Profiling in resolving inflammatory exudates identifies novel anti-inflammatory and pro-resolving mediators and signals for termination
L. V. Norling,
From the Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
From the Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA
Charles N. Serhan, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur (HIM 829), Boston, MA 02115, USA. (fax: +1-617-525-5017; e-mail: email@example.com).
Abstract. Norling LV, Serhan CN (Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA). Profiling in resolving inflammatory exudates identifies novel anti-inflammatory and pro-resolving mediators and signals for termination (Review). J Intern Med 2010; 268:15–24.
A highly orchestrated inflammatory response and its completion, termed resolution, are essential for ongoing health. Thus, complete understanding of the cellular and molecular events that govern natural resolution is vital. Using an unbiased systems approach to profile self-limited inflammatory exudates, we identified a novel genus of specialized pro-resolving lipid mediators (SPMs) comprised of three new families coined the resolvins, protectins and most recently the maresins biosynthesized from ω-3 fatty acids. These join the lipoxin- and aspirin-triggered lipoxins as anti-inflammatory and pro-resolving lipid mediators formed from arachidonic acid with the genus. SPMs have proven stereoselective, and control both the duration and magnitude of inflammation. Mapping these endogenous resolution circuits provides new avenues to probe the molecular basis of many widely occurring diseases where uncontrolled inflammation is characteristic. The focus of this JIM review is to depict recent advances from studies by the authors and colleagues on the biosynthesis and actions of these novel anti-inflammatory, pro-resolving and protective lipid mediators. Together these findings indicate that defective mechanisms and pathways in resolution may underlie our current appreciation of the inflammatory phenotype(s) that characterize some prevalent human diseases.
Current, widely used anti-inflammatory therapies are directed towards the inhibition of specific enzymes and/or antagonism of specific receptors . Both selective cyclooxygenase (COX) inhibitors and anti-tumour necrosis factor-α (TNF-α) are examples of this approach that are used with the goal of blocking the production of proinflammatory chemical mediators [1, 2]. Research efforts within our laboratory focus on profiling self-limited inflammation and uncovered novel mechanisms that terminate the local acute inflammatory response and stimulate resolution with the return of the tissue to homeostasis. Identification of these biochemical and cellular processes indicates that resolution, once considered a passive process, is actually an active programmed process at the tissue level in vivo; for recent reviews, see refs [3, 4].
As natural resolution of acute, self-limited, inflammatory responses proved to be an active process, rather than targeting inhibition or antagonism of inflammation, research in the author’s laboratory addresses the potential use of endogenous agonists to stimulate key endogenous regulatory or set points within the control of mechanisms that naturally resolve inflammation. This approach can now open a new understanding of the mechanisms underlying inflammatory disease as well as a new approach in molecular pharmacology, namely resolution pharmacology. The essential ω-3 fatty acids, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are precursors to a new genus of potent lipid mediators (LMs) that are both pro-resolving and anti-inflammatory (specialized pro-resolving mediators: SPM) and serve a physiological role defining programmed resolution. This review highlights recent advances on the actions of this novel genus of endogenous chemical mediators that govern resolution of acute inflammation and its termination.
Complete resolution: the ideal outcome
An acute inflammatory response, initiated from infection or tissue damage is characterized macroscopically by the cardinal signs of inflammation, that is, heat, redness, swelling and pain . These symptoms are accompanied by an established set of microscopic events, including oedema and the accumulation of leucocytes, specifically polymorphonuclear leucocytes (PMNs), followed by monocytes that differentiate locally to macrophages . Inflammatory reactions are generally protective and serve to maintain tissue homeostasis, although if uncontrolled they become deleterious to the host, progressing to chronic inflammation, scarring and fibrosis (Fig. 1). In nearly all cases, the fundamental cause of tissue damage is excessive leucocyte accumulation. In self-limited resolving inflammatory reactions leucocyte recruitment is coupled with the release of local factors that prevent further or excessive trafficking of leucocytes allowing for resolution [7, 8]. Early in the inflammatory response, proinflammatory mediators such as prostaglandins and leukotrienes play an important role . Indeed, monkeys fed a diet lacking essential fatty acids were defective at mounting an efficient inflammatory response, highlighting the importance of arachidonate-derived eicosanoids . Functionally, both neutrophil chemotaxis and superoxide generation in response to N-formyl-methionyl-leucyl-phenylalanine were markedly abrogated. The progression from acute inflammation to chronic inflammation as in many widely occurring human diseases such as periodontal disease, arthritis  and cardiovascular disease , to name a few, is widely viewed as an excess of proinflammatory mediators . Although mononuclear cells can sometimes contribute to proinflammatory responses, they are also critical in wound healing, tissue repair and remodelling in a noninflammatory, nonphlogistic manner . Hence, it is possible that problems associated with mounting the endogenous pro-resolving circuits and local autacoids de novo could underlie some of the aberrant mechanisms in chronic inflammation.
