Matrix metalloproteinase-13 (MMP13) is a Zn2؉ -dependent protease that catalyzes the cleavage of type II collagen, the main structural protein in articular cartilage. Excess MMP13 activity causes cartilage degradation in osteoarthritis, making this protease an attractive therapeutic target. However, clinically tested MMP inhibitors have been associated with a painful, joint-stiffening musculoskeletal side effect that may be due to their lack of selectivity. In our efforts to develop a disease-modifying osteoarthritis drug, we have discovered MMP13 inhibitors that differ greatly from previous MMP inhibitors; they do not bind to the catalytic zinc ion, they are noncompetitive with respect to substrate binding, and they show extreme selectivity for inhibiting MMP13. By structurebased drug design, we generated an orally active MMP13 inhibitor that effectively reduces cartilage damage in vivo and does not induce joint fibroplasias in a rat model of musculoskeletal syndrome side effects. Thus, highly selective inhibition of MMP13 in patients may overcome the major safety and efficacy challenges that have limited previously tested non-selective MMP inhibitors. MMP13 inhibitors such as the ones described here will help further define the role of this protease in arthritis and other diseases and may soon lead to drugs that safely halt cartilage damage in patients.The National Institutes of Health has estimated that more than 20 million adults in the United States suffer from osteoarthritis (OA), 3 a debilitating disease in which the protective cushion of cartilage is destroyed, resulting in pain and reduced mobility. A critical step in OA pathology is breakdown of the main structural protein of articular cartilage, type II collagen. This triple helical protein is resistant to most proteases but is efficiently recognized and degraded by the Zn 2ϩ -dependent enzyme, collagenase-3, known as matrix metalloproteinase-13 (MMP13) (1-3). MMP13 catalyzes the hydrolysis of type II collagen at a unique site resulting in 3 ⁄4-and 1 ⁄4-length polypeptide products (2-6). MMP13 is not found in normal adult tissues but is expressed in the joints and articular cartilage of OA patients (4 -8). In addition, regulated expression of human MMP13 in hyaline and joint cartilages induces OA in genetically modified mice (9). Furthermore, a MMP inhibitor that preferentially inhibits MMP13 has been shown to block the degradation of explanted human osteoarthritic cartilage (5). Based on these findings, it is likely that MMP13 is the direct cause of irreversible cartilage damage in OA.The clinical development of drugs that inhibit the actions of MMPs has been plagued by the association of a painful, joint-stiffening tendonitis-like side effect, termed "musculoskeletal syndrome" (MSS), with these inhibitors (10, 11). Such joint side effects are not unique to humans. Rats dosed with non-selective MMP inhibitors (i.e. compounds that inhibit several or all MMPs) also display MSS-like side effects such as soft tissue fibroplasias, inflammation, and pain (...

Objective. To characterize the clinical and histopathologic changes in a rat model of broad-spectrum matrix metalloproteinase (MMP)-induced musculoskeletal syndrome (MSS), and to facilitate research into the causes and treatments of MSS in humans.Methods. Male Lewis rats weighing 150-180 gm were administered 10-30 mg of the broad-spectrum MMP inhibitor marimastat over a 2-week period via surgically implanted subcutaneous osmotic pumps. The animals were monitored and scored for the onset and severity of MSS, using clinical and histologic parameters.Results. Marimastat-treated rats exhibited various clinical signs, including compromised ability to rest on their hind feet, high-stepping gait, reluctance or inability to move, and hind paw swelling. Histologically, marimastat-treated rat joints were characterized by soft tissue and bone changes, such as increased epiphyseal growth plate, synovial hyperplasia, and increased cellularity in the joint capsule and extracapsular ligaments. The severity of MSS, as judged by clinical criteria (2 blinded observers using 3 clinical parameters), paw volume, and histologic score, was nearly identical. The observed changes were indistinguishable from those reported for primate models and mimic MSS in humans.Conclusion. This simple and sensitive model of MSS is an attractive alternative for studying the pathology of MSS.

Specimens from four regions of oral mucosa (palate, buccal mucosa, lateral border of the tongue, and the floor of the mouth) and of abdominal skin were taken from 58 individuals at autopsy, for determination of permeability constants (Kp) to tritium-labeled water. Comparisons between fresh specimens and those stored at -80 degrees C revealed no significant effect on Kp as a result of freezing; similar results were found with use of specimens from corresponding regions of the pig. Values for Kp were significantly different for all of the tissue regions examined and ranged from 44 +/- 4 x 10(-7) cm/min for skin to 973 +/- 33 x 10(-7) cm/min for the floor of the mouth, which was the most permeable region. Similar differences were evident among corresponding regions of porcine oral mucosa and skin. Moreover, the Kp values obtained for human tissues were not significantly different from those of the pig, except for the floor of the mouth, which was more permeable in human than in pig tissue. The results reveal interesting differences in the permeability of human oral mucosa that might be related to susceptibility to mucosal disease in those conditions where local extrinsic etiological agents are implicated.

