Education & Certifications
Bachelor of Arts, Washington University, Biology (2006)
New Haven, CT
We previously demonstrated that carboxypeptidase B (CPB) protects against joint erosion in rheumatoid arthritis by inactivating complement component C5a. We also found that levels of CPB are abnormally high in the synovial fluid of individuals with another joint disease, osteoarthritis (OA). We undertook this study to investigate whether CPB plays a role in the pathogenesis of OA.We compared the development of OA in CPB-deficient (Cpb2(-/-) ) mice and wild-type mice by subjecting them to medial meniscectomy and histologically assessing cartilage damage, osteophyte formation, and synovitis in the stifle joints 4 months later. We measured levels of proCPB, proinflammatory cytokines, and complement components in synovial fluid samples from patients with symptomatic and radiographic knee OA. Finally, we used enzyme-linked immunosorbent assay, flow cytometry, and hemolytic assays to assess the effect of CPB on formation of membrane attack complex (MAC)-a complement effector critical to OA pathogenesis.Cpb2(-/-) mice developed dramatically greater cartilage damage than did wild-type mice (P < 0.01) and had a greater number of osteophytes (P < 0.05) and a greater degree of synovitis (P < 0.05). In synovial fluid samples from OA patients, high levels of proCPB were associated with high levels of proinflammatory cytokines and complement components, and levels of proCPB correlated positively with those of MAC. In in vitro complement activation assays, activated CPB suppressed the formation of MAC as well as MAC-induced hemolysis.Our data suggest that CPB protects against inflammatory destruction of the joints in OA, at least in part by inhibiting complement activation.
View details for DOI 10.1002/art.38213
View details for PubMedID 24449579
BACKGROUND/PURPOSE: Patients with rheumatoid arthritis (RA) are at increased risk for cardiovascular disease, an observation not explained by traditional cardiac risk factors and generally limited to those with RA-associated autoantibodies such as rheumatoid factor and anti-citrullinated protein antibodies (ACPA). We hypothesized that citrullinated proteins within the atherosclerotic plaque can be targeted by ACPA, forming stimulatory immune complexes which propagate the progression of atherosclerosis. METHODS AND RESULTS: Protein lysates prepared from atherosclerotic segments of human aorta were investigated for the presence of citrulline-modified proteins and specifically citrullinated fibrinogen (cFb) by immunoprecipitation and/or immunoblotting followed by mass spectrometry. Immunohistochemistry was performed in coronary artery plaques for the presence of citrullinated proteins and the PAD4 enzyme. Serum levels of anti-cyclic citrullinated peptide (CCP), anti-citrullinated vimentin (cVim), and anti-cFb antibodies were measured in 134 women with seropositive RA previously characterized for the presence of subclinical atherosclerosis by electron beam CT scan (EBCT). Western analysis of atherosclerotic plaque lysates demonstrated several citrullinated proteins and the presence of cFb was confirmed by immunoprecipitation, and mass spectrometry. Immunohistochemistry demonstrated co-localization of citrullinated proteins and the PAD4 enzyme within the coronary artery plaque. In age-adjusted regression models, antibodies targeting cFb and cit-vimentin, but not CCP2, were associated with an increased aortic plaque burden. CONCLUSION: Citrullinated proteins are prevalent within the atherosclerotic plaque, and certain ACPAs are associated with atherosclerotic burden. These observations suggest that targeting of citrullinated epitopes, specifically cFb, within the atherosclerotic plaque could provide a mechanism for accelerated atherosclerosis observed in patients with RA. © 2013 American College of Rheumatology.
