Iveta Grants

ADOA3R as a Therapeutic Target in Experimental Colitis: Proof by Validated High-density Oligonucleotide Microarray Analysis

&NA; Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)‐(3‐iodobenzyl)‐adenosine‐5‐N‐methyluronamide (IB‐MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine‐based drugs in experimental inflammatory bowel disease. High‐density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS‐treated (7 days), and oral IB‐MECA‐treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin‐trap monitoring of free radicals. Remarkable colitis‐induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis‐related dysregulation. Downregulation occurs in membrane transporter, mitogen‐activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox‐sensitive genes. Oral IB‐MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB‐MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB‐MECA blocked the colitis‐induced upregulation (≤20‐fold) of Bzrp, P2X1R, P2X4R, P2X7R, P2Y2R, P2Y6R, and A2aR/A2bR but not A1R or A3R genes or downregulated P2X2R, P2Y1R, and P2Y4R. Real‐time SYBR green polymerase chain reaction validated gene chip data for both induction of colitis and treatment with IB‐MECA for >90% of genes tested (33 of 37 genes). We conclude that our validated high‐density oligonucleotide microarray analysis is a powerful technique for molecular gene dysregulation studies to assess the beneficial effects of purine‐based or other drugs in experimental colitis. ADOA3R is new potential therapeutic target for inflammatory bowel disease.

Age-related alterations in the activation and expression of phosphotyrosine kinases and protein kinase C (PKC) among human B cells ☆

Age-related changes in the functional properties of human B cells have been reported by several groups, but little is known about the early biochemical events and signaling pathways that might be altered during aging. The present investigation examined whether differences in the activation of protein tyrosine kinases (PTK) and in the expression of protein kinase C (PKC) enzymatic activity could be identified in B cells from 16 elderly subjects (mean 77 years) compared to B cells from 15 young subjects (mean 33 years). B cells from young subjects stimulated with the surface immunoglobulin (sIg) crosslinkers anti-IgM or Staphylococcus aureus Cowan I (SAC) demonstrated rapid increases in PTK mediated de novo tyrosine phosphorylation of endogenous proteins. In comparison, stimulated B cells from elderly subjects were reduced 22-46% in tyrosine phosphorylations. Quantitation of the enzymatic levels and activation/translocation of PKC activity among resting and sIg stimulated B cells showed that B cells from approximately 50% of elderly subjects had significant reductions compared to B cells from young subjects. Further analyses of the expression of PTK and PKC enzymatic activity by stimulated B cells from elderly subjects demonstrated that aging was associated with greater heterogeneity in PKC expression and that defects in PKC enzymatic activity could coexist with relatively normal PTK activity. Thus, these data suggest that aging can alter the expression of PTK/PKC enzymatic activity in human B cells and that these age-related alterations might perturb the balance between PKC-dependent and -independent signaling pathways.

New bioinformatics approach to analyze gene expressions and signaling pathways reveals unique purine gene dysregulation profiles that distinguish between CD and UC

Background: Expression of purine genes is modulated by inflammation or experimental colitis and altered expression leads to disrupted gut function. We studied purine gene dysregulation profiles in inflammatory bowel disease (IBD) and determined whether they can distinguish between Crohns disease (CD) and ulcerative colitis (UC) using Pathway Analysis and a new Comparative Analysis of Gene Expression and Selection (CAGES) method. Methods: Raw datasets for 22 purine genes and 36 probe‐sets from National Center for Biotechnology Information (NCBI) GEO (Gene Expression Omnibus) (http://www.ncbi.nlm.nih.gov/projects/geo/) were analyzed by National Cancer Institute (NCI) Biological Resources Branch (BRB) array tools for random‐variance of multiple/36 t‐tests in colonic mucosal biopsies or peripheral blood mononuclear cells (PBMCs) of CD, UC or control subjects. Dysregulation occurs in 59% of purine genes in IBD including ADORA3, CD73, ADORA2A, ADORA2B, ADAR, AMPD2, AMPD3, DPP4, P2RY5, P2RY6, P2RY13, P2RY14, and P2RX5. Results: In CD biopsies, expression of ADORA3, AMPD3, P2RY13, and P2RY5 were negatively correlated with acute inflammatory score, Crohns Disease Activity Index (CDAI) or disease chronicity; P2RY14 was positively correlated in UC. In mucosal biopsies or PBMCs, CD and UC were distinguished by unique patterns of dysregulation (up‐ or downregulation) in purine genes. Purine gene dysregulation differs between PBMCs and biopsies and possibly between sexes for each disease. Ingenuity Pathway Analysis (IPA) revealed significant associations between alterations in the expression of CD73 (upregulation) or ADORA3 (downregulation) and inflammatory or purine genes (≤10% of 57 genes) as well as G‐protein coupled receptors, cAMP‐dependent, and inflammatory pathways; IPA distinguishes CD from UC. Conclusion: CAGES and Pathway Analysis provided novel evidence that UC and CD have distinct purine gene dysregulation signatures in association with inflammation, cAMP, or other signaling pathways. Disease‐specific purine gene signature profiles and pathway associations may be of therapeutic, diagnostic, and functional relevance.

