Abstract. Previous work in our laboratory has shown that microvascular pericytes sort muscle and nonmuscle actin isoforms into discrete cytoplasmic domains (Herman, I. M., and P. A.

Abstract. We characterized the form and distribution of muscle and nonmuscle actin within retinal pericytes. Antibodies with demonstrable specificities for the actin isoforms were used in localization and immunoprecipitation experiments to identify those cellular domains that were enriched or deficient in one or several actin isoforms. Living pericyte behavior was monitored with phase-contrast video microscopy before fixation to identify those cellular areas that might preferentially be stained with either of the fluorescent antiactins or phallotoxins. Antibody and phallotoxin staining of pericytes revealed that nonmuscle actin is present within membrane ruffles, pseudopods, and stress fibers. In contrast, muscle actin could be convincingly localized in stress fibers, but not within specific motile areas of pericyte cytoplasm. To confirm and quantitatively extend the results obtained by fluorescence microscopy, nonionic and ionic detergents were used to selectively extract the motile or immobilized (stress fiber-containing) regions of biosynthetically labeled pericyte cytoplasm. Immunoprecipitated actins that were present within these discrete cellular domains were subjected to isoelectric focusing in urea-polyacrylamide gels before fluorographic analysis. Scanning laser densitometry of the focused actins could not reveal any detectable ot-actin within those ~-and 3,-actin-enriched motile regions extracted with nonionic detergents. Moreover, when pericyte stress fibers are completely dissolved by ionic detergent lysis, three actin isoforms can be quantified to be present in a ratio of 1:2.75:3 (tx:B:3,). These biochemical findings on biosynthetically labeled and immunoprecipitated pericyte actins confirm the fluorescent localization studies. While the regulatory events governing this actin sorting are unknown, it seems possible that such events may play important roles in controlling cell shape, adhesion, or the promotion of localized cell spreading.THOUGH the pericyte has been described in the literature for more than a century (31), little information has been accumulated regarding its exact function within the microvasculature. Mesodermal in origin, the pericyte is found surrounding the endothelial cells of capillaries and postcapillary venules. It has been implicated in the regulation of intraocular pressures, the selectivity of the bloodbrain barrier (3), and the minute-to-minute control of capillary vasomotion (34, 35). Moreover, intimate associations between the endothelial cell and the pericyte may be important in mediating the alterations in morphology, motility, and metabolism of the microvascular endothelial cells observed during angiogenesis (9,20), in response to injury (9), or in association with the diseased state (5, 8).Recently, vascular cell typing using antibodies specific for contractile proteins has been demonstrated to be an effective This work was presented, in part, at the 26th annual American Society for Cell Biology meeting held December 7-11, 1986 in Washington, DC. David DeNofrio's p...

Objective. To assess the efficacy and safety of oral decernotinib (VX-509; Vertex Pharmaceuticals) monotherapy in a 12-week, randomized, double-blind, placebo-controlled, dose-ranging study of patients with rheumatoid arthritis (RA).Methods. Two hundred four adults with active RA who had been unsuccessfully treated with >1 diseasemodifying antirheumatic drug were administered placebo tablets or decernotinib twice a day at dosages of 25 mg, 50 mg, 100 mg, or 150 mg. Primary measures of efficacy at week 12 were the response rate according to the American College of Rheumatology 20% improvement criteria (ACR20) and mean change from baseline in the Disease Activity Score in 28 joints using the C-reactive protein level (DAS28-CRP).Results. At week 12, the ACR20 response rates were 39.0%, 61.0%, 65.0%, and 65.9% in the 25-mg, 50-mg, 100-mg, and 150-mg groups, respectively, and were significantly higher in the 50-mg group (P ‫؍‬ 0.007) and the 100-mg and 150-mg groups (P ‫؍‬ 0.002) as compared to the response rates in the placebo group (29.3%). The mean change from baseline in DAS28-CRP was greater in the 50-mg, 100-mg, and 150-mg groups as compared to the placebo group (P < 0.001). Decernotinib treatment resulted in higher ACR50 and ACR70 response rates, more patients with DAS28-CRP scores <2.6, and improvements in the Health Assessment Questionnaire disability index as compared to placebo. The most common adverse events in any decernotinib group were nausea (6.1%), headache (4.3%), an increase in levels of alanine aminotransferase (4.3%), and hypercholesterolemia (3.7%). In the groups receiving decernotinib, there was an increased risk of infections and increased liver transaminase levels.Conclusion. Decernotinib was efficacious in improving clinical signs and symptoms of RA at week 12 at dosages of 50-150 mg twice a day. Infections and increases in liver transaminase and lipid levels were noted as potential safety signals.