View details for DOI 10.1002/art.37961
View details for Web of Science ID 000322155000007
Osteoarthritis (OA) has traditionally been classified as a noninflammatory arthritis; however, the dichotomy between inflammatory and degenerative arthritis is becoming less clear with the recognition of a plethora of ongoing immune processes within the OA joint and synovium. Synovitis is defined as inflammation of the synovial membrane and is characteristic of classical inflammatory arthritidies. Increasingly recognized is the presence of synovitis in a significant proportion of patients with primary OA, and based on this observation, further studies have gone on to implicate joint inflammation and synovitis in the pathogenesis of OA. However, clinical OA is not one disease but a final common pathway secondary to many predisposing factors, most notably age, joint trauma, altered biomechanics, and obesity. How such biochemical and mechanical processes contribute to the progressive joint failure characteristic of OA is tightly linked to the interplay of joint damage, the immune response to perceived damage, and the subsequent state of chronic inflammation resulting in propagation and progression toward the phenotype recognized as clinical OA. This review will discuss a wide range of evolving data leading to our current hypotheses regarding the role of immune activation and inflammation in OA onset and progression. Although OA can affect any joint, most commonly the knee, hip, spine, and hands, this review will focus primarily on OA of the knee as this is the joint most well characterized by epidemiologic, imaging, and translational studies investigating the association of inflammation with OA.
View details for DOI 10.1177/1759720X12467868
View details for PubMedID 23641259
Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of 'wear and tear'. Although low-grade inflammation is detected in osteoarthritis, its role is unclear. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in complement component 5 (C5), C6 or the complement regulatory protein CD59a, we show that complement, specifically, the membrane attack complex (MAC)-mediated arm of complement, is crucial to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints from C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Further, MAC colocalized with matrix metalloprotease 13 (MMP13) and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints has a key role in the pathogenesis of osteoarthritis.
View details for DOI 10.1038/nm.2543
View details for Web of Science ID 000297978000042
View details for PubMedID 22057346
Widely accessible small animal models suitable for the study of hepatitis C virus (HCV) in vivo are lacking, primarily because rodent hepatocytes cannot be productively infected and because human hepatocytes are not easily engrafted in immunodeficient mice.We report here on a novel approach for human hepatocyte engraftment that involves subcutaneous implantation of primary human fetal hepatoblasts (HFH) within a vascularized rat collagen type I/human fibronectin (rCI/hFN) gel containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) in severe combined immunodeficient X beige (SCID/bg) mice. Maturing hepatic epithelial cells in HFH/Bcl-2-HUVEC co-implants displayed endocytotic activity at the basolateral surface, canalicular microvilli and apical tight junctions between adjacent cells assessed by transmission electron microscopy. Some primary HFH, but not Huh-7.5 hepatoma cells, appeared to differentiate towards a cholangiocyte lineage within the gels, based on histological appearance and cytokeratin 7 (CK7) mRNA and protein expression. Levels of human albumin and hepatic nuclear factor 4alpha (HNF4alpha) mRNA expression in gel implants and plasma human albumin levels in mice engrafted with HFH and Bcl-2-HUVEC were somewhat enhanced by including murine liver-like basement membrane (mLBM) components and/or hepatocyte growth factor (HGF)-HUVEC within the gel matrix. Following ex vivo viral adsorption, both HFH/Bcl-2-HUVEC and Huh-7.5/Bcl-2-HUVEC co-implants sustained HCV Jc1 infection for at least 2 weeks in vivo, based on qRT-PCR and immunoelectron microscopic (IEM) analyses of gel tissue.The system described here thus provides the basis for a simple and robust small animal model of HFH engraftment that is applicable to the study of HCV infections in vivo.
View details for DOI 10.1371/journal.pone.0009987
View details for PubMedID 20376322
Fatty acid-induced triacylglycerol synthesis produces triacylglycerol droplets with a protein coat that includes perilipin 3/TIP47 and perilipin 4/S3-12. This study addresses the following two questions. Where do lipid droplets emerge, and how are their coat proteins recruited? We show that perilipin 3- and perilipin 4-coated lipid droplets emerge along the endoplasmic reticulum (ER). Blocking membrane trafficking with AlF(4)(-) during fatty acid-induced triacylglycerol synthesis drove perilipin 3 to the tubular ER. Forskolin, which like AlF(4)(-) activates adenylate cyclase, did not redistribute perilipin 3, but when added together with AlF(4)(-) perilipin 3 was recruited to lipid droplets rather than the ER. Thus inhibiting trafficking with AlF(4)(-) redistributed perilipin 3 differently under conditions of triacylglycerol synthesis (fatty acid addition) versus hydrolysis (forskolin) suggesting a shared acylglycerol-mediated mechanism. We tested whether diacylglycerol (DG), the immediate precursor of triacylglycerol and its first hydrolytic product, affects the distribution of perilipin 3. Stabilizing DG with the DG lipase inhibitor RHC80267 enhanced the perilipin 3 recruited to lipid droplets and raised DG levels in this fraction. Treating cells with a membrane-permeable DG recruited perilipin 3 to the ER. Stabilizing DG, by blocking its hydrolysis with RHC80267 or its acylation with triacsin C, enhanced recruitment of perilipin 3 to the ER. Expressing the ER enzyme DGAT1, which removes DG by converting it to triacylglycerol, attenuated perilipin 3 DG-induced ER recruitment. Membrane-permeable DG also drove perilipin 4 and 5 onto the ER. Together the data suggest that these lipid droplet proteins are recruited to DG-enriched membranes thereby linking lipid coat proteins to the metabolic state of the cell.
