A potential role for Dkk-1 in the pathogenesis of osteosarcoma predicts novel diagnostic and treatment strategies.

Canonical Wnt signaling is an osteo-inductive signal that promotes bone repair through acceleration of osteogenic differentiation by progenitors. Dkk-1 is a secreted inhibitor of canonical Wnt signaling and thus inhibits osteogenesis. To examine a potential osteo-inhibitory role of Dkk-1 in osteosarcoma (OS), we measured serum Dkk-1 in pediatric patients with OS (median age, 13.4 years) and found it to be significantly elevated. We also found that Dkk-1 was maximally expressed by the OS cells at the tumor periphery and _in vitro_ Dkk-1 and RANKL are co-expressed by rapidly proliferating OS cells. Both Dkk-1 and conditioned media from OS cells reduces osteogenesis by human mesenchymal cells and by immuno-depletion of Dkk-1, or by adding a GSK3[beta] inhibitor, the effects of Dkk-1 were attenuated. In mice, we found that the expression of Dkk-1 from implanted tumors was similar to the human tumor biopsies in that human Dkk-1 was present in the serum of recipient animals. These data demonstrate that systemic levels of Dkk-1 are elevated in osteosarcoma. Furthermore, the expression of Dkk-1 by the OS cells at the periphery of the tumor probably contributes to its expansion by inhibiting repair of the surrounding bone. These data demonstrate that Dkk-1 may serve as a prognostic or diagnostic marker for evaluation of OS and furthermore, immuno-depletion of Dkk-1 or administration of GSK3[beta] inhibitors could represent an adjunct therapy for this disease

Expression of a human cartilage procollagen gene (COL2A1) in mouse 3T3 cells.

Expression in a recombinant system has been difficult to obtain for any of the major fibrillar collagens that require processing by eight or more post-translational enzymes. Here, two DNA constructs were designed so that the promoter region of the gene for the pro-alpha 1(I) chain of human type I procollagen drove expression of the human type II procollagen gene in mouse NIH 3T3 cells, a culture line that normally synthesizes type I procollagen but not any cartilage-specific protein such as type II procollagen. Both constructs were expressed as both mRNA and protein. In clones expressing the construct at high levels, the steady-state levels of mRNA and the production of type II procollagen were comparable to the mRNA levels and production of type I procollagen from the endogenous mouse genes. Comparison of clones containing the two constructs demonstrated that sequences extending 80 base pairs beyond the major polyadenylation signal of the gene are not in themselves sufficient for correct termination and 3\u27 processing of RNA transcripts. The results strongly suggest that specific sequences present in a downstream 3.5-kilobase SphI/SphI fragment determine the termination of the transcription. Of special importance is that the system will make it possible to examine the consequences of mutations in the human type II procollagen gene on the processing of RNA transcripts and on the functional properties of the protein simply by using the genomic DNA from leukocytes or other non-cartilaginous sources

Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/β-catenin signaling

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a matrix metalloproteinase (MMP)-independent regulator of growth and apoptosis in various cell types. The receptors and signaling pathways that are involved in the growth factor activities of TIMP-1, however, remain controversial. RNA interference of TIMP-1 has revealed that endogenous TIMP-1 suppresses the proliferation, metabolic activity, and osteogenic differentiation capacity of human mesenchymal stem cells (hMSCs). The knockdown of TIMP-1 in hMSCs activated the Wnt/β-catenin signaling pathway as indicated by the increased stability and nuclear localization of β-catenin in TIMP-1–deficient hMSCs. Moreover, TIMP-1 knockdown cells exhibited enhanced β-catenin transcriptional activity, determined by Wnt/β-catenin target gene expression analysis and a luciferase-based β-catenin– activated reporter assay. An analysis of a mutant form of TIMP-1 that cannot inhibit MMP indicated that the effect of TIMP-1 on β-catenin signaling is MMP independent. Furthermore, the binding of CD63 to TIMP-1 on the surface of hMSCs is essential for the TIMP-1–mediated effects on Wnt/β-catenin signaling. An array analysis of microRNAs (miRNAs) and transfection studies with specific miRNA inhibitors and mimics showed that let-7f miRNA is crucial for the regulation of β-catenin activity and osteogenic differentiation by TIMP-1. Let-7f was up-regulated in TIMP-1–depleted hMSCs and demonstrably reduced axin 2, an antagonist of β-catenin stability. Our results demonstrate that TIMP-1 is a direct regulator of hMSC functions and reveal a regulatory network in which let-7f modulates Wnt/β-catenin activity

Differential patterns of PMN-elastase and type III procollagen peptide in knee joint effusions due to acute and chronic sports injuries

In 38 traumatic knee joint effusions the proteolytic enzyme PMN-elastase (PMN-E) and the repair marker procollagen III aminoterminal peptide (PIIINP) were determined. According to the period between trauma and first aspiration of the effusion, the patients were divided into 3 groups. Group I (17 patients; period between trauma and first aspiration not longer than 72 hours) showed high concentrations of PMN-E (up to 5400 ng/ml) and low concentrations of PIIINP (<13 U/ml). Group II (11 patients; aspiration within 4 to 14 days) had mean PMN-E and PIIINP concentrations of 125.6 ng/ml and 52.1 U/ ml, respectively. In group III (10 patients, aspiration after 14 days) mean PMN-E concentration was 123.8 ng/ml and mean PIIINP concentration was 63.4 U/ml. Graphic depiction of PMN-E and PIIINP levels in each individual sample as a function of time between trauma and fluid collection revealed highly increasing PMN-E levels during the first 24 posttraumatic hours, followed by rapidly decreasing levels within 72 hours post trauma, and no change after the 4th posttraumatic day. In contrast, PIIINP increased continuously up to the first posttraumatic week and stayed at high levels up to 90 days (end of the observation period). The differential patterns of PMN-E and PIIINP concentration in knee joint effusions may be useful in estimating the period between trauma and first treatment (aspiration of effusion) and should, therefore, be helpful in detecting degenerative lesions, which seem to be characterized by low PMN-E concomitantly with high PIIINP levels

