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Background Morphea is an autoimmune, sclerosing skin disorder. Despite the recent emphasis on immune dysregulation in morphea, the role of autoantibodies in morphea pathogenesis or utility as biomarkers are poorly defined. Methods Autoantigen microarray was used to profile autoantibodies from the serum of participants from the Morphea in Adults and Children (MAC) cohort. Clinical and demographic features of morphea patients with myelin basic protein (MBP) autoantibodies were compared to those without. MBP immunohistochemistry staining was subsequently performed in morphea skin to assess for perineural inflammation in areas of staining. Immunofluorescence staining on mouse brain tissue was also performed using patient sera and mouse anti-myelin basic protein antibody to confirm the presence of MBP antibodies in patient sera. Results Myelin basic protein autoantibodies were found in greater frequency in morphea (n = 50, 71.4%) compared to systemic sclerosis (n = 2, 6.7%) and healthy controls (n = 7, 20%). Patients with MBP antibodies reported pain at higher frequencies. Morphea skin biopsies, highlighted by immunohistochemistry, demonstrated increased perineural inflammation in areas of MBP expression. Immunofluorescence staining revealed an increased fluorescence signal in myelinated areas of mouse brain tissue (i.e. axons) when incubated with sera from MBP antibody-positive morphea patients compared to sera from MBP antibody-negative morphea patients. Epitope mapping revealed target epitopes for MBP autoantibodies in morphea are distinct from those reported in MS, and included fragments 11–30, 41–60, 51–70, and 91–110. Conclusions A molecular classification of morphea based on distinct autoantibody biosignatures may be used to differentially classify morphea. We have identified anti-MBP as a potential antibody associated with morphea due to its increased expression in morphea compared to healthy controls and systemic sclerosis patients.

Tyrosine kinases are enzymes that catalyze the phosphorylation of tyrosine residues on protein substrates. They are key components of signaling pathways that drive an array of cellular responses including proliferation, differentiation, migration, and survival. Specific tyrosine kinases have recently been identified as critical to the pathogenesis of several autoimmune and inflammatory diseases. Small-molecule inhibitors of tyrosine kinases are emerging as a novel class of therapy that may provide benefit in certain patient subsets. In this review, we highlight tyrosine kinase signaling implicated in inflammatory dermatologic diseases, evaluate strategies aimed at inhibiting these aberrant signaling pathways, and discuss prospects for future drug development.

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the presence of anticitrullinated protein Abs, although the exact targets and role of anticitrullinated protein autoimmunity in the pathogenesis of RA remain to be defined. Fibrinogen, which can be citrullinated, has recently emerged as a candidate autoantigen. To determine whether autoimmunity against fibrinogen can mediate inflammatory arthritis, we immunized a variety of common mouse strains with fibrinogen and found that DBA/1 and SJL mice developed an inflammatory and erosive arthritis. Mice with fibrinogen-induced arthritis (FIA) possess fibrinogen-reactive T cells that produce the proinflammatory cytokines IL-6, IL-17, TNF-α, and IFN-γ. FIA can be adoptively transferred with either plasma or fibrinogen-specific T cells from diseased mice. Mice with FIA possess rheumatoid factor, circulating immune complexes, and anticyclic citrullinated peptide Abs, all of which are characteristic of human RA. These observations demonstrate that fibrinogen is arthritogenic in mice and that the pathogenesis of FIA is mediated by both autoantibodies and fibrinogen-reactive T cells.

