigate the association between inflammatory biochemical parameters and serum concentrations of IFX during induction treatment with a primary nonresponse in a prospective cohort of Crohn's disease (CD) patients. RESULTS: Of the 35 patients included, 8 (22.8%) had primary nonresponse at the end of induction. Induction IFX levels were lower among primary nonresponders at weeks 6 and 14 (week 6: median IFX level 7.3 vs. 11.2 µg/mL, respectively, p = 0.090; week 14: median IFX level 1.5 vs. 4.7 µg/mL, respectively, p = 0.020). FC levels were higher in patients with primary nonresponse versus primary response at weeks 0, 6, and 14 (week 0: median FC level 1,830 vs. 410 µg/g, -respectively, p = 0.030; week 6: median FC level 1,150 vs. 230 µg/g, respectively, p = 0.074; week 14: median FC level 1,210 vs. 208 µg/g, respectively, p = 0.060). For the multivariate analysis, the median IFX level at week 14 and median FC level at week 0 were independently associated with primary nonresponse. A significant inverse correlation was determined between FC level at week 0 and IFX level at week 14 (Spearman's rho correlation, 0.440; p < 0.05). CONCLUSIONS: IFX levels (at week 14) and baseline FC levels could predict primary nonresponse after induction IFX therapy in patients with CD. A high baseline inflammatory load might modify the pharmacokinetic processes of anti-tumor necrosis factor drugs. Drug level monitoring and measurement of baseline inflammatory parameters could improve the efficacy of IFX in the induction therapy of patients with active CD.

Different Genetic Expression Profiles of Oxidative Stress and Apoptosis-Related Genes in Crohn's Disease.

BACKGROUND/AIMS: Increased oxidative stress and decreased immune cell apoptosis have been reported to be important factors in the pathogenesis of Crohn's disease (CD). Our aim was to characterize the genetic expression of molecules implicated in the regulation of oxidative stress and apoptosis in peripheral white mononuclear cells of 18 healthy volunteers (controls) and 20 patients at the onset of CD (active CD [aCD]): 10 who achieved remission (inactive CD [iCD]) and 10 who did not present a complete and deep response to treatment (aCD-T). METHODS: mRNA expression was measured by the Agena MassARRAY quantitative gene expression analysis application. The genes analyzed were Fas-receptor (FASR), Fas-ligand (FASL), signal transducer and activator of transcription 1 (STAT1), nuclear factor kappa-light-chain--enhancer of activated B cells (NFKB1), apoptosis signal-regulating kinase 1 (ASK1), serine/threonine-protein kinase H1 (PSKH1), ATP-binding cassette sub-family B1 (ABCB1) and peptidylprolyl isomerase D (PPID). RESULTS: During a CD flare, we found specific upregulated expression of the genes STAT1 and PSKH1, whereas ABCB1 and FASL were downregulated. In the patients with iCD, FASR and NFKB1 were upregulated. The expression levels of NFKB1, STAT1 and ABCB1 did not show any difference in patients with aCD at the onset of the disease and after treatment (aCD-T). The expression levels of PPID and ASK1 did not show any differences in the patients with aCD, iCD and the controls. We have also reviewed the cellular function and role of these genes in CD. CONCLUSIONS: These findings contribute to improving the understanding of the pathogenesis of CD and highlight potential genes involved.

Adsorptive granulocyte/monocyte apheresis use in severe ulcerative colitis and determination of changes in plasma cytokines.

Despite controversy regarding the use of granulocyte/monocyte adsorption (GMA) in inflammatory bowel disease, some studies have shown favorable outcomes when it is used in steroid-dependent patients with ulcerative colitis (UC). The mechanisms responsible for such outcomes are not well characterized, but changes in immune cell populations and cytokine levels have been suggested to play an important role. We report the cases of 3 patients with chronically active severe UC who underwent GMA due to an inadequate response to standard and rescue therapy, as well as changes to their plasma cytokine profile. All the patients presented severe UC that was only partially responsive to various immunosuppressive drugs, and they were, therefore, referred for colectomy; however, all 3 refused this option, which led to the compassionate use of GMA as a last therapeutic resort. Following GMA treatment, rapid normalization of the clinical, endoscopic and laboratory parameters was observed in all the patients. Despite having achieved a good response, most cytokines remained at high concentrations after GMA, and only two, IL-6 and IL-8, showed a clear decrease throughout the GMA sessions. In view of this outcome, we hypothesize that GMA can help to lower the inflammatory load, thereby enhancing the effect of biologic drugs. To confirm this hypothesis and explore further indications for GMA, we propose the need for research directed toward the characterization of immune cell populations and their specific cytokine production rather than global cytokine assessment.

