John Rediske

Pain related behaviour in two models of osteoarthritis in the rat knee

Abstract Osteoarthritis (OA) is a major healthcare burden, with increasing incidence. Pain is the predominant clinical feature, yet therapy is ineffective for many patients. While there are considerable insights into the mechanisms underlying tissue remodelling, there is poor understanding of the link between disease pathology and pain. This is in part owing to the lack of animal models that combine both osteoarthritic tissue remodelling and pain. Here, we provide an analysis of pain related behaviours in two models of OA in the rat: partial medial meniscectomy and iodoacetate injection. Histological studies demonstrated that in both models, progressive osteoarthritic joint pathology developed over the course of the next 28 days. In the ipsilateral hind limb in both models, changes in the percentage bodyweight borne were small, whereas marked mechanical hyperalgesia and tactile allodynia were seen. The responses in the iodoacetate treated animals were generally more robust, and these animals were tested for pharmacological reversal of pain related behaviour. Morphine was able to attenuate hyperalgesia 3, 14 and 28 days after OA induction, and reversed allodynia at days 14 and 28, providing evidence that this behaviour was pain related. Diclofenac and paracetamol were effective 3 days after arthritic induction only, coinciding with a measurable swelling of the knee. Gabapentin varied in its ability to reverse both hyperalgesia and allodynia. The iodoacetate model provides a basis for studies on the mechanisms of pain in OA, and for development of novel therapeutic analgesics.Abbreviations: OA: osteoarthritis; DRGs: dorsal root ganglia; NSAIDs: non‐steroidal anti‐inflammatory drugs; PWT: paw withdrawal thresholds; p.o.: orally; s.c.: sub cutaneously.

Inflammation-Induced Chondrocyte Hypertrophy Is Driven by Receptor for Advanced Glycation End Products

The multiligand receptor for advanced glycation end products (RAGE) mediates certain chronic vascular and neurologic degenerative diseases accompanied by low-grade inflammation. RAGE ligands include S100/calgranulins, a class of low-molecular-mass, calcium-binding polypeptides, several of which are chondrocyte expressed. Here, we tested the hypothesis that S100A11 and RAGE signaling modulate osteoarthritis (OA) pathogenesis by regulating a shift in chondrocyte differentiation to hypertrophy. We analyzed human cartilages and cultured human articular chondrocytes, and used recombinant human S100A11, soluble RAGE, and previously characterized RAGE-specific blocking Abs. Normal human knee cartilages demonstrated constitutive RAGE and S100A11 expression, and RAGE and S100A11 expression were up-regulated in OA cartilages studied by immunohistochemistry. CXCL8 and TNF-α induced S100A11 expression and release in cultured chondrocytes. Moreover, S100A11 induced cell size increase and expression of type X collagen consistent with chondrocyte hypertrophy in vitro. CXCL8-induced, IL-8-induced, and TNF-α-induced but not retinoic acid-induced chondrocyte hypertrophy were suppressed by treatment with soluble RAGE or RAGE-specific blocking Abs. Last, via transfection of dominant-negative RAGE and dominant-negative MAPK kinase 3, we demonstrated that S100A11-induced chondrocyte type X collagen expression was dependent on RAGE-mediated p38 MAPK pathway activation. We conclude that up-regulated chondrocyte expression of the RAGE ligand S100A11 in OA cartilage, and RAGE signaling through the p38 MAPK pathway, promote inflammation-associated chondrocyte hypertrophy. RAGE signaling thereby has the potential to contribute to the progression of OA.

Phase I study of the anti-CD40 humanized monoclonal antibody lucatumumab (HCD122) in relapsed chronic lymphocytic leukemia

Abstract Lucatumumab is a fully humanized anti-CD40 antibody that blocks interaction of CD40L with CD40 and also mediates antibody-dependent cell-mediated cytotoxicity (ADCC). We evaluated lucatumumab in a phase I clinical trial in chronic lymphocytic leukemia (CLL). Twenty-six patients with relapsed CLL were enrolled on five different dose cohorts administered weekly for 4 weeks. The maximally tolerated dose (MTD) of lucatumumab was 3.0 mg/kg. Four patients at doses of 4.5 mg/kg and 6.0 mg/kg experienced grade 3 or 4 asymptomatic elevated amylase and lipase levels. Of the 26 patients enrolled, 17 patients had stable disease (mean duration of 76 days, range 29–504 days) and one patient had a nodular partial response for 230 days. Saturation of CD40 receptor on CLL cells was uniform at all doses post-treatment but also persisted at trough time points in the 3.0 mg/kg or greater cohorts. At the MTD, the median half-life of lucatumumab was 50 h following the first infusion, and 124 h following the fourth infusion. In summary, lucatumumab had acceptable tolerability, pharmacokinetics that supported chronic dosing and pharmacodynamic target antagonism at doses of 3.0 mg/kg, but demonstrated minimal single-agent activity. Future efforts with lucatumumab in CLL should focus on combination-based therapy.

