A randomized, double-blind, placebo-controlled assessment of BMS-936558, a fully human monoclonal antibody to programmed death-1 (PD-1), in patients with chronic hepatitis C virus infection.

UNLABELLED: Expression of the programmed death 1 (PD-1) receptor and its ligands are implicated in the T cell exhaustion phenotype which contributes to the persistence of several chronic viral infections, including human hepatitis C virus (HCV). The antiviral potential of BMS-936558 (MDX-1106) - a fully human anti-PD-1 monoclonal immunoglobulin-G4 that blocks ligand binding - was explored in a proof-of-concept, placebo-controlled single-ascending-dose study in patients (N = 54) with chronic HCV infection. Interferon-alfa treatment-experienced patients (n = 42) were randomized 5∶1 to receive a single infusion of BMS-936558 (0.03, 0.1, 0.3, 1.0, 3.0 mg/kg [n = 5 each] or 10 mg/kg [n = 10]) or of placebo (n = 7). An additional 12 HCV treatment-naïve patients were randomized to receive 10 mg/kg BMS-936558 (n = 10) or placebo (n = 2). Patients were followed for 85 days post-dose. Five patients who received BMS-936558 (0.1 [n = 1] or 10 mg/kg) and one placebo patient achieved the primary study endpoint of a reduction in HCV RNA ≥0.5 log10 IU/mL on at least 2 consecutive visits; 3 (10 mg/kg) achieved a >4 log10 reduction. Two patients (10 mg/kg) achieved HCV RNA below the lower limit of quantitation (25 IU/mL), one of whom (a prior null-responder) remained RNA-undetectable 1 year post-study. Transient reductions in CD4(+), CD8(+) and CD19(+) cells, including both naïve and memory CD4(+) and CD8(+) subsets, were observed at Day 2 without evidence of immune deficit. No clinically relevant changes in immunoglobulin subsets or treatment-related trends in circulating cytokines were noted. BMS-936558 exhibited dose-related exposure increases, with a half-life of 20-24 days. BMS-936558 was mostly well tolerated. One patient (10 mg/kg) experienced an asymptomatic grade 4 ALT elevation coincident with the onset of a 4-log viral load reduction. Six patients exhibited immune-related adverse events of mild-to-moderate intensity, including two cases of hyperthyroidism consistent with autoimmune thyroiditis. Further investigation of PD-1 pathway blockade in chronic viral disease is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT00703469.

Mechanical injury potentiates proteoglycan catabolism induced by interleukin-6 with soluble interleukin-6 receptor and tumor necrosis factor alpha in immature bovine and adult human articular cartilage.

OBJECTIVE: Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNFalpha. METHODS: Injured or uninjured cartilage disks were incubated with or without TNFalpha and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6-blocking antibody Fab fragment and subjected to injury and TNFalpha treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. RESULTS: In bovine cartilage samples, injury combined with TNFalpha and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNFalpha exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6-blocking Fab significantly reduced proteoglycan degradation. CONCLUSION: Our findings indicate that mechanical injury potentiates the catabolic effects of TNFalpha and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFalpha appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6-blocking Fab.

Mechanical injury of cartilage explants causes specific time-dependent changes in chondrocyte gene expression.

OBJECTIVE: Joint injury in young adults leads to an increased risk of developing osteoarthritis (OA) later in life. This study was undertaken to determine if injurious mechanical compression of cartilage explants results in changes at the level of gene transcription that may lead to subsequent degradation of the cartilage. METHODS: Cartilage was explanted from the femoropatellar groove of newborn calves. Levels of messenger RNA encoding matrix molecules, proteases, their natural inhibitors, transcription factors, and cytokines were assessed in free swelling control cultures as compared with cartilage cultures at 1, 2, 4, 6, 12, and 24 hours after application of a single injurious compression. RESULTS: Gene-expression levels measured in noninjured, free swelling cartilage varied over 5 orders of magnitude. Matrix molecules were the most highly expressed of the genes tested, while cytokines, matrix metalloproteinases (MMPs), aggrecanases (ADAMTS-5), and transcription factors showed lower expression levels. Matrix molecules showed little change in expression after injurious compression, whereas MMP-3 increased approximately 250-fold, ADAMTS-5 increased approximately 40-fold, and tissue inhibitor of metalloproteinases 1 increased approximately 12-fold above the levels in free swelling cultures. Genes typically used as internal controls, GAPDH and beta-actin, increased expression levels approximately 4-fold after injury, making them unsuitable for use as normalization genes in this study. The expression levels of tumor necrosis factor alpha and interleukin-1beta, cytokines known to be involved in the progression of OA, did not change in the chondrocytes after injury. CONCLUSION: Changes in the level of gene expression after mechanical injury are gene specific and time dependent. The quantity of specific proteins may be altered as a result of these changes in gene expression, which may eventually lead to degradation at the tissue level and cause a compromise in cartilage structure and function.