View details for DOI 10.1074/jbc.M109.013995
View details for Web of Science ID 000271378400023
View details for PubMedID 19748893
Immunodeficient mice bearing components of a human immune system present a novel approach for studying human immune responses. We investigated the number, phenotype, developmental kinetics, and function of developing human immune cells following transfer of CD34(+) hematopoietic stem cell (HSC) preparations originating from second trimester human fetal liver (HFL), umbilical cord blood (UCB), or granulocyte colony-stimulating factor-mobilized adult blood (G-CSF-AB) delivered via intrahepatic injection into sublethally irradiated neonatal NOD-scid/gammac(-/-), Balb/c-Rag1(-/-)gammac(-/-), and C.B-17-scid/bg mice. HFL and UCB HSC provided the greatest number and breadth of developing cells. NOD-scid/gammac(-/-) and Balb/c-Rag1(-/-)gammac(-/-) harbored human B and dendritic cells as well as human platelets in peripheral blood, whereas NOD-scid/gammac(-/-) mice harbored higher levels of human T cells. NOD-scid/gammac(-/-) mice engrafted with HFL CD34(+) HSC demonstrated human immunological competence evidenced by white pulp expansion and increases in total human immunoglobulin following immunization with T-dependent antigens and delayed-type hypersensitivity-infiltrating leukocytes in response to antigenic challenge. In conclusion, we describe an encouraging base system for studying human hematopoietic lineage development and function utilizing human HFL or UCB HSC-engrafted NOD-scid/gammac(-/-) mice that is well suited for future studies toward the development of a fully competent humanized mouse model.
View details for DOI 10.1016/j.humimm.2009.06.005
View details for Web of Science ID 000270577900005
View details for PubMedID 19524633
Multilayer nanofilms, formed by the layer-by-layer (LbL) adsorption of positively and negatively charged polyelectrolytes, are promising substrates for tissue engineering. We investigate here the attachment and function of hepatic cells on multilayer films in terms of film composition, terminal layer, rigidity, charge, and presence of biofunctional species. Human hepatocellular carcinoma (HepG2) cells, adult rat hepatocytes (ARH), and human fetal hepatoblasts (HFHb) are studied on films composed of the polysaccharides chitosan (CHI) and alginate (ALG), the polypeptides poly(l-lysine) (PLL) and poly(l-glutamic acid) (PGA), and the synthetic polymers poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS). The influence of chemical cross-linking following LbL assembly is also investigated. We find HepG2 to reach confluence after 7 days of culture on only 2 of 18 candidate multilayer systems: (PAH-PSS)(n) (i.e. nPAH-PSS bilayers) and cross-linked (PLL-ALG)(n)-PLL. Cross-linked PLL-ALG and PLL-PGA films support attachment and function of ARH, independently of the terminal layer, provided collagen is adsorbed to the top of the film. (PAH-PSS)(n), cross-linked (PLL-ALG)(n), and cross-linked (PLL-PGA)(n)-PLL films all support attachment, layer confluence, and function of HFHb, with the latter film promoting the greatest level of function at 8 days. Overall, film composition, terminal layer, and rigidity are key variables in promoting attachment and function of hepatic cells, while film charge and biofunctionality are somewhat less important. These studies reveal optimal candidate multilayer biomaterials for human liver tissue engineering applications.
View details for DOI 10.1016/j.biomaterials.2008.06.027
View details for Web of Science ID 000260025100007
View details for PubMedID 18653230