Ines Moret

Stability of PEI–DNA and DOTAP–DNA complexes: effect of alkaline pH, heparin and serum

DNA complexes formed with nonviral vectors such as polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) are widely used in gene therapy. These complexes prevent the interaction of DNA with the fluorescent probes usually employed to quantify DNA. We thus studied the procedures for DNA quantification from DNA complexes as well as their stability in the presence of DNase or mouse, rat and human sera. Release of the DNA from its complexes was accomplished by increasing the pH of the medium (from 7.3 to 13.4) or by adding heparin. The stability against degradation was tested in vitro, by incubating the complexes at 37 degrees C in the presence of DNase I and sera from the three species. Both high pH and heparin were able to release DNA from its complexes. Naked DNA formed aggregates with serum proteins that delayed electrophoresis migration, and this effect was reversed in the presence of heparin. However, these aggregates did not protect DNA from digestion by serum DNase, and the DNA digesting ability of serum was: mouse>rat>human. The DNA from the complexes was resistant to degradation by DNase I, although a low proportion of DNA from the complexes was partially digested, as determined by electrophoresis. In contrast, PEI-DNA and DOTAP-DNA complexes were stable in the presence of all sera. Heparin and high pH release DNA from its complexes. The order of DNA degradation is: mouse>rat>human, but DOTAP and PEI avoid degradation of DNA by serum compounds.

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 Crohns 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 (&Dgr;&ggr;m) 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 (H2O2) and nitric oxide, &Dgr;&ggr;m, 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 H2O2 production were significantly higher during active CD but returned to control levels in remission. &Dgr;&ggr;m 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 H2O2 production. The inhibition of &Dgr;&ggr;m 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. Inflamm Bowel Dis 2010

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.

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.

Tu1913 Differential Regulation of Oxidative Stress and Apoptosis Related Genes During Active and Inactive Crohn's Disease (CD)

Background: An increase of oxidative stress (OxS) and a decrease of apoptosis have been reported as key factors in Crohns disease (CD) pathogenesis. We have previously shown that OxS in CD depends on an increased production of H2O2 which detoxification is independent of catalase (CAT) enzyme because CAT showed permanently inhibited in CD patients1. CAT could be more implicated in regulating cellular processes, as apoptosis, than in detoxifying H2O2. Aims: To characterize the genetic expression of CAT and other OxS and apoptosis related genes in CD at beginning of the disease and when remission has been achieved. Methods: Blood samples were obtained from 18 healthy subjects (H), 20 patients at onset of CD and yet to begin any specific medication (active CD, aCD) and from 10 patients who achieved clinical, analytical and morphological remission (inactive CD, iCD). Patients were diagnosed according to Leonard-Jones criteria and disease activity was scored based on the Harvey-Bradshaw index, serological markers of inflammation (CRP, fibrinogen) and evaluation of morphological lesions (magnetic resonance enterography, colonoscopy or capsule endoscopy). PWMC were isolated by Histopaque sedimentation. Total RNA from leukocytes was extracted according to manufacturers protocol (LeukoLOCKTM; Ambion). mRNA expression was measured by Sequenoms MassARRAY® quantitative gene expression analysis application. The genes analyzed were: CAT, SOD2, NOS2A, STAT1, NFKB1, PKCgamma, PKCzeta, PSKH1, PPID, ABCB1, ASK1, FASR, FASLG, TERT, IL-2, IL23R. Results: Genes differentially expressed in CD are summarized in the table. Specific upregulated genes during flair were SOD2, STAT1, and PSKH1, and down-regulated were FASL and ABCB1. In iCD, FASR and NFKB1 were up-regulated. The down-regulation of CAT gene expression was persistent. Conclusions: Up regulation of SOD2, STAT1 and PSKH1 only during a flare, indicates the relation between OxS and aCD. Down regulation of FASL and ABCB1 shows an apoptotic and multidrug resistance alteration at onset of the disease. Increased expression of FASR and NFKB1 in iCD could indicate that apoptosis are genetically affected in CD. Permanent inhibition of CAT activity in CD patients correlates to a persistent down regulation of the CAT-gene mRNA. The implications of CAT inhibition in regulating cellular processes such as apoptosis should be studied. 1) Beltran B, et al. Inflamm Bowel Dis. 2010 16:76-86.

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.

MicroRNAs as Novel Biomarkers in IBD: Characterization and Current Status

The pathophysiology of the Inflammatory Bowel Disease (IBD) has been intensively investigated but is still unknown. The theory that IBD is the result of an inadequate activation of the immune system to a luminal factor occurring in genetically predisposed subjects is the most widely accepted to date. MicroRNAs (miRNAs) are a class of small noncoding RNAs which regulate gene expression at post-transcriptional level and are involved in the regulation of many biological processes, as well as in the induction of several cancers, chronic inflammatory diseases and autoimmune diseases. The currently evidence has demonstrated that miRNAs are differentially expressed in diverse circumstances of the IBD course. These molecules open a new opportunity to employ as a non-invasive biomarker for diagnosis, prognosis and follow up. Knowing the role of miRNA in IBD will improve our knowledge of the pathogenesis. In addition, the development of miRNA-based therapeutics technologies supposes a qualitative advance in the management of IBD.