Immunological Mechanisms of Adsorptive Cytapheresis in Inflammatory Bowel Disease.

Ulcerative colitis and Crohn's disease are the two main forms of inflammatory bowel disease (IBD). The study of immunological pathways involved in the onset of IBD is of fundamental importance to identify potential biological markers of disease activity and specific targets for therapy. Removing excess and activated circulating leukocytes with adsorptive cytapheresis has been shown to be a potentially effective treatment for patients with an inflamed bowel. Adsorptive cytapheresis is a non-pharmacological approach for active IBD, in which known sources of inflammatory cytokines such as activated myeloid lineage leucocytes are selectively depleted from the circulatory system. The decrease in inflammatory load caused by removing these cells is thought to enhance drug therapy and thereby promote disease remission. The benefit of cytapheresis appears to rest upon its ability to reduce levels of certain immune cell populations; however, whether this depletion results in further changes in lymphocyte populations and cytokine production needs further clarification. In this review, we aim to summarize existing evidence on the role of cytapheresis in patients with IBD, its effect on cytokine levels and cellular populations, and to discuss its potential impact on disease activity.

Metagenomic Analysis of Crohn's Disease Patients Identifies Changes in the Virome and Microbiome Related to Disease Status and Therapy, and Detects Potential Interactions and Biomarkers.

BACKGROUND: The aim of this study was to survey the bacterial and viral communities in different types of samples from patients with Crohn's disease (CD) at different stages of the disease to relate their distribution with the origin and progression of this disorder. METHODS: A total of 42 fecal samples and 15 biopsies from 20 patients with CD and 20 healthy control individuals were collected for bacterial 16S rRNA gene profiling and DNA/RNA virome metagenomic analysis through 454 pyrosequencing. Their composition, abundance, and diversity were analyzed, and comparisons of disease status, patient status, and sample origin were used to determine statistical differences between the groups. RESULTS: Bacterial composition and relative abundance in new-onset patients with CD differed markedly from control individuals. Individual variability and sample origin had a stronger impact on viral communities than the disease, contrary to what was observed for bacterial populations although increased numbers of overrepresented viruses were observed in feces from patients with CD. Correlation-based networks were constructed to show potential relations between bacteria and between those and viruses. CONCLUSIONS: The bacterial community reflects the disease status of individuals more accurately than their viral counterparts. However, numerous viral biomarkers specifically associated with CD disease were identified. Because viruses can modulate bacterial communities, the correlation networks between both communities constitute a step forward in unraveling their interactions under normal and CD disease conditions.

Lung inflammatory pattern and antibiotic treatment in pneumonia.

BACKGROUND: In community-acquired pneumonia host inflammatory response against the causative microorganism is necessary for infection resolution. However an excessive response can have deleterious effects. In addition to antimicrobial effects, macrolide antibiotics are known to possess immunomodulatory properties. METHODS: A prospective study was performed on 52 admitted patients who developed an inadequate response after 72 hours of antibiotic treatment - non-responders community-acquired pneumonia - (blood and bronchoalveolar lavage), and two control groups: 1) community-acquired pneumonia control (blood) and 2) non-infection control (blood and bronchoalveolar lavage). Cytokine profiles (interleukin (IL)-6, IL-8, IL-10), tumour necrosis factor α and clinical outcomes were assessed. RESULTS: Non-responders patients treated with macrolide containing regimens showed significantly lower levels of IL-6 and TNF-α in bronchoalveolar lavage fluid and lower IL-8 and IL-10 in blood than those patients treated with non-macrolide regimens. Clinical outcomes showed that patients treated with macrolide regimens required fewer days to reach clinical stability (p < 0.01) and shorter hospitalization periods (p < 0.01). CONCLUSIONS: After 72 hours of antibiotic effect, patients who received macrolide containing regimens exhibited lower inflammatory cytokine levels in pulmonary and systemic compartments along with faster stabilization of infectious parameters.