Evaluation of medium supplemented with insulin-transferrin-selenium for culture of primary bovine calf chondrocytes in three-dimensional hydrogel scaffolds.

Insulin-transferrin-selenium (ITS) was investigated as a complete or partial replacement for fetal bovine serum (FBS) during in vitro culture of bovine calf chondrocytes in hydrogel scaffolds. Chondrocyte-seeded agarose and self-assembling peptide hydrogels were maintained in Dulbecco's modified Eagle's medium plus 10% FBS, 1% ITS plus 0.2% FBS, or 1% ITS and evaluated for biosynthesis, cell division, and surface outgrowth of fibroblastic-like cells and fibrous capsule formation over several weeks of culture. In peptide hydrogels, cells cultured in ITS plus 0.2% FBS medium exhibited high rates of biosynthesis and showed similar cell division trends as seen in 10% FBS cultures. ITS medium alone did not support glycosaminoglycan accumulation beyond 5 days of culture, and cell division was less than that in both serum-containing cultures. Extensive cellular outgrowth and fibrous capsule formation were observed in 10% FBS medium, whereas little outgrowth was observed in ITS plus 0.2% FBS and none was seen in ITS medium alone. In agarose hydrogels, chondrocyte biosynthesis and cell division in ITS medium were similar to that in 10% serum culture over 5 weeks, and cellular outgrowth was eliminated. Taken together, ITS was suitable as a partial (peptide) or complete (agarose) substitute for serum, and also provided the benefit of reducing or eliminating cell outgrowth and fibrous capsule formation on the hydrogel surface.

Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds.

Dynamic mechanical loading has been reported to affect chondrocyte biosynthesis in both cartilage explant and chondrocyte-seeded constructs. In this study, the effects of dynamic compression on chondrocyte-seeded peptide hydrogels were analyzed for extracellular matrix synthesis and retention over long-term culture. Initial studies were conducted with chondrocyte-seeded agarose hydrogels to explore the effects of various non-continuous loading protocols on chondrocyte biosynthesis. An optimized alternate day loading protocol was identified that increased proteoglycan (PG) synthesis over control cultures maintained in free-swelling conditions. When applied to chondrocyte-seeded peptide hydrogels, alternate day loading stimulated PG synthesis up to two-fold higher than that in free-swelling cultures. While dynamic compression also increased PG loss to the medium throughout the 39-day time course, total PG accumulation in the scaffold was significantly higher than in controls after 16 and 39 days of loading, resulting in an increase in the equilibrium and dynamic compressive stiffness of the constructs. Viable cell densities of dynamically compressed cultures differed from free-swelling controls by less than 20%, demonstrating that changes in PG synthesis were due to an increase in the average biosynthesis per viable cell. Protein synthesis was not greatly affected by loading, demonstrating that dynamic compression differentially regulated the synthesis of PGs. Taken together, these results demonstrate the potential of dynamic compression for stimulating PG synthesis and accumulation for applications to in vitro culture of tissue engineered constructs prior to implantation.

Mechanisms and kinetics of glycosaminoglycan release following in vitro cartilage injury.

