Mitali Purohit *, Carol Artlett *, Sihem Sassi Gaha *, James D. Thacker 1* , Richard F. Rest* - PowerPoint PPT Presentation

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Mitali Purohit *, Carol Artlett *, Sihem Sassi Gaha *, James D. Thacker 1* , Richard F. Rest* PowerPoint Presentation
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Mitali Purohit *, Carol Artlett *, Sihem Sassi Gaha *, James D. Thacker 1* , Richard F. Rest*

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Mitali Purohit *, Carol Artlett *, Sihem Sassi Gaha *, James D. Thacker 1* , Richard F. Rest*

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  1. PDAG IS HOMOLOGOUS TO TRPC1 ABSTRACT To control disease caused by emerging antibiotic-resistant pathogens, there is a constant quest to develop novel therapeutic agents and adjuncts to antimicrobials. We have pursued a broad-spectrum strategy that involves boosting the innate immune system to combat a broad range of infectious agents. In the search of such immunomodulatory molecules, we screened the low-molecular weight inflammatory proteome of mammalian plasma and serum and identified a unique lipopeptide – 1-peptidyl-2,3-diacylglyceride (PDAG). PDAG stimulates the innate immune response resulting in activation of tissue resident immune cells, recruitment of phagocytic cells, and clearance of pathogens. Serum-derived PDAG stimulates significant release from whole human blood of proinflammatory cytokines IL-6, IL-8, MCP-1, MIP-1α and MIP-1β. The cytokine profile induced by PDAG differs from that induced by LPS, indicating a lack of endotoxin contamination of the natural product. In addition, PDAG stimulates isolated human macrophages and THP-1 macrophage-like cells to release IL-6 and IL-8 in a dose and time dependent manner. Interestingly, PDAG also stimulates cultured human fibroblasts to secrete IL-6. We determined whether PDAG activity was due to the entire molecule, PDAG peptide or DAG. Neither DAG nor synthetic PDAG peptide alone induce cytokine release from whole blood. PDAG is an exciting new and unique immunoregulatory molecule that has a plethora of possible clinical uses. [This work is funded by Drexel University College of Medicine.] The PDAG peptide sequence is homologous with an extra-cellular loop of TRPC-1, a transmembrane Ca2+ channel protein. Figure 1: Putative domain structure and topology of TRPC1. Percent identity between TRPC1 and at least one other member of the TRP family is indicated. The red circle indicates the PDAG peptide sequence. Wes, et al. Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9652-9656 The evolution and rapid spread of resistant microorganisms are significant problems in nosocomial infections and are of increasing importance in community acquired infections. There is an urgent need for alternatives to antimicrobials that can either have direct microbicidal activity or that can exert their effects by stimulating the innate immune system. Immunomodulatory peptides, such as the defensins, stimulate a number of effects on innate immune cells, including monocytes, macrophages, and neutrophils to act as chemoattractants, promote histamine release from mast cells, and enhance phagocytosis at the site of infection. In our investigation of the low-molecular weight inflammatory proteome in mammalian plasma and serum we identified a new immunoactive lipopeptide with the general structure : wherein X1 is a linear peptide and X2 and X3 are arachidonic and stearic acid respectively. The peptide is covalently linked to the diacylglycerol by an ester bond at the C-terminus of the peptide. We hypothesize that 1-peptidyl-2,3-diacylglycerol, “PDAG”, activates the innate immune cascade, activates tissue resident immune cells at the site of injury, recruits professional phagocytic cells to local tissue, clears pathogens, and abates disease progression. PDAG Controlled induction of cytokines and chemokines Delayed cell migration and recruitment Modified from NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 4 APRIL 2007 Characterization of a Unique, Naturally-occurring Immunomodulatory Lipopeptide: 1-peptidyl-2,3-diacylglyceride (PDAG) Mitali Purohit*, Carol Artlett*, SihemSassiGaha*, James D. Thacker1*, Richard F. Rest* *Department of Microbiology and Immunology, Drexel University College of Medicine 1TherimuneX Pharmaceuticals, Inc., Doylestown, PA PDAG protects in a mouse peritonitis model Diverse cells and mediators are involved in innate immunity. We wanted to investigate the effects of PDAG on fibroblasts and keratinocytes which are excellent sources of cytokines and chemokines during inflammatory response to infections. PDAG Untreated Figure 6: Swiss Webster mice were challenged with Salmonella typhimurium (5x103 cfu/mouse) by intraperitoneal injection. Mice were treated with natural PDAG in normal saline (1.5 mg /mouse; n=15) or saline alone (n=15) by subcutaneous injection a day before bacterial challenge. Mortality was monitored daily for ten days. By day 10, all mice in the control group were dead where as there was 20% death in the PDAG treated group. Administration of PDAG did not cause a cytokine storm. Lymphopenia, neutrophilia, and monocytosis were observed in the infected control mice, whereas circulating neutrophils and monocytes in PDAG treated mice were at or near normal levels. The bacterial load in the spleens of the mice was 50% less in PDAG treated mice on day 1 (data not shown). PDAG induces cytokine release from human fibroblasts PDAG INDUCES CYTOKINE & CHEMOKINE RELEASE FROM HUMAN BLOOD INTRODUCTION SUMMARY Figure 4: Normal human fibroblasts were cultured in DMEM with 10% FBS at 37oC, with or without natural PDAG. RNA was purified from treated and control fibroblasts, and assayed for the indicated cytokines or signaling molecules by quantitative RT PCR. Values are normalized to -actin expression. Figure 2: Fresh heparinized human blood was diluted five-fold with RPMI 1640 medium and incubated with dilutions of purified natural PDAG for 13 or 24 hrs. After incubation, samples were centrifuged and plasma was assayed using the Excelarray Human Inflammation kit, or Chemotaxis kit (Thermo Fisher Scientific). PDAG induced a dose and time dependent release of cytokines. FUTURE STUDIES • Determine whether PDAG stimulates neutrophils, monocytes or macrophages to express antibacterial activities in vitro. • Determine the cellular origin of PDAG and conditions for its production or release. • Determine what pathways are activated by PDAG in non-immune cells. • Determine if PDAG induces an antimicrobial effect in vivo. PDAG induces IL-6 and IL-8 release from macrophages ISOLATION OF NATURAL PDAG PDAG induces cytokine release from human keratinocytes Dialyze Caprine serum Reduce dialysate volume by lyophilization Extract lyophilized fraction with chloroform/ methanol and remove solids Prepare purified PDAG by preparative reversed phase HPLC. PDAG samples are >99% chromatographically pure. Figure 3: PMA-differentiated THP-1 cells in RPMI, 10% HI FBS and 50 µM beta-mercaptoethanol were treated with 1:100 natural PDAG for 24 hrs. Supernatants were assayed using the Excelarray Human Inflammation kit. LPS is used as a positive control. Similar results were seen with human monocyte derived macrophages. Figure 5: PDAG treatment of keratinocytes induces increased expression of IL-6 and IL-8 as determined by RT-PCR. Values are normalized to β-actin. Determine PDAG composition by mass spectrometry and sequence the PDAG peptide FUNDING: This research has been funded by Drexel University College of Medicine in a cooperative agreement with TherimuneX Pharmaceuticals, Inc.