Enhanced COMP catabolism detected in serum of patients with arthritis and animal disease models through a novel capture ELISA.

OBJECTIVE: The study aimed determining whether assessment of cartilage oligomeric matrix protein (COMP) degradation products could serve as a serological disease course and therapeutic response predictor in arthritis. METHODS: We generated a panel of monoclonal antibodies against COMP fragments and developed a novel capture enzyme-linked immunosorbent assay (ELISA) for detecting COMP fragments in patients with osteoarthritis (OA) and rheumatoid arthritis (RA). This test was also used to monitor COMP fragments in surgically-induced OA, collagen-induced arthritis (CIA), and tumor necrosis factor (TNF) transgenic animal models. RESULTS: Compared with a commercial COMP ELISA kit that detected no significant difference in COMP levels between OA and control groups, a significant increase of the COMP fragments were noted in the serum of OA patients assayed by this newly established ELISA. In addition, serum COMP fragment levels were well correlated with severity in OA patients and the progression of surgically-induced OA in murine models. Furthermore, the serum levels of COMP fragments in RA patients, mice with CIA, and TNF transgenic mice were significantly higher when compared with their controls. Interestingly, treatment with TNFα inhibitors and methotrexate led to a significant decrease of serum COMP fragments in RA patients. Additionally, administration of Atsttrin [Tang, et al., Science 2011;332(6028):478] also resulted in a significant reduction in COMP fragments in arthritis mice models. CONCLUSION: A novel sandwich ELISA is capable of reproducibly measuring serum COMP fragments in both arthritic patients and rodent arthritis models. This test also provides a valuable means to utilize serum COMP fragments for monitoring the effects of interventions in arthritis.

Comparative analysis with collagen type II distinguishes cartilage oligomeric matrix protein as a primary TGFß-responsive gene.

OBJECTIVE: This study aims to investigate the regulation of expression of Cartilage oligomeric matrix protein (COMP), which is predominately expressed by chondrocytes and functions to organize the extracellular matrix. Mutations in COMP cause two skeletal dysplasias: pseudoachondroplasia and multiple epiphyseal dysplasia. The mechanism controlling COMP expression during chondrocyte differentiation is still poorly understood. DESIGN: Primary human bone marrow-derived stem cells were induced to differentiate into chondrocyte by pellet cultures. We then compared the temporal expression of COMP with the well-characterized cartilage-specific Type II collagen (Col2a1), and their response to transforming growth factor (TGF)ß and Sox trio (Sox5, 6, and 9) stimulation. RESULTS: COMP and Col2a1 expression are differentially regulated by three distinct mechanisms. First, upregulation of COMP mRNA precedes Col2a1 by several days during chondrogenesis. Second, COMP expression is independent of high cell density but requires TGF-ß1. Induction of COMP mRNA by TGF-ß1 is detected within 2h in the absence of protein synthesis and is blocked by specific inhibitors of the TGFß signaling pathway; and therefore, COMP is a primary TFGß-response gene. Lastly, while Col2a1 expression is intimately controlled by the Sox trio, overexpression of Sox trio fails to activate the COMP promoter. CONCLUSION: COMP and Col2a1 expression are regulated differently during chondrogenesis. COMP is a primary response gene of TGFß and its fast induction during chondrogenesis suggests that COMP is suitable for rapidly accessing the chondrogenic potential of stem cells.

Increased friction coefficient and superficial zone protein expression in patients with advanced osteoarthritis.