Complete termination of an acute inflammatory insult is pertinent for restoration of tissue homeostasis and is necessary for ongoing health. Key to this process is the complete removal of leucocytes from inflammatory sites without leaving remnants of the host’s combat between leucocytes, invading microbes, and/or other initiators of inflammation. In our laboratory we have focused on the question ‘How is the acute inflammatory response regulated?’ It was widely believed and argued that simple dilution of proinflammatory mediators was enough to switch off or ‘burn out’ inflammation, with the subsequent responses ending naturally or passively. Evidence from our laboratory and collaborators now indicates that the resolving phase of inflammation is not merely a passive process as once believed, but actively takes place as a programmed response at the tissue level [reviewed in refs 3, 4], which is analogous to programmed cell death .
Specialized pro-resolving LMs
A new genus of autacoids coined SPMs that possess potent anti-inflammatory, pro-resolving and protective properties have been uncovered by monitoring the self-limited inflammatory response in experimental animal models of disease. These include the lipoxins from arachidoinc acid (AA) , and the ω-3-derived resolvins, protectins and the newly identified maresins. These novel families of endogenous lipid-derived mediators were originally isolated from self-limited inflammatory murine exudates captured during the natural resolution phase. Each of these local chemical mediators is actively biosynthesized via distinct cellular and in some cases transcellular enzymatic pathways yielding unique stereospecific molecules. SPM are potent agonists that control the duration and magnitude of inflammation by acting on specific receptors on separate cell populations to stimulate the overall resolution of inflammation .
Identifying the mediators and modulators of resolution
An unbiased systems approach was taken to analyse the endogenous mediators and mechanisms in place to actively resolve inflammation. For this, the murine dorsal air pouch system proved ideal because self-limited inflammatory exudates could be obtained [7, 8]. This system permitted direct analysis of exudate in terms of lipidomics (bioactive products, as well as their inactive precursors and further metabolites), proteomics and cellular composition by monitoring leucocyte trafficking. Importantly, utilizing this approach made it possible to determine when and where different local mediators were biosynthesized and activated during resolution, namely their temporal and spatial differential analyses [3, 18].
Lipoxin A4 (LXA4) and LXB4 were the first anti-inflammatory and pro-resolving LMs recognized, signalling the resolution of acute contained inflammation . Lipoxins are lipoxygenase-derived eicosanoids, derived enzymatically from AA, an ω-6 fatty acid that is released and mobilized during inflammation . In human systems, they are biosynthesized via transcellular metabolic events engaged during leucocyte interactions with mucosal cells, that is, epithelia of the gastrointestinal tract or bronchial tissue, and within the vasculature during platelet–leucocyte interactions . Aspirin has an unexpected impact within resolution. In humans, aspirin ‘jump-starts’ this process by its ability to trigger the endogenous biosynthesis of LMs [20–22].
