Margaret Fleming - Academia.edu (original) (raw)

Papers by Margaret Fleming

In asthma, mast cells infiltrate the airway smooth muscle cell layer and secrete proinflammatory ... more In asthma, mast cells infiltrate the airway smooth muscle cell layer and secrete proinflammatory and profibrotic agents that contribute to airway remodeling. To study the effects of mast cell activation on smooth muscle cell-dependent matrix contraction, we developed coculture systems of human airway smooth muscle cells (HASM) with primary human mast cells derived from circulating progenitors or with the HMC-1 human mast cell line. Activation of primary human mast cells by IgE receptor cross-linking or activation of HMC-1 cells with C5a stimulated contraction of HASM-embedded collagen gels. Contractile activity could be transferred with conditioned medium from activated mast cells, implicating involvement of soluble factors. Cytokines and proteases are among the agents released by activated mast cells that may promote a contractile response. Both IL-13 and IL-6 enhanced contraction in this model and the activity of IL-13 was ablated under conditions leading to expression of the inhibitory receptor IL-13R␣2 on HASM. In addition to cytokines, matrix metalloproteinases (MMPs), and serine proteases induced matrix contraction. Inhibitor studies suggested that, although IL-13 could contribute to contraction driven by mast cell activation, MMPs were critical mediators of the response. Both MMP-1 and MMP-2 were strongly expressed in this system. Serine proteases also contributed to contraction induced by mast cell-activating agents and IL-13, most likely by mediating the proteolytic activation of MMPs. Hypercontractility is a hallmark of smooth muscle cells in the asthmatic lung. Our findings define novel mechanisms whereby mast cells may modulate HASM-driven contractile responses.

Arthritis & Rheumatology

OBJECTIVE Use a highly potent and selective small molecule inhibitor of interleukin-1 associated ... more OBJECTIVE Use a highly potent and selective small molecule inhibitor of interleukin-1 associated kinase (IRAK) 4, PF-06650833, to demonstrate its role in autoimmune pathophysiology in vitro, in vivo and in the clinic. METHODS Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through stimulation of primary human macrophages (MΦ) with anti-citrullinated protein antibody (ACPA) immune complexes (IC), RA fibroblast-like synoviocyte (-FLS) cultures stimulated with toll-like receptor (TLR) ligands, as well as additional human primary cell co-cultures. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells (DC), B cells and PBMC stimulated with TLR ligands and SLE patient IC. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNASeq data generated with whole blood samples from a Phase 1 multiple ascending dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected rats from CIA. In a phase 1 clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacology pertinent to SLE by reducing whole blood interferon (IFN) gene signature expression in healthy volunteers. CONCLUSION These data demonstrate that inhibition of IRAK4 kinase activity can reduce markers of inflammation in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.

Chronic Obstructive Pulmonary Diseases: Journal of the COPD Foundation

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European journal of medicinal chemistry, Jan 10, 2018

Many diseases are believed to be driven by pathological levels of reactive oxygen species (ROS) a... more Many diseases are believed to be driven by pathological levels of reactive oxygen species (ROS) and oxidative stress has long been recognized as a driver for inflammatory disorders. Apoptosis signal-regulating kinase 1 (ASK1) has been reported to be activated by intracellular ROS and its inhibition leads to a down regulation of p38-and JNK-dependent signaling. Consequently, ASK1 inhibitors may have the potential to treat clinically important inflammatory pathologies including renal, pulmonary and liver diseases. Analysis of the ASK1 ATP-binding site suggested that Gln756, an amino acid that rarely occurs at the GK+2 position, offered opportunities for achieving kinase selectivity for ASK1 which was applied to the design of a parallel medicinal chemistry library that afforded inhibitors of ASK1 with nanomolar potency and excellent kinome selectivity. A focused optimization strategy utilizing structure-based design resulted in the identification of ASK1 inhibitors with low nanomolar p...

