Targeting of ADAMTS5's ancillary domain with the recombinant mAb CRB0017 ameliorates disease progression in a spontaneous murine model of osteoarthritis.

OBJECTIVE: ADAMTS5 (aggrecanase-2) has been demonstrated to be crucial in the development of osteoarthritis (OA), by use of several mouse mutants carrying either truncated, catalytically inactive enzymes or aggrecanase-resistant mutant aggrecan. We have selected recombinant monoclonal antibodies directed against ADAMTS5, by using Intracellular Antibody Capture Technology (IACT). CRB0017 revealed very high affinity for the enzyme in Biacore analyses and very good specificity in a panel of binding assays. Therefore, we tested CRB0017 in a relevant spontaneous OA model, the STR/ort mouse. DESIGN: STR/ort male mice were recruited at 5 months of age, and treated intra-articularly in each knee with CRB0017 1.2 µg, CRB0017 12 µg, or vehicle. After 6 weeks, the intra-articular administration of CRB0017 was repeated with the same doses. After 3 months from recruitment, the animals were sacrificed and the femorotibial joints processed for histology and scored in a blind fashion according to both Mankin's and the OARSI methods. RESULTS AND CONCLUSIONS: All histological scores were significantly decreased in the CRB0017 12 µg/knee group compared to vehicle, while administration of CRB0017 1.2 µg was associated with a trend to a decrease in the same parameters. Therefore, CRB0017 administered twice in 3 months could modify the course of OA in the STR/ort mouse, by delaying cartilage breakdown as assessed histologically. The procedure of blind scoring of the histological samples clearly showed that knee intra-articular administration of CRB0017, an anti-ADAMTS5 antibody, dose-dependently improved disease progression in a relevant animal model of OA.

The neuronal microtubule-associated protein tau is a substrate for caspase-3 and an effector of apoptosis.

We have identified a class of tau fragments inducing apoptosis in different cellular contexts, including a human teratocarcinoma-derived cell line (NT2 cells) representing committed human neuronal precursors. We have found a transition point inside the tau molecule beyond which the fragments lose their ability to induce apoptosis. This transition point is located around one of the putative caspase-3 cleavage sites. This is the only site that can be effectively used by caspase-3 in vitro, releasing the C-terminal 19 amino acids of tau. These results establish tau as a substrate for an apoptotic protease that turns tau itself into an effector of apoptosis. Accordingly, tau may be involved in a self-propagating process like what has been predicted for the pathogenesis of different neurodegenerative disorders.

Selection of antibodies for intracellular function using a two-hybrid in vivo system.

Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.

A new approach to retrain gait in stroke patients through body weight support and treadmill stimulation.

BACKGROUND AND PURPOSE: A new gait training strategy for patients with stroke proposes to support a percentage of the patient's body weight while retraining gait on a treadmill. This research project intended to compare the effects of gait training with body weight support (BWS) and with no body weight support (no-BWS) on clinical outcome measures for patients with stroke. METHODS: One hundred subjects with stroke were randomized to receive one of two treatments while walking on a treadmill: 50 subjects were trained to walk with up to 40% of their body weight supported by a BWS system with overhead harness (BWS group), and the other 50 subjects were trained to walk bearing full weight on their lower extremities (no-BWS group). Treatment outcomes were assessed on the basis of functional balance, motor recovery, overground walking speed, and overground walking endurance. RESULTS: After a 6-week training period, the BWS group scored significantly higher than the no-BWS group for functional balance (P = 0.001), motor recovery (P = 0.001), overground walking speed (P = 0.029), and overground w alking endurance (P = 0.018). The follow-up evaluation, 3 months after training, revealed that the BWS group continues to have significantly higher scores for overground walking speed (P = 0.006) and motor recovery (P = 0.039). CONCLUSIONS: Retraining gait in patients with stroke while a percentage of their body weight was supported resulted in better walking abilities than gait training while the patients were bearing their full weight. This novel gait training strategy provides a dynamic and integrative approach for the treatment of gait dysfunction after stroke.

