Rajesh Somasundaram

Bone marrow stromal cell interaction reduces Syndecan-1 expression and induces kinomic changes in myeloma cells

CD138 (Syndecan 1) is a heparan sulfate proteoglycan that concentrates heparan sulfate-binding growth factors on the surface of normal and malignant plasma cells (multiple myeloma, MMC). Recent studies have shown the presence of a CD138-negative fraction of MMC within myelomatous bone marrow (BM). We employed kinome array technology to characterize this fraction at a molecular level, using a myeloma cell line model. Compared to CD138-positive cells, CD138-negative MMC showed (i) a reduced activity of kinases involved in cell cycle progression, in agreement with a decreased labeling index and (ii) reduced Rho signaling to F-actin. Interestingly, CD138 mRNA and protein expression was reduced upon interaction of MM cells with stromal cell lines and primary mesenchymal cultures, which was accompanied by the acquisition of an increased Bcl6/Blimp1 ratio. Co-culture induced an increased activity of kinases involved in adhesion and a decreased S-phase transition in both CD138-positive and -negative fractions. In addition, CD138-negative MMC demonstrated an increased STAT3 and ERK1/2 activation compared to CD138+ MMC, in agreement with a lower sensitivity to compound exposure. The presence of a less mature, more resistant CD138-negative myeloma cell fraction within bone marrow microniches might contribute to high incidence of relapse of Myeloma patients.

Linking risk conferring mutations in NCF4 to functional consequences in Crohn's disease

We read with interest the paper from Muise et al in which they describe a rare variant in the NCF2 gene, which demonstrates a diminished RAC2 binding capacity.1 The NCF2 encoded protein p67phox is one of the components of the NADPH oxidase complex which drives the production of reactive oxygen species (ROS) during the bactericidal response of innate immune cells. Output of disturbed granulocytic ROS as a result of impaired functioning of this enzyme complex has been shown in a number of diseases, including myelodysplasia (MDS) and chronic granulomatous disease.2 3 As Muise and colleagues point out, these diseases have been linked to development of a colitis resembling that seen in Crohns disease (CD), suggesting a potential role for impaired ROS production in CD pathology. Genome-wide association studies (GWAS) are a promising tool to identify genetic variants …

Role of defective autophagia and the intestinal flora in Crohn disease.

The precise mechanisms underlying the development of Crohn disease (CD) remain controversial, but sufficient data have been collected to suggest that an uncontrolled immune response within the intestinal mucosa leads to inflammation in a genetically susceptible host. Although lack of mucosal regulatory T cells causes colitis in humans and experimental rodents, patients with CD have more rather than less regulatory activity in the intestine, apparently excluding defects in tolerance as the cause of CD. Genome-wide association studies have identified many gene variants that confer susceptibility and which seem associated to diminished functioning of especially innate immunity. In apparent agreement, CD patients are impaired with respect to innate immune responses and controlling bacterial flora in the intestine. Furthermore, severe genetic deficiencies in innate immunity, like e.g., lack of NADP oxidase activity or diminished function of the Wiskott Aldrich syndrome protein are associated with colitis in mice and men, and are often mistakenly diagnosed as CD. Thus we favor the view that the primary defect in CD is a lack in innate immunity, causing second tier immunological defenses to combat otherwise easily controlled bacterial breaches of the mucosal barrier.

Analysis of SHIP1 expression and activity in Crohn’s disease patients

Background SH2 domain containing inositol-5-phosphatase (SHIP1) is an important modulator of innate and adaptive immunity. In mice, loss of SHIP1 provokes severe ileitis resembling Crohn’s disease (CD), as a result of deregulated immune responses, altered cytokine production and intestinal fibrosis. Recently, SHIP1 activity was shown to be correlated to the presence of a CD-associated single nucleotide polymorphism in ATG16L1. Here, we studied SHIP1 activity and expression in an adult cohort of CD patients. Methods SHIP1 activity, protein and mRNA expression in peripheral blood mononuclear cells from CD patients in clinical remission were determined by Malachite green assay, Western blotting and qRT-PCR respectively. Genomic DNA was genotyped for ATG16L1 rs2241880. Results SHIP1 protein levels are profoundly diminished in a subset of patients; however, SHIP1 activity and expression are not correlated to ATG16L1 SNP status in this adult cohort. Conclusions Aberrant SHIP1 activity can contribute to disease in a subset of adult CD patients, and warrants further investigation.