OBJECTIVE: To quantify the concentration of superficial zone protein (SZP) in the articular cartilage and synovial fluid of patients with advanced osteoarthritis (OA) and to further correlate the SZP content with the friction coefficient, OA severity, and levels of proinflammatory cytokines. METHODS: Samples of articular cartilage and synovial fluid were obtained from patients undergoing elective total knee replacement surgery. Additional normal samples were obtained from donated body program and tissue bank sources. Regional SZP expression in cartilage obtained from the femoral condyles was quantified by enzyme-linked immunosorbent assay (ELISA) and visualized by immunohistochemistry. Friction coefficient measurements of cartilage plugs slid in the boundary lubrication system were obtained. OA severity was graded using histochemical analyses. The concentrations of SZP and proinflammatory cytokines in synovial fluid were determined by ELISA. RESULTS: A pattern of SZP localization in knee cartilage was identified, with load-bearing regions exhibiting high SZP expression. SZP expression patterns were correlated with friction coefficient and OA severity; however, SZP expression was observed in all samples at the articular surface, regardless of OA severity. SZP expression and aspirate volume of synovial fluid were higher in OA patients than in normal controls. Expression of cytokines was elevated in the synovial fluid of some patients. CONCLUSION: Our findings indicate a mechanochemical coupling in which physical forces regulate OA severity and joint lubrication. The findings of this study also suggest that SZP may be ineffective in reducing joint friction in the boundary lubrication mode at an advanced stage of OA, where other mechanisms may dominate the observed tribological behavior.

Zinc opposes genotoxicity of cadmium and vanadium but not of lead.

Protection by essential metals against the genotoxic effects of toxic elements is an open question. Here, human Hs27 dermal fibroblasts and B-mel melanoblasts were exposed for 10 days to (1 µM) zinc (Zn) or copper (Cu) or selenium (+ 4, Sei; + 6, Sea). Afterwards, cells were exposed for 3 days to subtoxic concentrations of lead (Pb, 100 µM) or vanadium (+ 5, V, 2 µM) or cadmium (Cd, 3 µM), slightly reducing, by themselves, cell proliferation and unaffecting cell viability and apoptosis. Genotoxic damage was evaluated by cytokinesis-block micronucleus assay (CBMN) and single cell gel electrophoresis (Comet assay, CA). CBMN and CA were preliminarly assessed following 3, 10 and 30 days of exposure to the above concentrations of Pb, V and Cd: Pb induced micronuclei (MN) formation in both Hs27 and B-mel cells, without determining direct DNA damage (as shown by CA); V did not reveal genotoxic effects on fibroblasts (as shown by CBMN and CA) but increased the frequency of MN and comets in melanoblasts; Cd induced a great number of MN and comets in fibroblasts but not in melanoblasts; all these effects did not differ after 3, 10 or 30 days of exposure to such elements so that Hs27 and B-mel cells were exposed to Pb,V and Cd for 3 days following pretreatment with (1 µM) Zn, Cu, Sei or Sea. By itself, the 10 day-exposure to (1 µM) Zn, Cu, Sei or Sea did not affect cell proliferation, viability, apoptosis and formation of MN or comets in either Hs27 or B-mel cells. Only Zn significantly reduced the Cd- and V-induced MN and comet formation in fibroblasts and melanoblasts, respectively; in these cells, however, Zn did not affect the Pb-induced MN formation. These results emphasize the role of Zn, in respect to other essential metals, in opposing the genotoxic effects of cancerogenic (Cd) or potentially cancerogenic elements (V).

The expression and regulation of nitric oxide synthase in human osteoarthritis-affected chondrocytes: evidence for up-regulated neuronal nitric oxide synthase.

