In Vitro Study of Novel Collagenase (XIAFLEX?) on Dupuytren's Disease Fibroblasts Displays Unique Drug Related Properties

Dupuytren''s disease (DD) is a benign, fibroproliferative disease of the palmar fascia, with excessive extracellular matrix (ECM) deposition and over-production of cytokines and growth factors, resulting in digital fixed flexion contractures limiting hand function and patient quality of life. Surgical fasciectomy is the gold standard treatment but is invasive and has associated morbidity without limiting disease recurrence. Injectable Collagenase Clostridium histolyticum (CCH) - Xiaflex® - is a novel, nonsurgical option with clinically proven in vivo reduction of DD contractures but with limited in vitro data demonstrating its cellular and molecular effects. The aim of this study was to delineate the effects of CCH on primary fibroblasts isolated from DD and non-DD anatomical sites (using RTCA, LDH, WST-1, FACS, qRT-PCR, ELISA and In-Cell Quantitative Western Blotting) to compare the efficacy of varying concentrations of Xiaflex® against a reagent grade Collagenase, Collagenase A. Results demonstrated that DD nodule and cord fibroblasts had greater proliferation than those from fat and skin. Xiaflex® exposure resulted in dose- and time-dependent inhibition of cellular spreading, attachment and proliferation, with cellular recovery after enzyme removal. Unlike Collagenase A, Xiaflex® did not cause apoptosis. Collagen expression patterns were significantly (p<0.05) different in DD fibroblasts across anatomical sites - the highest levels of collagen I and III were detected in DD nodule, with DD cord and fat fibroblasts demonstrating a smaller increase in both collagen expression relative to DD skin. Xiaflex® significantly (p<0.05) down-regulated ECM components, cytokines and growth factors in a dose-dependent manner. An in vitro scratch wound assay model demonstrated that, at low concentrations, Xiaflex® enabled a faster fibroblast reparatory migration into the wound, whereas, at high concentrations, this process was significantly (p<0.05) inhibited. This is the first report elucidating potential mechanisms of action of Xiaflex® on Dupuytren fibroblasts, offering a greater insight and a better understanding of its effect in DD.     

Effect of low-power helium-neon laser irradiation on 13-week immobilized articular cartilage of rabbits

Influence of low-power (632.8 nm, Helium-Neon, 13 J/cm2, three times a week) laser on 13-week immobilized articular cartilage was examined with rabbits knee model. Number of chondrocytes and depth of articular cartilage of experimental group were significantly higher than those of sham irradiated group. Surface morphology of sham-irradiated group had rough prominences, fibrillation and lacunae but surface morphology of experimental group had more similarities to control group than to sham irradiated group. There were marked differences between ultrastructure features of control group and experimental group in comparison with sham irradiated group. Low-power Helium-Neon laser irradiation on 13-week immobilized knee joints of rabbits neutrilized adverse effects of immobilization on articular cartilage.     

Ribosomal S6 kinase-1 modulates interleukin-1beta-induced persistent activation of NF-kappaB through phosphorylation of IkappaBbeta

Activation of NF-B requires the phosphorylation and degradation of its associated inhibitory proteins, IB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1 to induce persistent activation of NF-B in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-B activation. In cultured rat VSMCs, IL-1 activated ERK and induced degradation of both IB and IB, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-B p65. RSK1, a downstream kinase of ERK, was associated with an IB/NF-B complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1 decreased IB in the RSK1/IB/NF-B complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1-induced IB decrease without influencing ether ERK phosphorylation or the earlier IB degradation. By using recombinant wild-type and mutant IB proteins, both active ERK2 and RSK1 were found to directly phosphorylate IB, but only active RSK1 phosphorylated IB on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IB and contributes to the persistent activation of NF-B by IL-1. extracellular signal-regulated kinase; in vitro phosphorylation assay; recombinant proteins; small interference RNA; vascular smooth muscle cell     

DARPin Ec1-LMWP protein scaffold in targeted delivery of siRNA molecules through EpCAM cancer stem cell marker

Molecular Biology Reports - This study is to investigate the binding ability of Designed Ankyrin Repeat Proteins type Ec1that was fused to Low Molecular Weight Protamine (DARPin Ec1-LMWP) protein...     

Photobiomodulation of a flowable matrix in a human skin ex vivo model demonstrates energy‐based enhancement of engraftment integration and remodeling

The use of dermal substitutes to treat skin defects such as ulcers has shown promising results, suggesting a potential role for skin substitutes for treating acute and chronic wounds. One of the main drawbacks with the use of dermal substitutes is the length of time from engraftment to graft take, plus the risk of contamination and failure due to this prolonged integration. Therefore, the use of adjuvant energy‐based therapeutic modalities to augment and accelerate the rate of biointegration by dermal substitute engraftments is a desirable outcome. The photobiomodulation (PBM) therapy modulates the repair process, by stimulating cellular proliferation and angiogenesis. Here, we evaluated the effect of PBM on a collagen‐glycosaminoglycan flowable wound matrix (FWM) in an ex vivo human skin wound model. PBM resulted in accelerated rate of re‐epithelialization and organization of matrix as seen by structural arrangement of collagen fibers, and a subsequent increased expression of alpha‐smooth muscle actin (α‐SMA) and vascular endothelial growth factor A (VEGF‐A) leading to an overall improved healing process. The use of PBM promoted a beneficial effect on the rate of integration and healing of FWM. We therefore propose that the adjuvant use of PBM may have utility in enhancing engraftment and tissue repair and be of value in clinical practice.      