Differential expression of the α- and β-isoforms of protein kinase C in peripheral blood T and B cells from young and elderly adults

Abstract The expression of α- and β-isoforms of protein kinase C (PKC) was analyzed in the peripheral blood T and B cells from 11 elderly and young humans. Immunoblot analysis with isoenzyme specific antibodies showed that T cells from five of 11 elderly subjects exhibited selective reductions in PKCα which was

Impact of disrupting adenosine A3 receptors (A3−/−AR) on colonic motility or progression of colitis in the mouse

Background: Pharmacological studies suggest that adenosine A3AR influences motility and colitis. Functional A3−/−AR knockout mice were used to prove whether A3AR activation is involved in modulating either motility or colitis. Methods: A3AR was probed by polymerase chain reaction (PCR) genotyping, Western blot, and immunochemistry. Motility was assessed in vivo by artificial bead‐expulsion, stool‐frequency, and FITC‐dextran transit. Colitis was induced with dextran sodium sulfate (DSS) in A3−/−AR or wildtype (WT) age‐ and sex‐matched controls. Progression of colitis was evaluated by histopathology, changes in myeloperoxidase (MPO), colon length, CD4+‐cells, weight‐loss, diarrhea, and the guaiac test. Results: Goat anti‐hu‐A3 antiserum identified a 66 kDa immunogenic band in colon. A3AR‐immunoreactivity is expressed in SYN+‐nerve varicosities, s‐100+‐glia, and crypt cells, but not 5‐HT+ (EC), CD4+ (T), tryptase+ (MC), or muscle cells. A3AR immunoreactivity in myenteric ganglia of distal colon ⟩ proximal colon by a ratio of 2:1. Intestinal transit and bead expulsion were accelerated in A3−/−AR mice compared to WT; stool retention was lower by 40%–60% and stool frequency by 67%. DSS downregulated A3AR in epithelia. DSS histopathology scores indicated less mucosal damage in A3−/−AR mice than WT. A3−/−AR phenotype protected against DSS‐induced weight loss, neutrophil (MPO), or CD4+‐T cell infiltration, colon shortening, change in splenic weight, diarrhea, or occult‐fecal blood. Conclusions: Functional disruption of A3AR in A3−/−AR mice alters intestinal motility. We postulate that ongoing release of adenosine and activation of presynaptic‐inhibitory A3AR can slow down transit and inhibit the defecation reflex. A3AR may be involved in gliotransmission. In separate studies, A3−/−AR protects against DSS colitis, consistent with a novel hypothesis that A3AR activation contributes to development of colitis. (Inflamm Bowel Dis 2010)

Purinergic Autocrine Regulation of Mechanosensitivity and Serotonin Release in a Human EC Model: ATP-gated P2X3 Channels in EC are Downregulated in Ulcerative Colitis