Classically, osteoarthritis (OA) has been considered a noninflammatory disease. However, the detection of selected inflammatory mediators in osteoarthritic fluid, in the absence of significant inflammatory cell infiltrate, is increasingly appreciated. We sought to identify the inflammatory component in human OA-affected cartilage that may be involved in cartilage damage/destruction. Using Western blot analysis and an antibody to the conserved region of nitric oxide synthase (NOS), we have observed up-regulation of NOS, one of the "key players" of inflammation, in chondrocytes of OA-affected patients. Remarkably, none of the cartilage samples examined from normal joints demonstrated detectable amounts of this NOS. Western blot analysis using the same alpha-NOS antibody indicated that this NOS from OA-affected cartilage (OA-NOS) was larger in size than (and distinct from) transfected human hepatocyte or murine inducible NOS (iNOS) (150 versus 133 kD) and similar in size to neuronal constitutive NOS (ncNOS). Antibodies specific for iNOS showed binding to murine and human iNOS but not to OA-NOS, endothelial constitutive NOS, or ncNOS. Antibodies specific for ncNOS bound to ncNOS and also to OA-NOS, but not to murine or human iNOS or endothelial constitutive NOS. Incubation of OA cartilage in serum-free medium resulted in spontaneous release, for up to 72 h, of substantial amounts of nitrite (up to approximately 80 microM/100 mg wet tissue), which could be inhibited by at least 80% with various inhibitors of iNOS, including inhibitors of protein synthesis and transcription factor NF-kappa B, but which (unlike murine macrophage iNOS) was not sensitive to hydrocortisone or TGF-beta. Exposure of OA-affected cartilage to interleukin 1 beta, tumor necrosis factor-alpha, and lipopolysaccharide resulted in approximately 20-50% augmentation of nitrite accumulation, which was also sensitive to cycloheximide and pyrrolidine dithiocarbamate. Hence, our data indicate that OA-NOS (based on immunoreactivity and molecular weight) is similar to ncNOS and that it releases nitric oxide, which may contribute to the inflammation and pathogenesis of cartilage destruction in OA.

The Human Right to Science and the Regulation of Human Germline Engineering.

There is currently no international consensus on how human germline engineering should be regulated. Existing national legislation fails to provide the governance framework necessary to regulate germline engineering in the CRISPR era. This is an obstacle to scientific and clinical advancements and inconsistent with human rights requirements. To move forward, we suggest that the human right to science is an ideal starting point for building consensus, at the national and international levels, on governing principles that promote responsible scientific and technological advancements. Regulatory frameworks must recognize the international nature of modern germline genome engineering research, the need for shared governance rather than tech-locked prohibitions, and the fact that humans are not their germline.

Specific involvement of PKC-epsilon in sensitization of the neuronal response to painful heat.

Pain is unique among sensations in that the perceived intensity increases, or sensitizes, during exposure to a strong stimulus. One important mediator of sensitization is bradykinin (BK), a peptide released as a consequence of tissue damage. BK enhances the membrane ionic current activated by heat in nociceptive neurons, using a pathway that involves activation of protein kinase C (PKC). We find that five PKC isoforms are present in sensory neurons but that only PKC-epsilon is translocated to the cell membrane by BK. The heat response is sensitized when constitutively active PKC-epsilon is incorporated into nociceptive neurons. Conversely, BK-induced sensitization is suppressed by a specific peptide inhibitor of PKC-epsilon. We conclude that PKC-epsilon is principally responsible for sensitization of the heat response in nociceptors by bradykinin.

Inhibition of ADAMTS-7 and ADAMTS-12 degradation of cartilage oligomeric matrix protein by alpha-2-macroglobulin.

OBJECTIVE: As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family, degrade cartilage oligomeric matrix protein (COMP) in vitro and are significantly induced in the cartilage and synovium of arthritic patients [Liu CJ, Kong W, Ilalov K, Yu S, Xu K, Prazak L, et al. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J 2006;20(7):988-90; Liu CJ, Kong W, Xu K, Luan Y, Ilalov K, Sehgal B, et al. ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein. J Biol Chem 2006;281(23):15800-8]. The purpose of this study was to determine (1) whether cleavage activity of ADAMTS-7 and ADAMTS-12 of COMP are associated with COMP degradation in osteoarthritis (OA); (2) whether alpha-2-macroglobulin (a(2)M) is a novel substrate for ADAMTS-7 and ADAMTS-12; and (3) whether a(2)M inhibits ADAMTS-7 or ADAMTS-12 cleavage of COMP. METHODS: An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1-beta (IL-1beta) with or without blocking antibodies; and in human chondrocytes treated with specific small interfering RNA (siRNA) to knockdown ADAMTS-7 or/and ADAMTS-12. Digestion of a(2)M by ADAMTS-7 and ADAMTS-12 in vitro and the inhibition of ADAMTS-7 or ADAMTS-12-mediated digestion of COMP by a(2)M were also analyzed. RESULTS: The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-alpha- or IL-1beta-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-alpha- or IL-1beta-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave a(2)M, giving rise to 180- and 105-kDa cleavage products, respectively. Furthermore, a(2)M inhibited both ADAMTS-7- and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner. CONCLUSION: Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo. Furthermore, a(2)M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, a(2)M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12.