We have recently cloned and characterized ankyrin-3 (also called ankyrinG), a new ankyrin that is widely distributed, especially in epithelial tissues, muscle, and neuronal axons (Peters, L.L., K.M. John, F.M. Lu, E.M. Eicher, A. Higgins, M. Yialamas, L.C. Turtzo, A.J. Otsuka, and S.E. Lux. 1995. J. Cell Biol. 130: 313–330). Here we show that in mouse macrophages, ankyrin-3 is expressed exclusively as two small isoforms (120 and 100 kD) that lack the NH2-terminal repeats. Sequence analysis of isolated Ank3 cDNA clones, obtained by reverse transcription and amplification of mouse macrophage RNA (GenBank Nos. U89274 and U89275), reveals spectrin-binding and regulatory domains identical to those in kidney ankyrin-3 (GenBank No. L40631) preceded by a 29–amino acid segment of the membrane (“repeat”) domain, beginning near the end of the last repeat. Antibodies specific for the regulatory and spectrin-binding domains of ankyrin-3 localize the protein to the surface of intracellular vesicles throughout the macrophage cytoplasm. It is not found on the plasma membrane. Also, epitope-tagged mouse macrophage ankyrin-3, transiently expressed in COS cells, associates with intracellular, not plasma, membranes. In contrast, ankyrin-1 (erythrocyte ankyrin, ankyrinR), which is also expressed in mouse macrophages, is located exclusively on the plasma membrane. The ankyrin-3–positive vesicles appear dark on phasecontrast microscopy. Two observations suggest that they are lysosomes. First, they are a late compartment in the endocytic pathway. They are only accessible to a fluorescent endocytic tracer (FITC-dextran) after a 24-h incubation, at which time all of the FITC-dextran– containing vesicles contain ankyrin-3 and vice versa. Second, the ankyrin-3–positive vesicles contain lysosomal-associated membrane glycoprotein (LAMP-1), a recognized lysosomal marker. This is the first evidence for the association of an ankyrin with lysosomes and is an example of two ankyrins present in the same cell that segregate to different locations.

Cytokines, growth factors, and other chemical messengers rely on a class of intracellular nonreceptor tyrosine kinases known as Janus kinases (JAKs) to rapidly transduce intracellular signals. A number of these cytokines are critical for lymphocyte development and mediating immune responses. JAK3 is of particular interest due to its importance in immune function and its expression, which is largely confined to lymphocytes, thus limiting the potential impact of JAK3 inhibition on nonimmune physiology. The aim of this study was to evaluate the potency and selectivity of the investigational JAK3 inhibitor VX-509,2-trifluoroethyl)butanamide] against JAK3 kinase activity and inhibition of JAK3-mediated signaling in vitro and JAK3-dependent physiologic processes in vivo. These results demonstrate that VX-509 potently inhibits JAK3 in enzyme assays (K i 5 2.5 nM 1 0.7 nM) and cellular assays dependent on JAK3 activity (IC 50 range, 50-170 nM), with limited or no measurable potency against other JAK isotypes or non-JAK kinases. VX-509 also showed activity in two animal models of aberrant immune function. VX-509 treatment resulted in dose-dependent reduction in ankle swelling and paw weight and improved paw histopathology scores in the rat collagen-induced arthritis model. In a mouse model of oxazolone-induced delayed-type hypersensitivity, VX-509 reduced the T cell-mediated inflammatory response in skin. These findings demonstrate that VX-509 is a selective and potent inhibitor of JAK3 in vitro and modulates proinflammatory response in models of immune-mediated diseases, such as collagen-induced arthritis and delayed-type hypersensitivity. The data support evaluation of VX-509 for treatment of patients with autoimmune and inflammatory diseases such as rheumatoid arthritis.

While several therapeutic options exist, the need for more effective, safe, and convenient treatment for a variety of autoimmune diseases persists. Targeting the Janus tyrosine kinases (JAKs), which play essential roles in cell signaling responses and can contribute to aberrant immune function associated with disease, has emerged as a novel and attractive approach for the development of new autoimmune disease therapies. We screened our compound library against JAK3, a key signaling kinase in immune cells, and identified multiple scaffolds showing good inhibitory activity for this kinase. A particular scaffold of interest, the 1H-pyrrolo[2,3-b]pyridine series (7-azaindoles), was selected for further optimization in part on the basis of binding affinity (Ki) as well as on the basis of cellular potency. Optimization of this chemical series led to the identification of VX-509 (decernotinib), a novel, potent, and selective JAK3 inhibitor, which demonstrates good efficacy in vivo in the rat host versus graft model (HvG). On the basis of these findings, it appears that VX-509 offers potential for the treatment of a variety of autoimmune diseases.

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The synthesis and characterization of a novel polycyclic azaindole based derivative is disclosed, and its binding to JAK2 is described. The compound is further evaluated for its ability to block the EPO/JAK2 signaling cascade in vitro and in vivo.