Background:Alterations in 5-hydroxytryptamine (HT) signaling in inflamed gut may contribute to pathogenesis of inflammatory bowel diseases. Adenosine 5′-triphosphate (ATP) regulates mucosal-mechanosensory reflexes and ATP receptors are sensitive to mucosal inflammation. Yet, it remains unknown whether ATP can modulate 5-HT signaling in enterochromaffin cells (EC). We tested the novel purinergic hypothesis that ATP is a critical autocrine regulator of EC mechanosensitivity and whether EC expression of ATP-gated P2X3-ion channels is altered in inflammatory bowel diseases. Methods:Laser confocal (fluo-4) Ca2+ imaging was performed in 1947 BON cells. Chemical stimulation or mechanical stimulation (MS) was used to study 5-HT or ATP release in human BON or surgical mucosal specimens, and purine receptors by reverse transcription-polymerase chain reaction, Western Blot, or P2X3-immunoreactivity in BON or 5-HT+ human EC (hEC) in 11 control and 10 severely inflamed ulcerative colitis (UC) cases. Results:ATP or MS triggered Ca2+-transients or 5-HT release in BON. ATP or adenosine diphosphate increased 5-HT release 5-fold. MS caused ATP release, detected after 5′ecto-ATPase inhibition by ARL67156. ARL67156 augmented and apyrase blocked Ca2+/5-HT mechanosensitive responses. 2-Methyl-thio-adenosine diphosphate 5′-monophosphate-evoked (P2Y1,12) or mechanically-evoked responses were blocked or augmented by a P2Y1,12 antagonist, MRS2179, in different cells or inhibited by U73122. A P2Y12 antagonist, 2MeSAMP, augmented responses. A P2X1,3 agonist, &agr;,&bgr;-MeATP, triggered Ca2+ responses, whereas a P2X1,2/3,3 antagonist, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP, blocked mechanical responses or cell-surface 5′ATP-TR labeling. In hEC, &agr;,&bgr;-MeATP stimulated 5-HT release. In UC, P2X3-immunoreactivity decreased from 15% to 0.2% of 5-HT+hECs. Human mucosa and BON expressed P2X1, P2X3, P2X4, P2X5, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12R-messenger RNA transcripts. Conclusions:ATP is a critical determinant of mechanosensation and 5-HT release via autocrine activation of slow P2Y1-phospholipase C/inositol-1,4,5-triphosphate-Ca2+ or inhibitory P2Y12-purinergic pathways, and fast ATP-gated P2X3-channels. UC downregulation of P2X3-channels (or A2B) is postulated to mediate abnormal 5-HT signaling.

Cyclic AMP modulation of human B cell proliferative responses: Role of cAMP-dependent protein kinases in enhancing B cell responses to phorbol diesters and lonomycin☆

The ability of cyclic AMP (cAMP) to modulate human B cell proliferative responses and the possible role of cAMP-dependent kinases (PKA) in cAMP modulation of proliferative responses were investigated. The addition of dibutyl cAMP (Bt2 cAMP) or the cAMP-elevating agent forskolin to B cells stimulated by crosslinking surface immunoglobulins (sIg) resulted in a concentration-dependent inhibition of proliferative responses. By contrast, Bt2 cAMP or forskolin enhanced the proliferative responses of B cells after direct stimulation by phorbol myristate acetate (PMA) and the calcium ionophore ionomycin. The inhibition and enhancement of B cell proliferative responses by Bt2 cAMP were observed at different incubation intervals and were not due to temporal shifts of optimal responses. Also, Bt2 cAMP caused only small changes in B cell RNA synthesis compared to modulation of proliferative responses. Exposure of B cells to Bt2 cAMP rapidly activated PKA. Blocking Bt2 cAMP activation of PKA with the kinase inhibitor HA1004 prevented Bt2 cAMP enhancement of B cell responses after direct stimulation by PMA and ionomycin. In reciprocal experiments, the kinase inhibitor H7 resulted in some inhibition of PKC activation but did not inhibit Bt2 cAMP activation of PKA or Bt2 cAMP enhancement of proliferative responses. Other experiments demonstrated that B cells treated with Bt2 cAMP had selective increases in the de novo phosphorylations of two endogenous substrates which reflected PKA activation. Furthermore, concentrations of HA1004 or H8 which inhibited Bt2 cAMP enhancement of proliferative responses also inhibited PKA phosphorylations of these substrates whereas H7 did not. Thus, elevations of cAMP can enhance or inhibit human B cell proliferative responses to different stimuli and the activation of PKA is important for cAMP enhancement of certain responses.

Molecular Signaling and Dysfunction of the Human Reactive Enteric Glial Cell Phenotype: Implications for GI Infection, IBD, POI, Neurological, Motility, and GI Disorders.