As critical regulators of numerous cell signaling pathways, tyrosine kinases are implicated in the pathogenesis of several diseases, including rheumatoid arthritis (RA). In the absence of disease, synoviocytes produce factors that provide nutrition and lubrication for the surrounding cartilage tissue; few cellular infiltrates are seen in the synovium. In RA, however, macrophages, neutrophils, T cells and B cells infiltrate the synovium and produce cytokines, chemokines and degradative enzymes that promote inflammation and joint destruction. In addition, the synovial lining expands owing to the proliferation of synoviocytes and infiltration of inflammatory cells to form a pannus, which invades the surrounding bone and cartilage. Many of these cell responses are regulated by tyrosine kinases that operate in specific signaling pathways, and inhibition of a number of these kinases might be expected to provide benefit in RA.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs and widespread vasculopathy. Current therapies for SSc focus on treating specific symptoms, but disease-modifying agents targeting the underlying pathogenesis are lacking. The pathogenesis of SSc involves activation of profibrotic pathways, with over-expression of the cytokines transforming growth factor (TGF)-β and platelet derived growth factor (PDGF). A recent report showed that SSc patients have autoantibodies against the PDGF receptor, which stimulate the production of reactive oxygen species and type I collagen expression(1). PDGF receptors are upregulated in the skin and bronchoalveolar lavage fluid of patients with SSc, and when activated, lead to fibroblast and myofibroblast proliferation(2, 3). PDGF participates in smooth muscle cell recruitment and mitogenic signaling that underlie the vasculopathy associated with pulmonary arterial hypertension (PAH), a complication of SSc associated with high mortality(4). In addition, stimulation of the TGF-β profibrotic pathway involves activation of c-Abl(2). Thus, the PDGF and TGF-β pathways are thought to contribute to the fibrotic and vascular complications in SSc. Imatinib mesylate (Gleevec, Novartis, East Hanover, New Jersey) is a small molecule that antagonizes specific tyrosine kinases that mediate fibrotic pathways, including c-Abl, a downstream mediator of TGF-β(2) and PDGF receptors(5). Imatinib has been shown to inhibit lung and dermal fibrosis in bleomycin-induced mouse models(6, 7), and the proliferation of synovial fibroblasts derived from patients with rheumatoid arthritis(8). Imatinib has also been reported to provide benefit in the treatment of refractory idiopathic PAH through its effects on vascular remodeling(9). We now describe two patients with early diffuse SSc who experienced clinical improvement in response to imatinib therapy and provide evidence that both c-Abl and PDGFR are targets of imatinib in scleroderma skin. Finally, we show that an imatinib-responsive gene signature is present in most cases of diffuse SSc.

Tyrosine kinases play a central role in the activation of signal transduction pathways and cellular responses that mediate the pathogenesis of rheumatoid arthritis. Imatinib mesylate (imatinib) is a tyrosine kinase inhibitor developed to treat Bcr/Abl-expressing leukemias and subsequently found to treat c-Kit-expressing gastrointestinal stromal tumors. We demonstrate that imatinib potently prevents and treats murine collagen-induced arthritis (CIA). We further show that micromolar concentrations of imatinib abrogate multiple signal transduction pathways implicated in RA pathogenesis, including mast cell c-Kit signaling and TNF-alpha release, macrophage c-Fms activation and cytokine production, and fibroblast PDGFR signaling and proliferation. In our studies, imatinib attenuated PDGFR signaling in fibroblast-like synoviocytes (FLSs) and TNF-alpha production in synovial fluid mononuclear cells (SFMCs) derived from human RA patients. Imatinib-mediated inhibition of a spectrum of signal transduction pathways and the downstream pathogenic cellular responses may provide a powerful approach to treat RA and other inflammatory diseases.

A fast and accurate method to quantify the new immunosuppressive JAK3 inhibitor CP-690,550 in whole blood using a dual-pump liquid chromatography-liquid chromatography-mass spectrometry (LC/LC-MS) system was developed and validated in nonhuman primate blood. Before injection, blood samples were prepared by precipitation with a reagent that included methanol and acetonitrile (30:70, vol/vol) along with the internal standard (CP-istd). Column-switching LC/LC-MS analysis used online extraction followed by separation on a C8 analytic column and MS detection of the [M + H] CP-690,550 (m/z = 313.1) and CP internal standard (m/z = 288.1). Linearity was always better than r = 0.99 (n = 7) for CP-690,550 (range 2.5-750 ng/mL), with a lower limit of quantification (LLOQ) of 2.5 ng/mL. The intrarun accuracy and precision ranged from 103.0% to 105.4% and 2.7% to 4.3%, respectively (n = 5), and the interday precision ranged from 8.7% to 11.1%, and the interday accuracy ranged from 98.1% to 103.8% of nominal values (n = 14). The injection repeatability for the method was 1.3% (n = 7). Except for the LLOQ, the intraday accuracy and precision in human blood were also within 15% (n = 5). The combination of simple sample preparation and short analytic run time of this sensitive procedure makes it effective for monitoring the concentration of CP-690,550 in whole blood in organ-transplant recipients.