A phase 1 study of lucatumumab, a fully human anti-CD40 antagonist monoclonal antibody administered intravenously to patients with relapsed or refractory multiple myeloma.

In this open‐label, multicentre, phase 1 study a fully human anti‐CD40 antagonist monoclonal antibody, lucatumumab, was evaluated in patients with relapsed/refractory multiple myeloma (MM). The primary objective was to determine the maximum tolerated dose (MTD) based on dose‐limiting toxicities (DLTs). Secondary objectives included safety, pharmacokinetics, pharmacodynamics and antimyeloma activity. Twenty‐eight patients, enrolled using a standard ‘3 + 3’ dose escalation, received one or two (n = 3) cycles of lucatumumab 1·0, 3·0, 4·5 or 6·0 mg/kg once weekly for 4 weeks. Common lucatumumab‐related adverse events were reversible, mild‐to‐moderate infusion reactions. Severe adverse events were anaemia, chills, hypercalcaemia and pyrexia (7% each). DLTs included grade 4 thrombocytopenia, grade 3 increased alanine aminotransferase and grade 4 increased lipase (n = 1 each). The MTD was 4·5 mg/kg. At doses ≥3·0 mg/kg, sustained receptor occupancy (≥87%), observed throughout weekly infusions up to 5 weeks after the last infusion, correlated with an estimated half‐life of 4–19 d. Twelve patients (43%) had stable disease, and one patient (4%) maintained a partial response for ≥8 months. These findings indicate that single‐agent lucatumumab was well tolerated up to 4·5 mg/kg with modest clinical activity in relapsed/refractory MM, warranting further study as a combination therapy.

Inducible nitric oxide synthase from human articular chondrocytes: cDNA cloning and analysis of mRNA expression

Human articular chondrocytes can be induced by IL-1 beta, TNF-alpha or LPS to release high levels of nitric oxide. Using degenerate PCR primers based on homologous regions from previously cloned NOS enzymes, a 1.9 kb cDNA fragment was amplified from IL-1 beta stimulated but not from resting chondrocytes. Screening of a lambda gt11 cDNA library, which was prepared from RNA of IL-1 beta activated chondrocytes, resulted in the isolation of the complete cDNA, encoding a protein of 1153 amino acids. Comparison of the cDNA sequence identified human chondrocyte iNOS to be almost identical to the sequence recently reported for the hepatocyte enzyme, differing in 12 amino acids. Northern blot analysis revealed, that stimulated chondrocytes express a single 4.5 kb iNOS mRNA species. IL-1 beta induction of iNOS mRNA was detectable by 6 h and continued to be elevated throughout a 72 h culture period. Screening of a human bone cDNA library identified this inducible NOS to be also expressed by bone cells.

The inducible production of nitric oxide by articular cell types

Nitric oxide (NO) may play a role in tissue remodeling associated with arthritis. The articular cell sources of human inducible NO synthesis, however, have not been defined. This study demonstrates that human articular chondrocytes in primary or organ culture, but not synovial fibroblasts, produce NO in response to catabolic cytokines such as interleukin-1 (IL-1). As measured by the accumulation of NO2- in culture medium, NO production by IL-1-stimulated chondrocytes was inhibited by the NO synthase inhibitor Ng-monomethyl-L-arginine (NMA) and dependent on the presence of exogenous L-arginine. Other inflammatory cytokines such as tumor necrosis factor, but not transforming growth factor-beta, induced chondrocyte NO synthesis. The stimulation of NO synthesis required both RNA and protein synthesis. Chondrocytes isolated from cartilage derived from osteoarthritic patients also produced large amounts of NO in response to IL-1. In beginning to define potential effects of NO on chondrocyte function, it is shown that IL-1 induced an increase in cyclic guanosine monophosphate (cGMP) which was inhibited by NMA. In summary, these results demonstrate that cytokine-induced production of NO is a response of human articular chondrocytes but not of synovial fibroblasts. A potential role of NO in cytokine-induced tissue remodeling in the joint is provided by the induction of cGMP.