Background:Clinical observations or animal studies implicate enteric glial cells in motility disorders, irritable bowel syndrome, inflammatory bowel disease, gastrointestinal (GI) infections, postoperative ileus, and slow transit constipation. Mechanisms underlying glial responses to inflammation in human GI tract are not understood. Our goal was to identify the “reactive human enteric glial cell (rhEGC) phenotype” induced by inflammation, and probe its functional relevance. Methods:Human enteric glial cells in culture from 15 GI-surgical specimens were used to study gene expression, Ca2+, and purinergic signaling by Ca2+/fluo-4 imaging and mechanosensitivity. A nanostring panel of 107 genes was designed as a read out of inflammation, transcription, purinergic signaling, vesicular transport protein, channel, antioxidant, and other pathways. A 24-hour treatment with lipopolysaccharide (200 &mgr;g/mL) and interferon-&ggr; (10 &mgr;g/mL) was used to induce inflammation and study molecular signaling, flow-dependent Ca2+ responses from 3 mL/min to 10 mL/min, adenosine triphosphate (ATP) release, and ATP responses. Results:Treatment induced a “rhEGC phenotype” and caused up-regulation in messenger RNA transcripts of 58% of 107 genes analyzed. Regulated genes included inflammatory genes (54%/IP10; IFN-&ggr;; CxCl2; CCL3; CCL2; C3; s100B; IL-1&bgr;; IL-2R; TNF-&agr;; IL-4; IL-6; IL-8; IL-10; IL-12A; IL-17A; IL-22; and IL-33), purine-genes (52%/AdoR2A; AdoR2B; P2RY1; P2RY2; P2RY6; P2RX3; P2RX7; AMPD3; ENTPD2; ENTPD3; and NADSYN1), channels (40%/Panx1; CHRNA7; TRPV1; and TRPA1), vesicular transporters (SYT1, SYT2, SNAP25, and SYP), transcription factors (relA/relB, SOCS3, STAT3, GATA_3, and FOXP3), growth factors (IGFBP5 and GMCSF), antioxidant genes (SOD2 and HMOX1), and enzymes (NOS2; TPH2; and CASP3) (P < 0.0001). Treatment disrupted Ca2+ signaling, ATP, and mechanical/flow-dependent Ca2+ responses in human enteric glial cells. ATP release increased 5-fold and s100B decreased 33%. Conclusions:The “rhEGC phenotype” is identified by a complex cascade of pro-inflammatory pathways leading to alterations of important molecular and functional signaling pathways (Ca2+, purinergic, and mechanosensory) that could disrupt GI motility. Inflammation induced a “purinergic switch” from ATP to adenosine diphosphate/adenosine/uridine triphosphate signaling. Findings have implications for GI infection, inflammatory bowel disease, postoperative ileus, motility, and GI disorders.

Neuropharmacology of Purinergic Receptors in Human Submucous Plexus: Involvement of P2X1, P2X2, P2X3 Channels, P2Y and A3 Metabotropic Receptors in Neurotransmission

RATIONALE The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. EXPERIMENTAL APPROACH LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). RESULTS Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. CONCLUSIONS Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.

Expression of an oncofetal protein (OFP) in rat and human leukemia cells

A unique oncofetal protein (OFP) previously identified in rat fetal tissue and rat and human solid tumors, is now shown to be present in rat and human leukemia cells by use of a monoclonal antibody-based assay. Using a highly specific anti-rat OFP monoclonal antibody OFP has been unquivocally immunolocalized to the cytoplasm of the rat leukemia cells. The factor is rapidly released to the circulation as 50 and 55 kD species which share the immunological determinants. When leukemia cells are transplanted to normal rats, OFP increases in the circulation in a biphasic manner which may be due to immune clearance since circulating anti-OFP antibodies have been demonstrated. Induction of differentiation in the human HL-60 leukemia cell line by 13-cis-retinoic acid caused a down regulation of OFP synthesis, both intra- and extra-cellular levels dropping to essentially zero. Induction of differentiation with dibutyryl cyclic AMP caused a cessation of secretion of OFP, with a marked increase in its intracellular concentration, a condition resembling the retention in fetal cells. Leukemia cells add to a growing list of tumors previously shown to produce OFP, suggesting that OFP is intimately involved in some facet of tumorigenesis.