Journal of Biological Chemistry

Edited by Luke O'Neill W. K. and R. H. were employees of Nodality, contracted by Pfizer to perfor... more Edited by Luke O'Neill W. K. and R. H. were employees of Nodality, contracted by Pfizer to perform SCNP studies with the IRAK4 inhibitor. All other authors were Pfizer employees. This article contains supplemental Figs. 1 and 2 and supplemental Table 1. Data are available in the Gene Expression Omnibus (GEO) database under accession no. GSE86234.

PloS one, 2016

The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are ... more The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tissue injury to mediate local tissue responses including inflammation and tissue remodeling. We found that in various models of kidney disease, Fn14 expression (mRNA and protein) is upregulated in the kidney. These models include: lupus nephritis mouse models (Nephrotoxic serum Transfer Nephritis and MRL.Faslpr/lpr), acute kidney injury models (Ischemia reperfusion injury and Folic acid injury), and a ZSF-1 diabetic nephropathy rat model. Fn14 expression levels correlate with disease severity as measured by disease histology. We have also shown for the first time the detection of soluble Fn14 (sFn14) in the urine and serum of mice. Importantly, we found the sFn14 levels are markedly increased in the diseased mice and are correlated with disease biomarkers including proteinuria and MCP-1. We have also detected sFn14 in human plasma and urine. Moreover, sFn14 levels, in uri...

D31. ANIMAL MODELS OF AIRWAY INFLAMMATION, 2010

/ Thematic Poster Session / Wednesday, May 19/8:15 AM-4:00 PM / Area D31 ANIMAL MODELS OF AIRWAY ... more / Thematic Poster Session / Wednesday, May 19/8:15 AM-4:00 PM / Area D31 ANIMAL MODELS OF AIRWAY INFLAMMATION ... E, Hall G (First Level), Morial Convention Center ... Implications For Acidic Mammalian Chitinase In Mouse Allergic Airway Disease

Protein Science, 2009

Acidic mammalian chitinase (AMCase) is a mammalian chitinase that has been implicated in allergic... more Acidic mammalian chitinase (AMCase) is a mammalian chitinase that has been implicated in allergic asthma. One of only two active mammalian chinases, AMCase, is distinguished from other chitinases by several unique features. Here, we present the novel structure of the AMCase catalytic domain, both in the apo form and in complex with the inhibitor methylallosamidin, determined to high resolution by X-ray crystallography. These results provide a structural basis for understanding some of the unique characteristics of this enzyme, including the low pH optimum and the preference for the b-anomer of the substrate. A triad of polar residues in the second-shell is found to modulate the highly conserved chitinase active site. As a novel target for asthma therapy, structural details of AMCase activity will help guide the future design of specific and potent AMCase inhibitors.

Journal of Interferon & Cytokine Research, 1995

Although the mouse IL-2 receptor (IL-2R) beta and gamma c subunits have been identified by molecu... more Although the mouse IL-2 receptor (IL-2R) beta and gamma c subunits have been identified by molecular cloning, the biochemical identity of these subunits has not yet been established. In the present study, the mouse IL-2R was biochemically characterized from cell lines expressing normal and aberrant IL-2R. Using novel monoclonal antibodies specific for the beta or gamma c subunits, we established that the M(r) of the beta chain is 90,000-100,000 and that of the gamma c subunit is 75,000-80,000. Analysis of transfected EL4 cells that expressed alpha, gamma c, and truncated beta subunits or mutant EL4 cells, which selectively lacked cell surface gamma c, revealed that no other material migrated to a position on SDS-PAGE characteristic of IL-2/IL-2R beta and IL-2/IL-2R gamma c cross-linked complexes, respectively. Thus, the beta and gamma c subunits appear to be the sole IL-2R constituents of these IL-2 cross-linked complexes. The IL-2/IL-2R gamma c, but not the IL-2/IL-2R beta, complex exhibited enhanced mobility after SDS-polyacrylamide gel electrophoresis under nonreducing conditions, suggesting a more compact structure for gamma c as a result of intrachain disulfide bonds. The primary posttranslational modification of the mouse beta and gamma c subunits is N-linked glycosylation. These biochemical studies reconcile past uncertainties concerning the subunit composition of the mouse IL-2R and are consistent with a model of the IL-2R containing only three subunits.