BACKGROUND Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common chain (gammac). Because mutations in genes encoding gammac or JAK3 result in immunodeficiency, we investigated the potential of a rationally designed inhibitor of JAK3, CP-690,550, to prevent renal allograft rejection in nonhuman primates. METHODS Life-supporting kidney transplantations were performed between mixed leukocyte reaction-mismatched, ABO blood group-matched cynomolgus monkeys. Animals were treated with CP-690,550 (n = 18) or its vehicle (controls, n = 3) and were euthanized at day 90 or earlier if there was allograft rejection. RESULTS Mean survival time (+/- standard error of mean) in animals treated with CP-690,550 (53 +/- 7 days) was significantly longer than in control animals (7 +/- 1 days, P=0.0003) and was positively correlated with exposure to the drug (r = 0.79, P < 0.01). Four treated animals were euthanized at 90 days with a normal renal function and low-grade rejection at final pathology. Occurrence of rejection was significantly delayed in treated animals (46 +/- 7 days from transplantation vs. 7 +/- 1 days in controls, P = 0.0003). Persistent anemia, polyoma virus-like nephritis (n = 2), and urinary calcium carbonate accretions (n = 3) were seen in animals with high exposure. Natural killer cell and CD4 and CD8 T-cell numbers were significantly reduced in treated animals. Blood glucose, serum lipid levels, and arterial blood pressure were within normal range in treated animals, and no cancers were demonstrated. CONCLUSIONS CP-690,550 is the first reported JAK3 inhibitor combining efficacy and good tolerability in a preclinical model of allotransplantation in nonhuman primates and thus has interesting potential for immunosuppression in humans.

Thepig-to-non-humansolidorgantransplantationmodelmostcloselyresemblesclinicalxenotrans-plantationofsolidorgans.Thefirsthurdleinthismodel is hyperacute rejection mediated by thedirect attack of xenogeneic naturally existingantibodiesincombinationwithcomplementacti-vation. In unsensitized Old World non-humanprimatesmost,ifnotall,naturallyexistinganti-bodies are directed to the Gala1-3Gal (Gal)epitopepresentonmostglycoproteinsandglycoli-pids. Hyperacute rejection can be managed byinhibitorsofcomplement,neutralizationofnaturalantibodies,ortheuseoforgansfrompigstrans-genic for human regulators of complementLamTT,Boeke-PurkisK,LauM,PaniaguaR,SchuurmanH-J,MorrisRE.Anti-pigantibodylevelsinnon-humanprimatesofvariousorigin.Xenotransplantation2004;11:332–339. BlackwellMunksgaard,2004Abstract:Background:Naturalanti-porcineantibodiesplayamajorroleinhyperacutesolidorganxenograftrejectioninthepig-to-non-humanprimatemodel.Workfromothergroupsandourexperienceintransplantationexperimentshasshownthatantibodylevelsarehighlyvariablebetweennon-humanprimatespecies,andthatextremelyhighlevelscanmediatehyperacuterejectioneveniforgansfromanimalstransgenicforhumandecay-acceleratingfactorareused.Methods:Serawereobtainedfromcynomolgusmonkeyswild-caughtinMauritius,captive-bredinthePhilippines,captive-bredinIndonesia(Indonesia-Ind),andoriginatingfromIndonesiabutcolony-bredinUSA(Indonesia-USA),frombaboonswild-caughtinKenya,andfromrhesusmonkeysoriginatingfromIndiabutcolony-bredinUSA(10animalsineachgroup).Antibodylevelsweredeterminedusingassaysforhaemolyticantibody(APA),IgMandIgGclassanti-Gala1-3Galantibody,andIgMandIgGclassanti-endothelialcellantibody.Results:CynomolgusmonkeysfromthePhilippinesandIndonesia-USAandrhesusmonkeysshowedmedianAPAandIgMantibodylevelsinthesamerangeasapooledhumanserumstandard,andmedianIgGlevelswellbelowthelevelinthisstandard.CynomolgusmonkeysfromMauritiusandIndonesia-IndshowedextremelyhighAPAlevels(med-iansevento10timesthehumanserumstandard):IgMclassantibodieswerealsohigher,whileIgGclassantibodieswereintherangeofthelevelinthehumanserumstandard.Antibodylevelsinbaboonswereinbetweenthesetwocategories.TheresultsoftheAPAassayshowedahighlystatisticallysignificantcorrelationwiththeassaysofIgManti-body,andthiswasalsothecasefortheIgMantibodyassays,indicativeoftheassessmentofthesameantibodiesintheseassays.ThesamewasobservedfortheassaysforIgGantibody.Takingbodyweightasanindicatorforage,therewasnorelationshipbetweenbodyweightandlevelsofantibodies.Conclusions:Naturalantibodylevelsshowasignificantvariationbetweenvariousgroupsofnon-humanprimates,withlevelsinsomegroupswellabovethoseinahumanserumstandard.