The murine dorsal air pouch was used to determine the formation and roles of endogenous LXA4 in the resolution of acute inflammation . Upon initiation of inflammation with TNF-α, there was a typical acute-phase response denoted by rapid PMN infiltration preceded by generation of local prostaglandins and leukotrienes. Unexpectedly, the eicosanoids then underwent what we have termed a ‘class switch’. As the exudate evolved, the eicosanoid profiles switched and the LMs made within that milieu changed with time . Within the inflammatory exudate, arachidonate-derived eicosanoids changed from initial production of prostaglandins and leukotrienes to lipoxins, which halted further recruitment of neutrophils (Fig. 1). This class switch was driven in part by COX-derived prostaglandins E2 and D2 that regulate the transcription of enzymes involved in lipoxin biosynthesis . Thus, with Sir John Savill we introduced the concept that ‘alpha signals omega’, the beginning signals the end in inflammation  because the appearance of lipoxins within inflammatory exudates was concomitant with the loss of PMN and resolution of inflammation . Within the inflammatory milieu, the frontiers of host defense, namely the neutrophils, undergo either apoptosis or necrotic cell death. As part of resolution, lipoxins signal macrophages to enhance engulfment of apoptotic PMN . Lipoxins are also potent chemoattractants, but in a nonphlogistic manner as they activate infiltration of mononuclear cells without stimulating release of proinflammatory chemokines or activation of proinflammatory gene pathways and products. These results suggested a dual action in that specific eicosanoids, namely lipoxins, actively reduced the entry of neutrophils to the site of inflammation whilst accelerating uptake of apoptotic neutrophils . Lipoxins are potent anti-inflammatory mediators that are formed and act in picogram to nanogram amounts within human tissues and in animal disease models . They have the specific pro-resolution actions of limiting PMN recruitment, chemotaxis and adhesion to the site of inflammation, acting essentially as a braking signal for PMN-mediated tissue injury [19, 22]. Notably, a stable lipoxin analogue, 16-phenoxy-LXA4 stimulates mononuclear cells to produce interleukin (IL)-1Ra, thus activating endogenous circuits to enhance resolution . Additionally, lipoxin analogues can activate endogenous antimicrobial defense mechanisms .
Novel local mediators are biosynthesized from ω-3 fatty acids
ω-3 polyunsaturated fatty acids (PUFA) are known to bestow protective clinical effects in the cardiovascular system, many inflammatory disorders and neural function [26–28]. However, the mechanism by which ω-3 PUFAs exert their biological effects has not been fully explored. To this end, research in our laboratory addressed the question ‘Are the essential ω-3 PUFAs such as EPA and DHA enzymatically converted to novel bioactive mediators?’ Accordingly, mice given ω-3 PUFA biosynthesized novel LMs during the resolution phase of acute inflammation as reviewed vide infra [7, 8].
Resolvins: resolution-phase interaction products
Resolvins are a family of new local mediators enzymatically generated within resolving inflammatory exudates. They were initially identified using a systems approach with LC-MS-MS-based lipidomics and informatics and subsequently complete structural elucidation of these bioactive mediators and related compounds was achieved [7, 8, 21, 29, 30]. The term resolvins or resolution-phase interaction products refers to endogenous compounds biosynthesized from the major ω-3 fatty acids EPA and DHA, denoted as E series (RvE) and D series (RvD) resolvins, respectively . Similar to lipoxins, resolvins are also produced by the COX-2 pathway in the presence of aspirin yielding ‘aspirin-triggered’ (AT) forms. Accruing evidence indicates that resolvins possess potent anti-inflammatory and immunoregulatory actions that include blocking the production of proinflammatory mediators and regulating leucocyte trafficking to inflammatory sites [reviewed in ref. 4] as well as clearance of neutrophils from mucosal surfaces . Specifically, resolvins limit PMN transendothelial migration in vitro and infiltration in vivo [8, 32].
Both RvD1 (7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) and AT-RvD1 (7S,8R,17R-trihydroxy-docosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid) stop PMN transmigration in a concentration-dependent manner. The potency of these compounds is notable, with concentrations as low as 10 nmol L−1 producing an ∼50% reduction in PMN transmigration. Experiments utilizing several inflammatory disease models have shown that RvD1 is a potent and stereoselective agonist of resolution [ref. 3 and references cited within]. Recent studies have established the stereochemistry and the stereoselective bioactions of RvD1, AT-RvD1, RvE1 (5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) and protectin D1 (PD1; vide infra) as well as their enzymatic inactivation [21, 30, 32].