E. coli-Produced BMP-2 as a Chemopreventive Strategy for Colon Cancer: A Proof-of-Concept Study

Colon cancer is a serious health problem, and novel preventive and therapeutical avenues are urgently called for. Delivery of proteins with anticancer activity through genetically modified bacteria provides an interesting, potentially specific, economic and effective approach here. Interestingly, bone morphogenetic protein 2 (BMP-2) is an important and powerful tumour suppressor in the colon and is thus an attractive candidate protein for delivery through genetically modified bacteria. It has not been shown, however, that BMP production in the bacterial context is effective on colon cancer cells. Here we demonstrate that transforming E. coli with a cDNA encoding an ileal-derived mature human BMP-2 induces effective apoptosis in an in vitro model system for colorectal cancer, whereas the maternal organism was not effective in this respect. Furthermore, these effects were sensitive to cotreatment with the BMP inhibitor Noggin. We propose that prevention and treatment of colorectal cancer using transgenic bacteria is feasible.

Sa1779 Adding Fuel to the Fire - Neutrophils As Antigen Presenting Cells in CrohnS Disease

BACKGROUND AND AIMS. Intestinal macrophages play a central role in the pathogenesis of inflammatory bowel disease (IBD). The phenotype of mucosal macrophages changes dramatically at the site of inflammation, where high numbers of CD33+CD68+ macrophages overexpress toll-like receptors, CD14 and triggering receptor expressed on myeloid cells 1 (TREM-1). TREM-1 is expressed on the surface of neutrophils and macrophages and contributes to the amplification of inflammatory responses by enhancing degranulation and secretion of pro-inflammatory mediators. After comparing the percentage of TREM-1-expressing macrophages in IBD versus control mucosa, we explored the ex vivo effects of an activating anti-TREM-1 antibody on the intestinal immune response in IBD. METHODS. Lamina propria mononuclear cells (LPMCs) were isolated from the inflamed colon of 11 IBD patients (5 ulcerative colitis and 6 Crohns disease), and from normal colon of 8 control subjects, and the expression of CD68, CD33 and TREM-1 was analysed by flow cytometry. TREM1 mRNA expression in CD33+ LPMCs was evaluated by qRT-PCR. IBD biopsies from inflamed mucosa were cultured ex vivo with an activating anti-TREM-1 antibody or its control isotype, and the production of pro-inflammatory cytokines, namely interleukin (IL)1beta, IL-6 and IL-8, was determined by ELISA. RESULTS. The percentage of mucosal CD33+CD68+ macrophages was significantly (p,0.05) higher both in Crohns disease and ulcerative colitis (17%±1.4 and 15%±7.6, respectively) in comparison to control subjects (6.0%±1.0). TREM-1 expression by mucosal macrophages was significantly (p,0.05) higher both in ulcerative colitis and in Crohns disease (6.4%±0.2 and 4.4%±0.6) than in control subjects (0.8%±0.1). TREM-1 transcripts were significantly (p,0.05) higher in CD33+ LPMCs from inflamed IBD compared to control subjects. TREM-1 activation significantly (p,0.01) increased IL-1beta, IL-6 and IL-8 production by IBD biopsies cultured ex vivo. CONCLUSIONS. TREM-1 is overexpressed on macrophages infiltrating inflamed IBD mucosa, and its activation amplifies the production of pro-inflammatory cytokines. Further studies using chromatin immunoprecipitation assays are underway in order to establish whether TREM-1 overexpression in IBD may derive from epigenetic changes.