Journal of Inflammation, 2013

The Journal of Immunology, 2009

Journal of Allergy and Clinical Immunology, 2010

Background: Matrix metalloproteinase (MMP)-12-mediated pathologic degradation of the extracellula... more Background: Matrix metalloproteinase (MMP)-12-mediated pathologic degradation of the extracellular matrix and the subsequent repair cycles influence the airway changes in patients with asthma and chronic obstructive pulmonary disease (COPD). The common serine variant at codon 357 of the MMP12 gene (rs652438) is associated with clinical manifestations consistent with more aggressive matrix degradation in other tissues. Objective: We sought to explore the hypothesis that MMP12 represents a novel therapeutic target in asthma. Methods: The role of the rs652438 variant on clinical phenotype was explored in young asthmatic patients and patients with COPD. Candidate MMP-12 inhibitors were identified on the basis of potency and selectivity against a panel of other MMPs. The role of MMP-12-specific inhibition was tested in vitro, as well as in animal models of allergic airway inflammation. Results: The odds ratio for having greater asthma severity was 2.00 (95% CI, 1.24-3.24; P 5 .004) when comparing asthmatic patients with at least 1 copy of the serine variant with those with none. The carrier frequency for the variant increased in line with asthma treatment step (P 5 .000). The presence of the variant nearly doubled the odds in favor of asthmatic exacerbations (odds ratio, 1.90; 95% CI, 1.19-3.04; P 5 .008) over the previous 6 months. The serine variant was also associated with increased disease severity in patients with COPD (P 5 .016). Prior administration of an MMP-12-specific inhibitor attenuated the early airway response and completely blocked the late airway response with subsequent Ascaris suum challenge in sheep. Conclusion: Studies on human participants with asthma and COPD show that the risk MMP12 gene variant is associated with disease severity. In allergen-sensitized sheep pharmacologic inhibition of MMP12 downregulates both early and late airway responses in response to allergic stimuli.

American Journal of …, 2010

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1993

Protein …, 2009

American Journal of Respiratory Cell and Molecular Biology, 2012

The expression of acidic mammalian chitinase (AMCase) is associated with Th2-driven respiratory d... more The expression of acidic mammalian chitinase (AMCase) is associated with Th2-driven respiratory disorders. To investigate the potentially pathological role of AMCase in allergic airway disease (AAD), we sensitized and challenged mice with ovalbumin or a combination of house dust mite (HDM) plus cockroach allergen. These mice were treated or not treated with small molecule inhibitors of AMCase, which significantly reduced allergen-induced chitinolytic activity in the airways, but exerted no apparent effect on pulmonary inflammation per se. Transgenic and AMCase-deficient mice were also submitted to protocols of allergen sensitization and challenge, yet we found little or no difference in the pattern of AAD between mutant mice and wild-type (WT) control mice. In a separate model, where mice were challenged only with intratracheal instillations of HDM without adjuvant, total bronchoalveolar lavage (BAL) cellularity, inflammatory infiltrates in lung tissues, and lung mechanics remained comparable between AMCase-deficient mice and WT control mice. However BAL neutrophil and lymphocyte counts were significantly increased in AMCase-deficient mice, whereas concentrations in BAL of IL-13 were significantly decreased compared with WT control mice. These results indicate that, although exposure to allergen stimulates the expression of AMCase and increased chitinolytic activity in murine airways, the overexpression or inhibition of AMCase exerts only a subtle impact on AAD. Conversely, the increased numbers of neutrophils and lymphocytes in BAL and the decreased concentrations of IL-13 in AMCase-deficient mice challenged intratracheally with HDM indicate that AMCase contributes to the Th1/Th2 balance in the lungs. This finding may be of particular relevance to patients with asthma and increased airway neutrophilia.