Estrés oxidativo en la enfermedad de Crohn / Oxidative stress in Crohn's disease

La enfermedad de Crohn (EC) se caracteriza por dar lugar a procesos de inflamación transmural que con mayor frecuencia se localizan en la región del íleon terminal. Aunque los mecanismos fisiopatológicos de la enfermedad no están todavía bien definidos, se ha observado que la respuesta inmunitaria no regulada está asociada a una producción elevada de especies reactivas de oxígeno (ERO). Estos elementos están relacionados con unos sistemas complejos denominados defensas antioxidantes (DAO) que tienen la función de regular los ERO, evitando así sus efectos dañinos. Sin embargo, se ha descrito ampliamente para la EC la presencia de un desequilibrio entre la producción de ERO y su eliminación por los elementos antioxidantes, originando lo que se denomina estrés oxidativo. Enmarcado en este contexto, a continuación se profundiza sobre los hallazgos más destacados relacionados con el estrés oxidativo en la mucosa intestinal y en la sangre periférica (AU)

Crohn’s disease (CD) is characterized by transmural inflammation that is most frequently located in the region of the terminal ileum. Although the physiopathological mechanisms of the disease are not yet well defined, the unregulated immune response is associated with high production of reactive oxygen species (ROS). These elements are associated with complex systems known as antioxidant defenses, whose function is ROS regulation, thereby preventing the harmful effects of these elements. However, the presence of an imbalance between ROS production and ROS elimination by antioxidants has been widely described and leads to oxidative stress. In this article, we describe the most significant findings on oxidative stress in the intestinal mucosa and peripheral blood

[Oxidative stress in Crohn's disease]. / Estrés oxidativo en la enfermedad de Crohn.

Crohn's disease (CD) is characterized by transmural inflammation that is most frequently located in the region of the terminal ileum. Although the physiopathological mechanisms of the disease are not yet well defined, the unregulated immune response is associated with high production of reactive oxygen species (ROS). These elements are associated with complex systems known as antioxidant defenses, whose function is ROS regulation, thereby preventing the harmful effects of these elements. However, the presence of an imbalance between ROS production and ROS elimination by antioxidants has been widely described and leads to oxidative stress. In this article, we describe the most significant findings on oxidative stress in the intestinal mucosa and peripheral blood.

Assessing an improved protocol for plasma microRNA extraction.

The first step in biomarkers discovery is to identify the best protocols for their purification and analysis. This issue is critical when considering peripheral blood samples (plasma and serum) that are clinically interesting but meet several methodological problems, mainly complexity and low biomarker concentration. Analysis of small molecules, such as circulating microRNAs, should overcome these disadvantages. The present study describes an optimal RNA extraction method of microRNAs from human plasma samples. Different reagents and commercially available kits have been analyzed, identifying also the best pre-analytical conditions for plasma isolation. Between all of them, the column-based approaches were shown to be the most effective. In this context, miRNeasy Serum/Plasma Kit (from Qiagen) rendered more concentrated RNA, that was better suited for microarrays studies and did not require extra purification steps for sample concentration and purification than phenol based extraction methods. We also present evidences that the addition of low doses of an RNA carrier before starting the extraction process improves microRNA purification while an already published carrier dose can result in significant bias over microRNA profiles. Quality controls for best protocol selection were developed by spectrophotometry measurement of contaminants and microfluidics electrophoresis (Agilent 2100 Bioanalyzer) for RNA integrity. Selected donor and patient plasma samples and matched biopsies were tested by Affymetrix microarray technology to compare differentially expressed microRNAs. In summary, this study defines an optimized protocol for microRNA purification from human blood samples, increasing the performance of assays and shedding light over the best way to discover and use these biomarkers in clinical practice.

Role of oxidative stress and antioxidant enzymes in Crohn's disease.