Inhibition of interleukin‐1α‐induced cartilage oligomeric matrix protein degradation in bovine articular cartilage by matrix metalloproteinase inhibitors: Potential role for matrix metalloproteinases in the generation of cartilage oligomeric matrix protein fragments in arthritic synovial fluid

OBJECTIVE To determine whether matrix metalloproteinases (MMPs) degrade cartilage oligomeric matrix protein (COMP) to produce fragments similar to those found in synovial fluid (SF) from patients with arthritis. METHODS COMP fragments were generated in vitro by treating (a) bovine articular cartilage with interleukin-1alpha (IL-1alpha), (b) purified bovine COMP with MMPs, and (c) articular cartilage with MMPs. The fragments generated in each case were analyzed by Western blot, using an antibody to the C-terminal heptadecapeptide of COMP. RESULTS IL-1alpha stimulation of cartilage resulted in a fragmentation of COMP, which was inhibited by MMP inhibitors CGS 27023A and BB-94. Isolated, recombinant MMPs rapidly degraded purified COMP, as well as COMP residing in cartilage. Several COMP fragments produced in vitro had similar electrophoretic mobility to those in SF of patients with arthritis. CONCLUSION MMPs may contribute to the COMP fragments found in vivo. Quantitation of MMP-specific fragments may be useful in the evaluation of MMP inhibitors in patients with arthritis.

Identification of a splice variant of neutrophil collagenase (MMP-8)

We have identified a splice variant of human neutrophil collagenase (MMP‐8) transcript (MMP‐8alt) that has a 91 bp insertion between codons for amino acid residues 34 and 35 of MMP‐8 cDNA. This splice variant encodes an open reading frame for a 444 residue protein, lacking a secretory signal sequence. Our data suggested that, as opposed to the original MMP‐8, the translation product of MMP‐8alt is not a secreted protein; nevertheless, it is enzymatically active. Further studies aimed at identifying the physiological substrates of MMP‐8alt protein may lead to uncover novel roles it plays in cellular physiology.

Characterization of the binding of bolton-hunter labeled [125I]C5a to human neutrophil, monocyte and U-937 cell membranes

The fifth component of the complement cascade, C5a, was iodinated using the Bolton-Hunter reagent. Results from the present study, using the high affinity ligand, [125I]Bolton-Hunter-labeled C5a ([125I]BH-C5a), revealed a single binding site on membranes prepared from human neutrophils, U-937 cells and human monocytes. Saturation studies demonstrated Bmax values in these cells of 11.5, 47.3 and 16.6 fmol/10(6) cells, respectively. The C5a receptor demonstrated a very high affinity for [125I]BH-C5a of approximately 4 pM in each cell type. Competition studies using analogs of C5a generated a similar order of potency in each of the cell types of C5a > or = C5a(1-74), Ser66Ala > C5a(1-73) > C5a(1-69). These studies indicate that [125I]BH-C5a labels a similar receptor in neutrophil, U-937 cell and monocyte membranes. Furthermore, C5a(1-73) produced shallow inhibition curves in competition experiments in each cell type. Computer analysis of the binding data revealed two components of binding. When 10 nM unlabeled C5a was used to initiate dissociation of [125I]BH-C5a binding in neutrophil membranes, two binding components were observed. In addition, dissociation of [125I]BH-C5a binding by 10 nM unlabeled C5a in the presence of 1 mM GppNHp decreased the percentage of binding to the slowly dissociating, high affinity binding component from 84 to 58%. These results suggest that multiple states of the C5a receptor exist.

Platelet activating factor stimulates intracellular calcium transients in human neutrophils: involvement of endogenous 5-lipoxygenase products.

Stimulation of human neutrophils with platelet activating factor (PAF) resulted in a transient elevation of free cytosolic calcium. Neutrophils exhibited a two‐component calcium response observed as a double peak when stimulated with > 5 nM PAF. In contrast, leukotriene B4 (LTB4), C5a, or formylmethionyl‐leucyl‐phenylalanine stimulated only a single‐peak calcium response. The double‐peak calcium response was not elicited in human monocytes or differentiated U937 cells, which demonstrated a single peak. Pretreatment of neutrophils with a 5‐lipoxygenase inhibitor or a specific LTB4‐receptor antagonist selectively blocked the second calcium peak. These results suggest that PAF‐mediated activation of human neutrophils results in the activation of the 5‐lipoxygenase and the subsequent generation of LTB4. This LTB4 in turn elicits a secondary rise in calcium, which contributes to the overall response of neutrophils to PAF. These results demonstrate how LTB4 participates in the cellular responses elicited by PAF in human neutrophils.