Characterization of the diffraction efficiency of polymer-liquid-crystal-polymer-slices gratings at all incidence angles.

Recently, a novel holographic diffraction grating made of polymer slices alternated to homogeneous films of nematic liquid crystal (POLICRYPS) was realized. We study the optical performance of the POLICRYPS gratings by both numerical simulations and experiments. Characterization of the grating at normal and conical reading mount are performed. The diffraction efficiency depends strongly on the angles of incidence. Besides, the characterization of the diffraction efficiency at Bragg angle incidence is studied. A uniform high diffraction efficiency is achieved when the incident wave satisfies the Bragg condition.

Surface damage to an Oxinium femoral head prosthesis after dislocation.

During open reduction of an irreducible anterior dislocation of a total hip replacement with an Oxinium femoral head, it was observed that the head had been significantly damaged. Gross and scanning electron microscopic examination revealed cracking, gouging, and delamination of the surface. Because of the risk which this poses for damaging the polyethylene acetabular liner, it is strongly recommended that patients with this type of prosthetic head be carefully monitored after a dislocation.

Power frequency magnetic field promotes a more malignant phenotype in neuroblastoma cells via redox-related mechanisms.

In accordance with the classification of the International Agency for Research on Cancer, extremely low frequency magnetic fields (ELF-MF) are suspected to promote malignant progression by providing survival advantage to cancer cells through the activation of critical cytoprotective pathways. Among these, the major antioxidative and detoxification defence systems might be targeted by ELF-MF by conferring cells significant resistance against clinically-relevant cytotoxic agents. We investigated whether the hyperproliferation that is induced in SH-SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field was supported by improved defence towards reactive oxygen species (ROS) and xenobiotics, as well as by reduced vulnerability against both H2O2 and anti-tumor ROS-generating drug doxorubicin. ELF-MF induced a proliferative and survival advantage by activating key redox-responsive antioxidative and detoxification cytoprotective pathways that are associated with a more aggressive behavior of neuroblastoma cells. This was coupled with the upregulation of the major sirtuins, as well as with increased signaling activity of the erythroid 2-related nuclear transcription factor 2 (NRF2). Interestingly, we also showed that the exposure to 50 Hz MF as low as 100 µT may still be able to alter behavior and responses of cancer cells to clinically-relevant drugs.

Peripheral pain mechanisms.

Our understanding of the cellular and molecular bases of transduction of painful stimuli has burgeoned in the past year, mainly as a result of studies on isolated sensory neurones in culture. The ion channels underlying neuronal responses to noxious heat, to protons and to ATP have recently been characterized. The typical increase in nociceptor sensitivity produced by tissue damage has been found to be mediated by at least two distinct mechanisms. In the first, bradykinin augments the current activated by heat through a mechanism that involves activation of protein kinase C. In a second sensitization mechanism, prostaglandin E2 alters the voltage threshold of several ion channels, including a novel tetrodotoxin-insensitive Na+ channel, in such a way that initiation of action potentials is facilitated.

Topical negative pressure in the treatment of infected wounds with exposed orthopaedic implants.

The use of TNP on infected open wounds with exposed orthopaedic implants has not yet been described in the literature. Here, its application on these wounds accelerated healing and enabled definitive wound closure to be undertaken.