There is increasing interest in oxidative stress being a potential aetiological factor and/or a triggering factor in Crohn's disease, rather than a concomitant occurrence during the pathogenesis of the disease. Recent research has shown that the immune mononuclear cells of Crohn's disease patients are induced to produce hydrogen peroxide (H2O2). Similarly, the regulation of antioxidant enzymes during disease in these cells has been unravelled, showing that SOD (superoxide dismutase) activity and GPx (glutathione peroxidase) activity is increased during active disease and returns to normal in remission phases. However, catalase remains constantly inhibited which supports the idea that catalase is not a redox-sensitive enzyme, but a regulator of cellular processes. ROS (reactive oxygen species) can be produced under the stimulus of different cytokines such as TNFα (tumour necrosis factor α). It has been shown in different experimental models that they are also able to regulate apoptosis and other cellular processes. The status of oxidative stress elements in Crohn's disease and their possible implications in regulating cellular processes are reviewed in the present paper.

Mitochondrial dysfunction, persistent oxidative damage, and catalase inhibition in immune cells of naïve and treated Crohn's disease.

BACKGROUND: Oxidative stress is considered a potential etiological factor for Crohn's disease (CD). We characterized the reactive oxygen species (ROS) generated in immune peripheral cells of CD patients, as well as their antioxidant enzyme status and the presence of oxidative damage. In addition, mitochondrial function (DeltaPsim) was analyzed to detect the possible origin of ROS. METHODS: Cells were obtained from patients at the onset of disease, prior to any treatment. Experiments were repeated when patients were in clinical remission. A set of experiments was carried out in a group of CD patients in persistent morphological remission. Controls were healthy volunteers who were not receiving any treatment at the time. The generation of superoxide, hydrogen peroxide (H(2)O(2)) and nitric oxide, DeltaPsim, superoxide dismutase (SOD) and catalase (CAT) activities, and concentrations of malondyaldehyde (MDA) and 8-oxo-deoxyguanosine (8-oxo-dG) were measured. RESULTS: SOD activity and H(2)O(2) production were significantly higher during active CD but returned to control levels in remission. DeltaPsim was inhibited during active CD and, although it returned to control levels, its recovery took longer than clinical remission. CAT activity was permanently inhibited during CD, independent of the disease activity. MDA and 8-oxo-dG were permanently elevated. CONCLUSIONS: Oxidative stress during active CD depends on H(2)O(2) production. The inhibition of DeltaPsim suggests that this organelle is a source of ROS. CAT is permanently inhibited in CD, the biological significance of which is under study. The persistent oxidative damage detected may have implications for the evolution of the disease.

Targeted oligonucleotide delivery in human lymphoma cell lines using a polyethyleneimine based immunopolyplex.

The efficacy of antisense gene therapy depends on efficient delivery of oligonucleotides into targeted cells. Although polyethyleneimine based polyplexes have been reported as good transfection reagents, they are inefficient in lymphoid cell transfection. We report the construction of an immunopolyplex, a targeted nonviral vector based on a polyplex backbone and its application for oligonucleotide transfer on human lymphoma cell lines. The salient characteristic of immunopolyplex lies in the possibility of easily replacing the targeting element (antibody), leaving the polyplex backbone intact. Furthermore, a study was made of the influence of endocytosis inhibitors on immunopolyplex activity. The capacity of the immunopolyplex for oligonucleotide transfer was studied in vitro using FITC-labeled oligonucleotides as fluorescent reporters, an anti-CD3 antibody as targeting element, and a CD3-positive cell line (Jurkat) as a target cell line, in the absence and presence of endocytosis inhibitors. A CD3-negative Jurkat-derived mutant cell line (J.RT3-T3.5) was used as control. A nine-fold increase in fluorescence in the CD3-positive above that in the CD3-negative cell line was observed, indicating that oligonucleotide transfer is mainly specific. Low fluorescence values were obtained in the presence of endocytosis inhibitor or with untargeted polyplexes. We conclude that the immunopolyplex is a good nonviral vector for specific oligonucleotide delivery. Abolition of immunopolyplex activity in the presence of endocytosis inhibitor suggests that targeted oligonucleotide transfer occurs through an endocytic pathway.