Does neurorehabilitation play a role in the recovery of walking in neurological populations?

This review demonstrates that neurorehabilitation approaches, based on recent neuroscience findings, can enhance locomotor recovery after a spinal cord injury or stroke. Findings are presented from more than 20 clinical studies conducted by numerous research groups on the effect of locomotor training using either body weight support (BWS), functional electrical stimulation (FES), pharmacological approaches or a combination of them. Among the approaches, only BWS-assisted locomotor training has been demonstrated to have a greater effect than conventional or locomotor training alone. However, when study results were combined and weighted for the number of subjects, the results indicated that there is a gradient of effects from small changes with the immediate application of FES or BWS to larger changes when locomotor training is combined with FES or BWS or pharmacological approaches. The findings of these studies suggest that these neurorehabilitation approaches do play a role in the recovery of walking in subjects with spinal cord injury or stroke. Several factors contribute to the potential for recovery including the site, etiology, and chronicity of the injury, as well as the type, duration, and specificity of the intervention and whether interventions are combined. Furthermore, how these neurorehabilitation approaches may take advantage of the plasticity process following neurological lesion is also discussed.

The effects of parallel bars, body weight support and speed on the modulation of the locomotor pattern of spastic paretic gait. A preliminary communication.

The effects of walking with and without parallel bars, providing 40% body weight support (BWS) and increasing speed on the gait pattern of spastic paretic subjects during treadmill locomotion were investigated. In asymmetrically involved subjects, walking without parallel bars led to a more symmetrical gait pattern with decreased compensation of the less involved side. This was accompanied by changes in electromyographic (EMG) and sagittal angular displacement profiles which favoured a more normal swing phase of the more involved limb. When symmetrically involved subjects walked without parallel bars, increases in EMG activity, with prolonged activation during the stance phase were noted, especially in the distal muscles. Providing 40% BWS facilitated gait when walking without parallel bars especially in the asymmetrically or severely involved subjects who showed marked difficulty at 0% BWS. Forty percent BWS led to a decrease in clonus associated with walking without parallel bars. Higher treadmill speeds increased clonus in some subjects while in others it only caused a small increase in EMG amplitude. Implications for gait training are discussed.

Potassium channels of pig articular chondrocytes are blocked by propofol.

The effect of propofol on the voltage-activated potassium channels in pig articular chondrocytes was investigated. Propofol was found to reversibly block the potassium channels in a dose-dependent manner. The blocking effect was voltage-independent and the Hill coefficient was 1.85 +/- 0.18. No changes either in the slope conductance or in the single channel kinetics were observed. The half-blocking concentration (Ec50) was 6.0 +/- 0.49 microM which is much lower than the concentrations used to observe the scavenging effect of the drug in an artificial synovial fluid. Interestingly, Ec50 found in our experiments is also smaller than the blood concentration of propofol used in anaesthesia. These results show that propofol may strongly affect the potassium channels in some non-excitable cells.

The effects of body weight support on the locomotor pattern of spastic paretic patients.

The effects of mechanically supporting a percentage of body weight on the gait pattern of spastic paretic subjects during treadmill locomotion was investigated. Electromyographic (EMG), joint angular displacement and temporal distance data were simultaneously recorded while 7 spastic paretic subjects walked at 0% and 40% body weight support (BWS) at their maximal comfortable treadmill speed. Forty percent BWS produced a general decrease in EMG mean burst amplitude for the lower limb muscles investigated with instances of more appropriate EMG timing in relation to the gait cycle. The joint angular displacement data at 40% BWS revealed straighter trunk and knee alignment during the weight bearing phase especially at initial foot-floor contact and midstance. An increase in single limb support time and a decrease in percentage total double support time were evident at 40% BWS. An increase in stride length and maximum comfortable walking speed was also seen with BWS. The use of BWS during treadmill locomotion as a therapeutic approach to retrain gait in neurologically impaired patients is discussed.