Correlation between the distal anterior femoral cortical axis and femoral rotational alignment: an anatomic study.

PURPOSE: To determine the correlation between the distal anterior femoral cortical axis (DAFCA) and the femoral rotational alignment/axis. METHODS: Magnetic resonance images (MRI) of 82 knees in 34 men and 23 women aged 16 to 47 (mean, 33.4) years were reviewed by a musculoskeletal radiologist. Their diagnoses included meniscal tear (n=4), chondromalacia (n=25), anterior cruciate ligament tears (n=11), and normal (n=42). In all patients the collateral ligaments were intact. The transepicondylar axis (TEA), posterior condylar axis (PCA), Whiteside line (WL), and joint line were drawn on the images, and the condylar twist angle (CTA), TEA-WL angle, DAFCA, epicondylar cortical angle (ECA), and condylar cortical angle (CCA) were measured. The correlations among ECA, CCA, and CTA (control) were assessed. RESULTS: The mean distances between the joint line and the TEA, PCA, and DAFCA were 30.8, 22.1, and 62.2 mm, respectively. The angles subtended by the intersection between the standard axes (TEA, PCA, and WL) and the DAFCA were determined. There was correlation between the CTA and ECA (r=0.34, p<0.05), between the ECA and the CCA (r=0.80, p<0.0001), and between the CTA and the CCA (r=- 0.19, p=0.08). CONCLUSION: There was correlation between the DAFCA and TEA and PCA; DAFCA can be used to determine the femoral rotational alignment when the standard landmarks are distorted by severe soft tissue and bone loss.

Antiproliferative effect and apoptotic response in vitro of human melanoma cells to liposomes containing the ribosome-inactivating protein luffin.

The present study describes the liposome-mediated delivery of the type 1 ribosome-inactivating protein luffin to human melanoma cells in vitro. Luffin from Luffa cylindrica seeds has been successfully incorporated into lecithin/cholesterol and lecithin/cholesterol/dicetylphosphate negatively charged liposomes. The exposure of melanoma cells to the two types of liposomes resulted in the inhibition of protein synthesis and cell growth; apoptotic cell death was verified by means of TUNEL reaction and quantitation of cytosolic oligonucleosome-bound DNA. The toxicity of encapsulated luffin varied with the lipid composition of the vesicles; the strongest effect was observed with lecithin/cholesterol liposomes. These results identify liposome-incorporated luffin as a possible alternative to immunotoxins for the treatment of human melanoma in situ.

Rapid synchrony of nuclear and cytosolic Ca2+ signals activated by muscarinic stimulation in the human tumour line TE671/RD.

The functional properties of muscarinic cholinergic receptors have been studied in single cells of the TE671/RD human line. Muscarinic stimulation causes large and quick elevations of cytosolic Ca2+ in the majority of the cells; these persist even in the absence of external Ca2+. Electrophysiological experiments reveal, in addition to the expected nicotinic current, the activation of a K(+)-specific current in response to muscarine. The cell nucleus appears freely permeable to the acid form of Fura-2 and the cytosolic Ca2+ changes easily spread into the nucleus, suggesting free diffusion through nuclear pores. Under appropriate Fura-2 loading conditions, fast (up to 0.5 Hz) Ca2+ oscillations can be observed, usually originating from a restricted cytosolic region. This phenomenon is reflected in fast oscillations of the Ca(2+)-activated K+ current.

Morphofunctional mitochondrial response to methylglyoxal toxicity in Bufo bufo embryos.