Background ISA TX 247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first in vivo study of the effects of ISA TX 247 on lymphocyte functions in non-human primates. Methods Groups of cynomolgus monkeys were treated orally twice daily for 7 days, each dose consisting of 25 mg/kg cyclosporine ( n = 5), 25 mg/kg ISA TX 247 ( n = 6) or 50 mg/kg ISA TX 247 ( n = 6). Levels of cyclosporine and ISA TX 247 in whole blood were measured by liquid chromatography/mass spectrometry. After mitogen stimulation, lymphocyte proliferation was assessed by tritium-labeled thymidine incorporation and by flow cytometry (expression of proliferating cell nuclear antigen in cells in S/G 2 M phase). Flow cytometry was also used to assess production of intracellular cytokines by T cells (interleukin-2, interferon-γ, tumor necrosis factor-α) and expression of T-cell surface activation antigens (CD25, CD71, CD11a, CD95, CD154). Results Trough ( C 14hr ) and peak ( C 3hr ) drug levels, as well as area under the concentration–time curve, were significantly higher for cyclosporine than ISA TX 247 (370 ng/ml vs 70 ng/ml, 877 ng/ml vs 303 ng/ml and 6,262 ng · h/ml vs 1,979 ng · h/ml, respectively). On Day 7 at C 14hr , lymphocyte proliferation had been suppressed by approximately 50% in all groups compared with proliferation before treatment. Three hours after dosing, lymphocyte proliferation was inhibited significantly more by ISA TX 247 (approximately 80%, with no differences between the two ISA TX 247 dose levels) than by cyclosporine (65% inhibition). Similar differences between the immunosuppressive effects of ISA TX 247 and cyclosporine were found for inhibition of expression of T-cell surface activation antigens. Despite lower ISA TX 247 exposures compared with cyclosporine, the cyclosporine treatment only rarely suppressed cytokine production more than treatment with ISA TX 247. Conclusions In non-human primates, ISA TX 247 produces a greater or similar inhibition of lymphocyte proliferation, expression of T-cell activation surface antigens, and cytokine production when compared with cyclosporine, despite ISA TX 247's lower blood levels and total exposure. We conclude that ISA TX 247 suppresses diverse T-cell functions more potently than cyclosporine in non-human primates in vivo.

Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolongedsurvival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associatedwith hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation andin other clinical settings.

Background Janus Kinase (JAK) 3 is a tyrosine kinase essential for proper signal transduction downstream of selected cytokine receptors and for robust T-cell and natural killer cells activation and function. JAK3 inhibition with CP-690,550 prevents acute allograft rejection. To provide further insight into the mechanisms of efficacy, we investigated the immunomodulatory effects of CP-690,550 in vitro and in vivo in nonhuman primates. Methods Pharmacodynamic assessments of lymphocyte activation, function, proliferation and phenotype were performed in three settings: in vitro in whole blood isolated from untransplanted cynomolgus monkeys (cynos), in vivo in blood from untransplanted cynos dosed with CP-690,550 for 8 days, and in vivo in blood from transplanted cynos immunosuppressed with CP-690,550. Cell surface activation markers expression, IL-2- enhanced IFN-gamma production, lymphocyte proliferation and immune cell phenotype analyzes were performed with multiparametric flow cytometry. Results In vitro exposure to CP-690,550 resulted in significant reduction of IL-2-enhanced IFN-gamma production by T-cells (maximum inhibition of 55-63%), T-cell surface expression of CD25 (50% inhibitory concentration (IC50); 0.18 microM) and CD71 (IC50; 1.6 microM), and T-cell proliferative capacities measured by proliferating cell nuclear antigen expression (IC50; 0.87 microM). Similar results were observed in animals dosed with CP-690,550. In addition, transplanted animals displayed significant reduction of NK cell (90% from baseline) and T-cell numbers whereas CD8 effector memory T-cell populations were unaffected. Conclusions Potent in vitro and in vivo immunomodulatory effects of the JAK3 inhibitor CP-690,550 likely contribute to its efficacy in the prevention of organ allograft rejection.

Background: Our introductory pig-to-cynomolgus monkey heart or kidney transplantation using organs from pigs transgenic for human decay-accelerating factor (hDAF), showed a high incidence of hyperacute rejection (HAR), which was ascribed to extraordinary high levels of anti-pig antibodies. We evaluated the efficacy of GAS914, a Galα1–3Gal trisaccharide linked to a poly-l-lysine backbone, in inhibition of HAR. Methods: hDAF transgenic heterotopic heart (n = 15) or life-supporting kidney (n = 8) transplantation included induction with cyclophosphamide or anti-thymocyte globulin, and maintenance with cyclosporine or tacrolimus, steroids and mycophenolate sodium/mofetil. Four doses of GAS914 were given before transplantation. Rejection was confirmed by graft histology, and anti-pig antibody levels were determined in various assays. Results: Four of six heart transplants without GAS914 treatment showed HAR. Nine subsequent transplants with GAS914 pre-treatment, did not show HAR (chi-square, P