Importantly, the actions of these endogenous SPMs are mediated through specific receptors. RvE1 acts as an agonist on at least two G-protein-coupled receptors (GPCRs), namely ChemR23 and as a partial agonist on the LTB4 receptor (BLT1) thus competing with LTB4 for binding [33, 34]. Recent research has revealed that RvE1 stimulates phosphorylation of Akt in a time- and dose-dependent manner via direct activation of ChemR23 . This agonist of resolution therefore displays a distinct mechanism of action compared with LXA4 that inhibits downstream tyrosine phosphorylation in eosinophils . Additionally, a recent study identified two separate GPCRs that RvD1 specifically binds on human leucocytes, namely the LXA4 receptor (ALX) and GPR32 an orphan receptor, which were validated using a GPCR β-arrestin-coupled system . Identification of receptors for other ω-3-derived SPM are yet to be uncovered, but are likely to be high-affinity GPCRs based on the potency of these newly discovered agonists of resolution. Thus, at least two GPCRs for each RvE1, RvD1 and LXA4 that are shared by AT-LXA4, have been identified. The concept that one ligand can act on a repertoire of receptors is not surprising in light of recent research on neuronal responses to formyl receptor-like peptides .
Protectins: protective mediators
DHA is also a precursor for the protectins, which are generated via a separate biosynthetic pathway. Protectins are distinguished by the presence of their conjugated triene-containing structure . The name ‘protectins’ was coined from the observed anti-inflammatory and protective actions in neural tissues and systems . The prefix neuroprotectin gives the tissue location of their generation and local actions, such as neuroprotectin D1 (NPD1) [30, 35]. Like resolvins, the protectins stop PMN infiltration [29, 30]. They are biosynthesized by and act on glial cells and reduce cytokine expression . NPD1 reduces retinal and corneal injury  and stroke damage , and improves corneal wound healing in mouse models  [see refs 3, 4 and references cited within]. DHA is well known for its important role in neuronal systems and, along with AA, is a major PUFA found in the retina.
Protectin D1 is also protective in acute models of inflammation including zymosan A-induced peritonitis [18, 30, 32]. To assess whether PD1 could reduce leucocyte infiltration, doses as low as 1 ng were injected. At 4 h, peritoneal lavages were collected and analysed. PD1 had a potent action, blocking >90% of further leucocyte infiltration, specifically stopping PMN migration and infiltration into the site in vivo . Studies were performed to determine whether the actions of PD1 and RvE1 were synergistic or additive by coinjection. Administration of RvE1 (10 ng) significantly reduced PMN infiltration, although the response was less than that obtained with PD1 (10 ng). In combination, the reduction was even greater, which suggests that there was an additive effect of PD1 and RvE1 in vivo in murine peritonitis.
Maresins: macrophage mediators in resolving inflammation
Macrophages are key cells in orchestrating resolution, tissue repair and homeostasis . As macrophages are pivotal, we sought evidence for macrophage-derived SPMs. Along these lines, using self-resolving inflammatory exudates and lipidomics, we identified a new pathway involving biosynthesis of potent anti-inflammatory and pro-resolving mediators from the essential fatty acid DHA by macrophages . In the resolution of murine peritonitis, exudates accumulated both 17-hydroxyDHA (17-HDHA), a known marker of 17S-D-series resolvin and protectin biosynthesis proceeds [8, 18], as well as accumulation of 14S-HDHA from endogenous DHA . These results suggested that 14S-HDHA may be a marker of a new pathway involved in the biosynthesis of bioactive mediators. Addition of either DHA or 14S-hydroperoxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid (14S-HpDHA) to either human or murine macrophages converted these substrates to novel dihydroxy-containing products that possessed potent anti-inflammatory and pro-resolving activity. The potency of these is in the range of RvE1 and PD1.
Stable isotope incorporation, intermediate trapping and characterization of physical and biological properties of the products demonstrated a novel 14-lipoxygenase pathway, generating bioactive 7,14-dihydroxy-docosa-4Z,8,10,12,16Z,19Z-hexaenoic acid from DHA that we coined maresin (macrophage mediator in resolving inflammation: MaR1), because they enhance resolution. These findings suggest that maresins and this new macrophage metabolome may be involved in some of the beneficial actions of DHA when utilized by resolving macrophages during inflammation-resolution, wound healing and host defense.
