Dissertation Defense Announcement
To:  The George Mason University Community

Candidate: Afshin Sohrabi
Program: PhD in Biosciences
Date:   Tuesday April 15, 2014
Time:   12:30 p.m.
Place:  George Mason University
Prince William Campus
             Occoquan Bldg., Room 110-H
Title: "Arthritic Cell-Based Assay Model for Measuring Inflammatory Response to Therapeutic Biological Agents"

Thesis Director: Dr. Ancha Baranova

Thesis Committee:  Dr. Alan Christensen, Dr. Patrick Gillevet, Dr. Kurt Langenbach
A copy of the dissertation will be available in the Mercer Library.  All are invited to attend the defense.

Rheumatoid arthritis (RA) is the commonest inflammatory joint disease with considerable morbidity and mortality. Conventional disease-modifying anti-rheumatic drugs like methotrexate and NSAIDs form the cornerstone of therapy. However, they have several limitations in terms of slow onset of action, adverse effects and modest remission and retention rates. Several cytokines are involved in the pathogenesis of RA. Biological agents that specifically inhibit the effects of tumor necrosis factor-α (TNF-α) or MMP-1 represent a major advancement in the treatment of RA. By targeting molecules that are directly involved in the pathogenesis of RA, these therapies are proving to be efficacious, highly specific and better tolerated than standard therapies. The use of these agents needs to be monitored carefully for possible side-effects, including the development of infections. Additional anti-cytokine agents for the treatment of RA are under further development. Thus finding a biological therapeutic drug that can down-regulate the major proinflammatory mediators secreted by synoviocytes that are involved in cartilage and bone erosion could protect against RA pathogenesis. Using an in vitro model, we aim to explore the anti-inflammatory properties of a naturally existing recombinant anti-alpha trypsin biological protein molecule termed rAAP on proinflammatory mediators such as TNF-α and MMP-1 levels via NF-κB/IkB-α signaling pathway, which are involved in cartilage degradation and bone erosion. A further goal of this study is to test if rAAP has the ability to act as an anti-inflammatory agent. First, we assessed the impact of the purified rAAP on TNF-α, IL-1β, IL-6, MMP1, PGE2, GM-CSG, and IL-8 expression in human synoviocytes stimulated with LPS for 1 hour first and treatment with rAAP for 17 hours. The nucleic acid sequence of the longest open reading frame of rat Serpina1A region was cloned it into a SUMO vector for protein expression. The rAAP was purified for subsequent testing. Using Q-PCR, we also discovered that rAAP inhibited the expression of TNF-α, Il-1β, IL-6, GM-CSF, MMP1, and COX-2. . Crystal Violet Cytotoxicity assay showed that rAAP protein molecule was not cytotoxic at 10µM rAAP with greater than 90% synoviocytes cell viability. rAAP affected NF-kB activation which plays a central role in the inflammatory responses to LPS activation. It occurs via phosphorylation of the inhibitory IkB-α protein at Ser32 and Ser36 (by a cellular kinase complex known as IKK) followed by proteasome-mediated degradation. Treatment after cell activation showed suppression of TNF-α levels to ~30% of untreated controls-suggesting that rAAP has TNF-α blocking properties. This information implies that rAAP can act as a prophylactic and protect against RA by modulating known activated RA inflammatory cascade. To help characterize the role of rAAP in inhibiting the production of proinflammatory mediators, we developed an in vitro model using human synoviocytes. In this study, LPS activated TNF-α, IL-1β, Il-6, and COX-2 expression within 30 minutes and induced the NF-kB/IkB-a signaling pathway within 5 minutes in cultured human synoviocytes.  LPS activation induced IkB-a degradation by 5 minutes and levels declined through 120 minutes.  These results suggest that LPS activated synoviocytes may use the same NF-kB/IkB-a signaling pathway as other well-known mediators.  Since the NF-kB/IkB-a signaling pathway is involved in cytokine production, it is likely that by blocking this pathway cytokine production would be inhibited.  Anti-inflammatory agents such as Aspirin have been shown to suppresses NF-kB accumulation and IkB-a degradation.  rAAP was potent enough to inhibit TNF-α, MMP-1 and prostaglandin E2 secretion by 17 hours after 1 hour of LPS activation. Induction of TNF-α, COX-2, and NF-kB/IkB-a signaling pathway were suppressed by rAAP.

Our findings show that LPS activation of cultured human synoviocytes induces an inflammatory response and that recombinant biological, rAAP, suppress this response.  The mechanism of action appears to be through the modulation of proinflammatory mediators such as TNF- α, IL-1β, LI-6, COX-2, MMP1, prostaglandin E2, GM-CSF, and IL-8. These observations suggest that the inhibition of LPS activated TNF-α cascade by rAAP and may help reduce the inflammation responsible for RA.  Our studies indicate that the LPS activated human synoviocyte model may help define the mechanisms involved in the inflammatory cascade observed in RA.