Background ISATX247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first study to compare the survival times of renal allografts in nonhuman primates treated with either ISATX247 or cyclosporine. Methods Adult, male cynomolgus monkeys were divided into blood-group compatible and mixed-lymphocyte, stimulation-mismatched, donor-recipient pairs. Heterotopic renal transplantation and bilateral native nephrectomies were performed. The monkeys were placed into either an ISATX247 or cyclosporine treatment group. Both groups were dosed twice daily to maintain a 12-hour drug-trough level of 150 ng/mL. Whole-blood concentrations of ISATX247 and cyclosporine, complete blood counts, and serum chemistry profiles were performed three times a week. Euthanasia was performed if the serum creatinine concentration became 7 or more mg/dL or a serious complication developed. Results The group receiving ISATX247 (n=8) survived significantly (P=0.0036) longer than the group receiving cyclosporine (n=7). The mean trough blood concentration of ISATX247 was 120 +/- 32 ng/mL and cyclosporine was 189 +/- 130 ng/mL. The average area under the curve 0-12 for ISATX247 was 6045 +/- 1679 ng/mL/hr and for cyclosporine was 4919 +/- 823 ng/mL/hr. The average calcineurin inhibition at trough blood concentrations was 80 +/- 11% for ISATX247 and 48 +/- 12% for cyclosporine. Conclusions Allografts in monkeys treated with ISATX247 survived significantly longer than those treated with cyclosporine. On the basis of survival times and degree of calcineurin inhibition, ISATX247 is a more potent immunosuppressive agent than cyclosporine in this nonhuman primate model of renal-allograft transplantation.

There has been much speculation on the potential use of kinase inhibitors, such as imatinib mesylate, for the treatment of autoimmune rheumatic diseases. Imatinib inhibits a select set of tyrosine kinases at submicromolar concentrations. In this Viewpoint, the authors discuss how imatinib could provide a potent therapeutic option for a variety of autoimmune and inflammatory diseases, for which current therapies are insufficient.

Introduction Tyrosine kinases are key mediators of multiple signaling pathways implicated in rheumatoid arthritis (RA). We previously demonstrated that imatinib mesylate--a Food and Drug Administration (FDA)-approved, antineoplastic drug that potently inhibits the tyrosine kinases Abl, c-Kit, platelet-derived growth factor receptor (PDGFR), and c-Fms--ameliorates murine autoimmune arthritis. However, which of the imatinib-targeted kinases is the principal culprit in disease pathogenesis remains unknown. Here we examine the role of c-Fms in autoimmune arthritis. Methods We tested the therapeutic efficacy of orally administered imatinib or GW2580, a small molecule that specifically inhibits c-Fms, in three mouse models of RA: collagen-induced arthritis (CIA), anti-collagen antibody-induced arthritis (CAIA), and K/BxN serum transfer-induced arthritis (K/BxN). Efficacy was evaluated by visual scoring of arthritis severity, paw thickness measurements, and histological analysis. We assessed the in vivo effects of imatinib and GW2580 on macrophage infiltration of synovial joints in CIA, and their in vitro effects on macrophage and osteoclast differentiation, and on osteoclast-mediated bone resorption. Further, we determined the effects of imatinib and GW2580 on the ability of macrophage colony-stimulating factor (M-CSF; the ligand for c-Fms) to prime bone marrow-derived macrophages to produce tumor necrosis factor (TNF) upon subsequent Fc receptor ligation. Finally, we measured M-CSF levels in synovial fluid from patients with RA, osteoarthritis (OA), or psoriatic arthritis (PsA), and levels of total and phosphorylated c-Fms in synovial tissue from patients with RA. Results GW2580 was as efficacious as imatinib in reducing arthritis severity in CIA, CAIA, and K/BxN models of RA. Specific inhibition of c-Fms abrogated (i) infiltration of macrophages into synovial joints of arthritic mice; (ii) differentiation of monocytes into macrophages and osteoclasts; (iii) osteoclast-mediated bone resorption; and (iv) priming of macrophages to produce TNF upon Fc receptor stimulation, an important trigger of synovitis in RA. Expression and activation of c-Fms in RA synovium were high, and levels of M-CSF were higher in RA synovial fluid than in OA or PsA synovial fluid. Conclusions These results suggest that c-Fms plays a central role in the pathogenesis of RA by mediating the differentiation and priming of monocyte lineage cells. Therapeutic targeting of c-Fms could provide benefit in RA.