RORγt is the key transcription factor controlling the development and function of CD4+ Th17 and CD8+ Tc17 cells. Across a range of human tumors, about 15% of the CD4+ T cell fraction in tumor-infiltrating lymphocytes are RORγ+ cells. To evaluate the role of RORγ in antitumor immunity, we have identified synthetic, small molecule agonists that selectively activate RORγ to a greater extent than the endogenous agonist desmosterol. These RORγ agonists enhance effector function of Type 17 cells by increasing the production of cytokines/chemokines such as IL-17A and GM-CSF, augmenting expression of co-stimulatory receptors like CD137, CD226, and improving survival and cytotoxic activity. RORγ agonists also attenuate immunosuppressive mechanisms by curtailing Treg formation, diminishing CD39 and CD73 expression, and decreasing levels of co-inhibitory receptors including PD-1 and TIGIT on tumor-reactive lymphocytes. The effects of RORγ agonists were not observed in RORγ−/− T cells, underscoring the selective on-target activity of the compounds. In vitro treatment of tumor-specific T cells with RORγ agonists, followed by adoptive transfer to tumor-bearing mice is highly effective at controlling tumor growth while improving T cell survival and maintaining enhanced IL-17A and reduced PD-1 in vivo. The in vitro effects of RORγ agonists translate into single agent, immune system-dependent, antitumor efficacy when compounds are administered orally in syngeneic tumor models. RORγ agonists integrate multiple antitumor mechanisms into a single therapeutic that both increases immune activation and decreases immune suppression resulting in robust inhibition of tumor growth. Thus, RORγ agonists represent a novel immunotherapy approach for cancer.

T-cell activation requires increased ATP and biosynthesis to support proliferation and effector function. Most models of T-cell activation are based on in vitro culture systems and posit that aerobic glycolysis is employed to meet increased energetic and biosynthetic demands. By contrast, T cells activated in vivo by alloantigens in graft-versus-host disease (GVHD) increase mitochondrial oxygen consumption, fatty acid uptake, and oxidation, with small increases of glucose uptake and aerobic glycolysis. Here we show that these differences are not a consequence of alloactivation, because T cells activated in vitro either in a mixed lymphocyte reaction to the same alloantigens used in vivo or with agonistic anti-CD3/anti-CD28 antibodies increased aerobic glycolysis. Using targeted metabolic 13 C tracer fate associations, we elucidated the metabolic pathway(s) employed by alloreactive T cells in vivo that support this phenotype. We find that glutamine (Gln)-dependent tricarboxylic acid cycle anaplerosis is increased in alloreactive T cells and that Gln carbon contributes to ribose biosynthesis. Pharmacological modulation of oxidative phosphorylation rapidly reduces anaplerosis in alloreactive T cells and improves GVHD. On the basis of these data, we propose a model of T-cell metabolism that is relevant to activated lymphocytes in vivo, with implications for the discovery of new drugs for immune disorders.

Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed by endothelial cells (ECs) that plays a role in tight junction formation, leukocyte adhesion, and transendothelial migration. In the current study, we investigated whether JAM-C is found in soluble form and whether soluble JAM-C (sJAM-C) mediates angiogenesis. We found that JAM-C is present in soluble form in normal serum and elevated in rheumatoid arthritis (RA) serum. The concentration of sJAM-C is also elevated locally in RA synovial fluid compared with RA serum or osteoarthritis synovial fluid. sJAM-C was also present in the culture supernatant of human microvascular ECs (HMVECs) and immortalized human dermal microvascular ECs, and its concentration was increased following cytokine stimulation. In addition, sJAM-C cleavage from the cell surface was mediated in part by a disintegrin and metalloproteinases 10 and 17. In functional assays, sJAM-C was both chemotactic and chemokinetic for HMVECs and induced HMVEC tube formation on Matrigel in vitro. Neutralizing anti–JAM-C Abs inhibited RA synovial fluid–induced HMVEC chemotaxis and sJAM-C–induced HMVEC tube formation on Matrigel. sJAM-C also induced angiogenesis in vivo in the Matrigel plug and sponge granuloma models. Moreover, sJAM-C–mediated HMVEC chemotaxis was dependent on Src, p38, and PI3K. Our results show that JAM-C exists in soluble form and suggest that modulation of sJAM-C may provide a novel route for controlling pathological angiogenesis.

The data showed that the formation of acetic acid-induced ulcer in rats is accompanied by an elevation of collagenase and gelatinase B that gradually tend to return to control values during the healing phase.

The distribution, density and activation of Langerhans cells (LC) has been established in biopsies of normal human buccal mucosa, hard palate, lateral border and dorsum of tongue, floor of mouth and lip taken from sudden death post mortems. LC were identified in cryostat sections with monoclonal antibodies against CD1, HLADR, HLADQ and HLADP using an immunoalkaline phosphatase technique. The use of post mortem material was validated by comparison with biopsies taken from volunteers. LC were predominantly situated in the basal and immediately suprabasal layers of the epithelium. In floor of mouth, lip, lateral border and dorsum of tongue the cells were found along the length of the epithelium. In buccal mucosa, although LC showed fundamentally a similar distribution, a tendency to cluster around the connective tissue papillae was also noted. In hard palate LC were found parallel to the surface in the mid zone of the epithelium. No evidence of LC free areas was found. Dorsum of tongue had the highest density of LC per mm epithelial surface length (28.3 cells per mm) which was significantly greater (P less than 0.05) than buccal mucosa (25.2) which in turn had significantly more cells (P less than 0.05) than lip (22.4). The lowest density of LC was found in lateral border of tongue (17.6), hard palate (17.6) and floor of mouth (16.7). These sites had significantly fewer cells than lip, buccal mucosa and dorsum of tongue (P less than 0.05). Class II MHC molecules are necessary for antigen presentation and in all sites except buccal mucosa there was no significant difference between the number of cells expressing CD1, HLADR, HLADQ and HLADP.(ABSTRACT TRUNCATED AT 250 WORDS)