OBJECTIVE: Acute joint injury leads to increased risk for osteoarthritis (OA). Although the mechanisms underlying this progression are unclear, early structural, metabolic, and compositional indicators of OA have been reproduced using in vitro models of cartilage injury. This study was undertaken to determine whether glycosaminoglycan (GAG) loss following in vitro cartilage injury is mediated by cellular biosynthesis, activation of enzymatic activity, or mechanical disruption of the cartilage extracellular matrix. METHODS: Immature bovine cartilage was cultured for up to 10 days. After 3 days, groups of samples were subjected to injurious mechanical compression (single uniaxial unconfined compression to 50% thickness, strain rate 100% per second). GAG release to the medium was measured, and levels were compared with those in location-matched, uninjured controls. The effects of medium supplementation with inhibitors of biosynthesis (cycloheximide), of matrix metalloproteinase (MMP) activity (CGS 27023A or GM 6001), and of aggrecanase activity (SB 703704) on GAG release after injury were assessed. RESULTS: GAG release from injured cartilage was highest during the first 4 hours after injury, but remained higher than that in controls during the first 24 hours postinjury, and was not affected by inhibitors of biosynthesis or degradative enzymes. GAG release during the period 24-72 hours postinjury was similar to that in uninjured controls, but the MMP inhibitor CGS 27023A reduced cumulative GAG loss from injured samples between 1 day and 7 days postinjury. Other inhibitors of enzymatic degradation or biosynthesis had no significant effect on GAG release. CONCLUSION: Injurious compression of articular cartilage induces an initially high rate of GAG release from the tissue, which could not be inhibited, consistent with mechanical damage. However, the finding that MMP inhibition reduced GAG loss in the days following injury suggests a potential therapeutic intervention.

Proteoglycan degradation after injurious compression of bovine and human articular cartilage in vitro: interaction with exogenous cytokines.

OBJECTIVE: Traumatic joint injury leads to an increased risk of osteoarthritis (OA), but the progression to OA is not well understood. We undertook this study to measure aspects of proteoglycan (PG) degradation after in vitro injurious mechanical compression, including up-regulation of enzymatic degradative expression and cytokine-stimulated degradation. METHODS: Articular cartilage tissue explants were obtained from newborn bovine femoropatellar groove and from adult normal human donor knee and ankle tissue. Following injurious compression of the cartilage, matrix metalloproteinase 3 (MMP-3) and MMP-13 messenger RNA (mRNA) expression levels were measured by Northern analysis, and PG loss to the medium after cartilage injury was measured in the presence and absence of added exogenous cytokine (interleukin-1alpha [IL-1alpha] or tumor necrosis factor alpha [TNFalpha]). RESULTS: During the first 24 hours after injury in bovine cartilage, MMP-3 mRNA levels increased 10-fold over the levels in control cartilage (n = 3 experiments), whereas MMP-13 mRNA levels were unchanged. PG loss was significantly increased after injury, but only by 2% of the total PG content and only for the first 3 days following injury. However, compared with injury alone or cytokine treatment alone, treatment of injured tissue with either 1 ng/ml IL-1alpha or 100 ng/ml TNFalpha caused marked increases in PG loss (35% and 54%, respectively, of the total cartilage PG content). These interactions between cytokine treatment and injury were statistically significant. In human knee cartilage, the interaction was also significant for both IL-1alpha and TNFalpha, although the magnitude of increase in PG loss was lower than that in bovine cartilage. In contrast, in human ankle cartilage, there was no significant interaction between injury and IL-1alpha. CONCLUSION: The cytokines IL-1alpha and TNFalpha can cause a synergistic loss of PG from mechanically injured bovine and human cartilage. By attempting to incorporate interactions with other joint tissues that may be sources of cytokines, in vitro models of mechanical cartilage injury may explain aspects of the interactions between mechanical forces and degradative pathways which lead to OA progression.

Development-associated differences in integrative cartilage repair: roles of biosynthesis and matrix.

A recurring problem in tissue transplantation therapies for articular cartilage defects is the lack of integration between the implant and the host cartilage. Previous studies have shown that in vitro integration between explants of calf cartilage is markedly higher than that between fetal cartilage, despite similarly high levels of deposition of newly synthesized collagen. The aim of this study was to determine if cellular biosynthesis and extracellular matrix each contribute to these development-associated differences in integrative repair in vitro. The approach taken was to examine integration between specific combinations of cartilage explants that were apposed for two weeks. The cartilage matrix showed different propensities for repair, as integration of calf live cartilage to calf devitalized cartilage was greater than that of calf live cartilage to fetal devitalized cartilage. An inhibiting factor appeared to be present in fetal cartilage matrix since guanidine treatment of fetal devitalized cartilage was able to enhance its integration. The difference between integration to living cartilage and integration to devitalized cartilage, for calf and fetal tissue, indicated that the biosynthetic contribution to integration by calf cartilage was greater than the biosynthetic contribution by fetal cartilage. Thus, the increasing level of integration between fetal and fetal cartilage, fetal and calf cartilage, and calf and calf cartilage appeared to reflect both biosynthetic and matrix differences. Therapeutic strategies to enhance integration to cartilage may thus target both the extracellular components and the cellular biosynthetic activities of implants and host cartilage.