The omega connection and substrate availability
To elucidate the role of EPA, DHA and AA during inflammation in vivo, we studied disease models in wild-type mice with mice that overexpress the Caenorhabditis elegans fat-1 gene to compare the effects of ω-3 and ω-6 PUFAs. This gene converts ω-6 PUFA into ω-3 resulting in elevated tissue levels of ω-3 PUFA within the fat-1 overexpressing mice. In a model of oxygen-induced retinopathy, a protective effect against pathological angiogenesis was found in the retina when there was a lower ratio of ω-6 : ω-3 PUFA. Wild-type mice lacking the fat-1 transgene had more extensive vaso-obliteration and more severe retinal neovascularization compared with fat-1 mice . Of interest, in mice fed ω-3 PUFA, biosynthetic markers of NPD1 and RvE1 were detected in the retinal tissues. In mice without ω-3 PUFA supplementation, administration of RvD1, RvE1 or NPD1 gave protection from vaso-obliteration and neovascularization , as well as suture-induced ocular inflammation . These fat-1 mice also have increased levels of resolvins and protectins in the colon and are protected from colitis . In evolving exudates, rapid appearance of unesterified ω-3 PUFA parallels the initial increase in oedema . Thus, ω-3 SPM precursors become available directly from the peripheral circulation, which contrasts with the pool or storage of AA that requires phospholipase A2 for liberation.
Pro-resolving actions of SPM in inflammatory disease models
Inflammatory bowel disorders, such as colitis, are characterized by a relapsing inflammatory process owing to mucosal damage and abnormal mucosal responses. In a well-studied experimental colitis model, whereby mice are challenged with an intrarectal antigenic hapten, 2,4,6-trinitrobenzene sulphonic acid (TNBS) to induce colitis, RvE1 was protective against bowel inflammation . One group of mice was administered RvE1 prior to the induction of colitis, which was compared with mice that received TNBS alone. With treatment of as little as 1 μg of RvE1 per mouse, there was dramatic reduction in mortality, weight loss and less severe histologic display of colitis, namely reduction in the associated inflammatory cells, such as PMN and lymphocytes compared with mice with TNBS-induced colitis. RvE1 is also protective in Porphyromonas gingivalis-induced periodontal disease in rabbits, where it appears to stimulate tissue regeneration of the periodontium . Another pro-resolving action of RvE1 was evident in a murine model of fatty liver disease, where RvE1 administration to obese mice significantly alleviated hepatic steatosis and restored the loss of insulin sensitivity .
The complete stereochemical assignment for RvD2 has recently been established as 7S,16R,17S-trihydroxy-4Z,8E,10Z,12E,14E,19Z-docosahexaenoic acid . The biosynthesis of RvD2 in human cells can occur within a single cell type or via transcellular biosynthesis as depicted in Fig. 2. Actively phagocytosing human PMNs convert the resolvin precursor 17S-HpDHA to RvD2 as determined by lipidomics based on liquid-chromatography tandem mass spectrometry . RvD2 proved very potent suggesting it may have a broad role in vivo and potential application in disease. Sepsis remains a clinical challenge with increasing prevalence and mortality rates. Infection progresses rapidly, unless contained and cleared by phagocytes, to chronic inflammation, and epithelial and endothelial barrier dysfunction, resulting in immune suppression, multiple-organ failure and death. Administration of ω-3 PUFA has shown favourable outcomes in sepsis , although the mechanism of action is still being studied. In an established mouse model of sepsis, initiated by ‘mid-grade’ caecal ligation and puncture (CLP) surgery, RvD2-methyl ester (RvD2-ME; 100 ng) was protective . After 12 h, mice that underwent CLP surgery had severe bacterial burden both locally within the peritoneum and systemically, which was accompanied by a significant leucocyte infiltrate to the peritoneal cavity. RvD2-ME treatment immediately following CLP significantly reduced both blood and peritoneal bacterial levels and dramatically limited local PMN influx. Septic mice were hypothermic 12 h after CLP and displayed a drastic decrease in activity levels, whereas RvD2 treated mice remained active within their cages and their body temperatures were similar to sham-operated control mice (Fig. 3a). The