Iron accumulation in deep cortical layers accounts for MRI signal abnormalities in ALS: correlating 7 Tesla MRI and pathology

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by cortical and spinal motor neuron dysfunction. Routine magnetic resonance imaging (MRI) studies have previously shown hypointense signal in the motor cortex on T(2)-weighted images in some ALS patients, however, the cause of this finding is unknown. To investigate the utility of this MR signal change as a marker of cortical motor neuron degeneration, signal abnormalities on 3T and 7T MR images of the brain were compared, and pathology was obtained in two ALS patients to determine the origin of the motor cortex hypointensity. Nineteen patients with clinically probable or definite ALS by El Escorial criteria and 19 healthy controls underwent 3T MRI. A 7T MRI scan was carried out on five ALS patients who had motor cortex hypointensity on the 3T FLAIR sequence and on three healthy controls. Postmortem 7T MRI of the brain was performed in one ALS patient and histological studies of the brains and spinal cords were obtained post-mortem in two patients. The motor cortex hypointensity on 3T FLAIR images was present in greater frequency in ALS patients. Increased hypointensity correlated with greater severity of upper motor neuron impairment. Analysis of 7T T(2)(*)-weighted gradient echo imaging localized the signal alteration to the deeper layers of the motor cortex in both ALS patients. Pathological studies showed increased iron accumulation in microglial cells in areas corresponding to the location of the signal changes on the 3T and 7T MRI of the motor cortex. These findings indicate that the motor cortex hypointensity on 3T MRI FLAIR images in ALS is due to increased iron accumulation by microglia.     

Proteolytic release of membrane-bound endo-(1,4)-beta-glucanase activity associated with cell wall softening in Achlya ambisexualis

Branching and other cell wall softening events in fungi and oomycetes are thought to involve the activity of secreted enzymes, which are packaged in membrane vesicles and delivered to sites of cell expansion, there to work in a carefully regulated manner upon the structure of the wall. Here we demonstrate a latent endo-(1,4)-beta-glucanase activity in a mixed membrane fraction of the oomycete Achlya ambisexualis, which can be released by cysteine proteases with an increase of apparent activity. In addition, a similar endogenous process is strongly inhibited by the cysteine protease inhibitor iodoacetamide, while inhibitors of other types of proteases have a much smaller effect. Detergent treatment of membranes releases two glucanases detectable by electrophoretic activity staining, with apparent molecular masses of about 164 and 35 kDa. Proteolysis produces several activity bands, with major species having apparent molecular masses of about 149, 133, 48, 35, and 25 kDa. The ca. 35- and 25-kDa bands migrate in parallel with glucanases secreted during wall softening in vivo. We propose that the initiation of wall softening in Achlya involves the proteolytic processing and solubilization of at least some secreted endoglucanases. We also propose that the solubilization component of this process functions not just to provide the enzymes with access to wall matrix substrates but also may provide a mechanism for the eventual termination of their biological function.     

Ruminal large and small particle kinetics in dairy cows fed red clover and grass silages harvested at two stages of growth

Passage, comminution and digestion rates of large and small particles were estimated using a rumen evacuation technique and total faecal collection with five lactating dairy cows in a 5 × 5 Latin square experiment. Two grass and two red clover silages harvested at early and late primary growth stages and a 1:1 mixture of late harvest grass and early harvest red clover were the dietary treatments. Cows received 9.0 kg supplementary concentrate per day. Ruminal contents and faeces were divided into large (>1.25 mm) and small (1.25–0.038 mm) particles by wet sieving. Indigestible neutral detergent fibre (iNDF) was determined by 12 days ruminal in situ incubation followed by neutral detergent extraction. Plant species did not affect ruminal particle size distribution, whereas advancing forage maturity decreased the proportion of large particles for both grass and red clover silage diets. Ruminal pool size of iNDF was higher (P<0.001) with red clover compared to grass silage diets. Ruminal passage rates of iNDF and potentially digestible NDF (pdNDF) increased with decreasing particle size (P<0.01). Passage rate of iNDF for small particles was slower (P<0.01) when red clover compared to grass silage diets were fed. Particle comminution rate in the rumen was slower (P<0.001) with red clover compared to grass silage diets and it increased (P<0.01) with advancing forage maturity. The contribution of particle comminution to ruminal mean retention time of iNDF in the ruminal large particle pool was smaller (P<0.01) in red clover compared to grass silage diets and it increased (P<0.05) with the mixed silage compared to the separate silages. Passage rate of pdNDF for both large and small particles was not affected by dietary treatments. Digestion rate of pdNDF for large particles was faster (P<0.001) with red clover compared to grass silage diets. Differences in ruminal passage and digestion rates of the large and small particles, in addition to differences in the passage and digestion rates of red clover compared to grass silage diets, emphasize the need to consider particle size and forage type in metabolic models predicting feed intake and fibre digestibility in ruminants.