Methylglyoxal (2-oxopropanal) is a reactive alpha-oxoaldehyde that can be formed endogenously mainly as a by-product of glycolytic pathway. It is a cytotoxic compound with significant antiproliferative properties as it can bind, under physiological conditions, to nucleic acids and proteins, forming stable adducts. We have recently shown that exogenous methylglyoxal (150-600 microM) is highly toxic for amphibian embryos where it produces, when added to the culture water, inhibition of cell proliferation in the early developmental stages, followed by severe malformations and strongly reduced embryonic viability. In this work we investigate the morphofunctional effect of methylglyoxal on the common toad B. bufo embryo mitochondria in order to verify if its dysmorphogenetic action might be also ascribed to impairment of mitochondrial functions. The mitochondria were isolated from embryos at the developmental stages of morula, neural plate and operculum complete and developing in the presence of 600 microM methylglyoxal. The results show that exogenous methylglyoxal is highly toxic at mitochondrial level, where it produces proliferation, swelling and membrane derangement. As a consequence, mitochondria from treated embryos show decreased oxidative phosphorylation efficiency, as indicated by the significant reduction both of the respiratory control index values and of the embryonic ATP content. On the basis of these data, it is possible that the methylglyoxal-induced embryonic malformations as well as the strongly reduced viability might be also ascribed to energy depletion.

Expression of cartilage oligomeric matrix protein by human synovium.

Human synovium was analyzed for the possible expression of cartilage oligomeric matrix protein (COMP). Immunostaining with polyclonal antiserum to COMP demonstrated positive staining within the synovial cells and immediately subjacent connective tissue, with less intense staining in the deeper connective tissue. Western blot analysis using either polyclonal or monoclonal antibodies to human COMP confirmed the presence of COMP by immunoreactive bands with the same molecular mass (approximately 110 kDa) as purified articular cartilage COMP. PCR using oligonucleotides that span human COMP exons 7-13 revealed identical amplification products from cDNA prepared from either human chondrocytes or synovium. Northern blot analysis using a biotinylated-probe to human COMP, spanning exons 12-13, also reveal an identical hybridization product to either human chondrocyte or synovium total RNA. Human synovium should be considered as a potential tissue source of COMP in any investigation of biological markers of cartilage metabolism.

Matrix metalloproteinases 19 and 20 cleave aggrecan and cartilage oligomeric matrix protein (COMP).

Matrix metalloproteinase (MMP)-19 and MMP-20 (enamelysin) are two recently discovered members of the MMP family. These enzymes are involved in the degradation of the various components of the extracellular matrix (ECM) during development, haemostasis and pathological conditions. Whereas MMP-19 mRNA is found widely expressed in body tissues, including the synovium of normal and rheumatoid arthritic patients, MMP-20 expression is restricted to the enamel organ. In this study we investigated the ability of MMP-19 and MMP-20 to cleave two of the macromolecules characterising the cartilage ECM, namely aggrecan and the cartilage oligomeric matrix protein (COMP). Both MMPs hydrolysed aggrecan efficiently at the well-described MMP cleavage site between residues Asn(341) and Phe(342), as shown by Western blotting using neo-epitope antibodies. Furthermore, the two enzymes cleaved COMP in a distinctive manner, generating a major proteolytic product of 60 kDa. Our results suggest that MMP-19 may participate in the degradation of aggrecan and COMP in arthritic disease, whereas MMP-20, due to its unique expression pattern, may primarily be involved in the turnover of these molecules during tooth development.

Degradation and repair of articular cartilage.

Approximately 95,000 total knee replacements and 41,000 other surgical procedures to repair cartilaginous defects of the knee are performed annually in the United States (1). The response of normal articular cartilage to injury or arthritic degeneration is often a sub-optimal repair; the biochemical and mechanical properties of the new tissue differ from the native cartilage, resulting in inadequate or altered function. It is believed that the chondrocytes from the surrounding areas, although perhaps capable of some limited migration at the damaged site, are not able to proliferate and produce the macromolecules necessary to create an organized matrix characteristic of normal articular cartilage (2,3). Current therapeutic options for articular cartilage injuries and degeneration have resulted in repair tissue which may be hyaline-like, but does not approximate the durability and function of the normal articular surface. Numerous studies have been performed to increase our understanding of the normal repair process of articular cartilage and its limitations, and to devise methods and materials to regenerate the joint surface.