proportion of mice that survived 7 days following ‘mid-grade’ CLP was approximately 36%. RvD2-ME was able to double the survival rates (Fig. 3b). Interestingly, whereas all of the mice that did not survive in the vehicle-treated group died by 36 h, of the mice that died in the RvD2-treated group, 25% lived until 48 h post-CLP suggesting that RvD2 may increase the ‘therapeutic window’ that could afford additional time for further interventions such as antibiotics. Further analysis of peritoneal exudates showed a ‘cytokine-storm’ of both proinflammatory cytokines and other mediators associated with detrimental outcomes in sepsis, as well as elevated proinflammatory LMs LTB4 and PGE2 (Fig. 3c,d). RvD2-ME significantly blunted overzealous cytokine production and proinflammatory LMs measured 12 h post-CLP. Dissection of mouse inguinal lymph nodes revealed that bacteria were disseminated throughout this organ in vehicle-CLP mice (Fig. 3e, left panel), whereas RvD2 enhanced phagocyte-dependent bacterial containment and clearance (Fig. 3e, right panel). Corroboratory results were obtained in vitro where human PMN exposed to RvD2 (1 and 10 nmol L−1) showed increased phagocytosis and killing of Escherichia coli . These novel findings highlight that RvD2 is protective in a model of mucosal barrier breakage and leak leading to sepsis. A recent study found that RvE1 is also anti-infective, enhancing clearance of bacteria from mouse lungs in a model of pneumonia, leading to increased survival . Thus, pro-resolving mediators display anti-inflammatory, antifibrotic and recently demonstrated anti-infective actions in several widely used laboratory models of inflammation (see Table 1). Together, these results indicate SPM, that is, resolvins are not immunosuppressive but rather enhance the innate antimicrobial systems in phagocytes and mucosal epithelial cells .
Table 1. Resolvins and protectins in animal disease modelsa
aThe actions of each of the main resolvins and protectins listed herein, that is, RvE1, RvD1, RvD2 and PD1, were confirmed with compounds prepared by total organic synthesis (see text and cited references for further details).
Reduces neutrophil infiltration; prevents connective tissue and bone loss; promotes healing of diseased tissues; regenerates lost soft tissue and bone
In summation, acute inflammation initiated by neutrophils in response to injury or infection is, ideally, a self-limited response that is protective for the host. Excessive uncontrolled inflammatory responses can lead to chronic disorders. Neutrophil-derived proinflammatory mediators, including leukotrienes and prostaglandins, can amplify this process and PGE2 and PGD2 activate a phenotypic switch of PMN LM profiles that induce LXA4. Within contained inflammatory exudates, we found that neutrophils can change phenotypes thus generating pro-resolving and protective mediators derived from essential fatty acids. There is an active catabasis to return tissues to a homeostatic healthy state from the battle of host defense during inflammatory episodes . Of interest, SPMs, such as protectins, resolvins and maresins, when administered in vivo can accelerate this process and the return to tissue homeostasis . These protective SPMs include the AA-derived lipoxins as well as ω-3 essential PUFA-derived resolvins, protectins and maresins. Each of these SPMs is temporally and spatially biosynthesized to actively regulate resolution by acting on specific receptors initiating anti-inflammatory and pro-resolving signals to terminate inflammation.
This short review was presented at the Birke Symposium on 3 February 2010; the authors dedicate this to Dr Jan Palmblad, a friend, colleague and connoisseur of LMs and phagocytes.
Conflict of interest statement
Resolvins are biotemplates for stable analogues. Patents on these are awarded and assigned to the Brigham and Women’s Hospital, and C.N.S. is the inventor. These analogue patents are licensed for clinical development. L.V.N. has no conflict of interest.
The authors thank Mary H. Small for expert assistance with manuscript preparation and the members of the laboratory and collaborators for their expertise and efforts in the reports referenced herein. Research in the authors’ laboratory reviewed here was supported by National Institutes of Health grants DK-074448 and GM-38765 (C.N.S) and Arthritis Research UK Fellowship 18445 (L.V.N).