Toxicity of tryptophan manganese(I) carbonyl (Trypto-CORM), against Neisseria gonorrhoeae

The potential for carbon monoxide-releasing molecules (CO-RMs) as antimicrobials represents an exciting prospective in the fight against antibiotic resistance. Trypto-CORM, a tryptophan-containing manganese(i) carbonyl, is toxic against E. coli following photo-activation. Here, we demonstrate that Trypto-CORM is toxic against Neisseria gonorrhoeae in the absence of photoactivation. Trypto-CORM toxicity was reversed by the high CO affinity globin leg-haemoglobin (Leg-Hb), indicating that the toxicity is due to CO release. Release of CO from Trypto-CORM in the dark was also detected with Leg-Hb (but not myoglobin) in vitro. N. gonorrhoeae is more sensitive to CO-based toxicity than other model bacterial pathogens, and may serve as a viable candidate for antimicrobial therapy using CO-RMs

Interphase chromosome positioning in in vitro porcine cells and ex vivo porcine tissues

Copyright @ 2012 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and 85 reproduction in any medium, provided the original author and source are credited. The article was made available through the Brunel University Open Access Publishing Fund.BACKGROUND: In interphase nuclei of a wide range of species chromosomes are organised into their own specific locations termed territories. These chromosome territories are non-randomly positioned in nuclei which is believed to be related to a spatial aspect of regulatory control over gene expression. In this study we have adopted the pig as a model in which to study interphase chromosome positioning and follows on from other studies from our group of using pig cells and tissues to study interphase genome re-positioning during differentiation. The pig is an important model organism both economically and as a closely related species to study human disease models. This is why great efforts have been made to accomplish the full genome sequence in the last decade. RESULTS: This study has positioned most of the porcine chromosomes in in vitro cultured adult and embryonic fibroblasts, early passage stromal derived mesenchymal stem cells and lymphocytes. The study is further expanded to position four chromosomes in ex vivo tissue derived from pig kidney, lung and brain. CONCLUSIONS: It was concluded that porcine chromosomes are also non-randomly positioned within interphase nuclei with few major differences in chromosome position in interphase nuclei between different cell and tissue types. There were also no differences between preferred nuclear location of chromosomes in in vitro cultured cells as compared to cells in tissue sections. Using a number of analyses to ascertain by what criteria porcine chromosomes were positioned in interphase nuclei; we found a correlation with DNA content.This study is partly supported by Sygen International PLC

Hematopoietic Cell Transplantation in Patients With Primary Immune Regulatory Disorders (PIRD): A Primary Immune Deficiency Treatment Consortium (PIDTC) Survey.

Primary Immune Regulatory Disorders (PIRD) are an expanding group of diseases caused by gene defects in several different immune pathways, such as regulatory T cell function. Patients with PIRD develop clinical manifestations associated with diminished and exaggerated immune responses. Management of these patients is complicated; oftentimes immunosuppressive therapies are insufficient, and patients may require hematopoietic cell transplant (HCT) for treatment. Analysis of HCT data in PIRD patients have previously focused on a single gene defect. This study surveyed transplanted patients with a phenotypic clinical picture consistent with PIRD treated in 33 Primary Immune Deficiency Treatment Consortium centers and European centers. Our data showed that PIRD patients often had immunodeficient and autoimmune features affecting multiple organ systems. Transplantation resulted in resolution of disease manifestations in more than half of the patients with an overall 5-years survival of 67%. This study, the first to encompass disorders across the PIRD spectrum, highlights the need for further research in PIRD management

Tracking of autologous adipose tissue-derived mesenchymal stromal cells with in vivo magnetic resonance imaging and histology after intralesional treatment of artificial equine tendon lesions - a pilot study

Background: Adipose tissue-derived mesenchymal stromal cells (AT-MSCs) are frequently used to treat equine tendinopathies. Up to now, knowledge about the fate of autologous AT-MSCs after intralesional injection into equine superficial digital flexor tendons (SDFTs) is very limited. The purpose of this study was to monitor the presence of intralesionally injected autologous AT-MSCs labelled with superparamagnetic iron oxide (SPIO) nanoparticles and green fluorescent protein (GFP) over a staggered period of 3 to 9 weeks with standing magnetic resonance imaging (MRI) and histology. Methods: Four adult warmblood horses received a unilateral injection of 10 × 106 autologous AT-MSCs into surgically created front-limb SDFT lesions. Administered AT-MSCs expressed lentivirally transduced reporter genes for GFP and were co-labelled with SPIO particles in three horses. The presence of AT-MSCs in SDFTs was evaluated by repeated examinations with standing low-field MRI in two horses and post-mortem in all horses with Prussian blue staining, fluorescence microscopy and with immunofluorescence and immunohistochemistry using anti-GFP antibodies at 3, 5, 7 and 9 weeks after treatment. Results: AT-MSCs labelled with SPIO particles were detectable in treated SDFTs during each MRI in T2*- and T1-weighted sequences until the end of the observation period. Post-mortem examinations revealed that all treated tendons contained high numbers of SPIO- and GFP-labelled cells. Conclusions: Standing low-field MRI has the potential to track SPIO-labelled AT-MSCs successfully. Histology, fluorescence microscopy, immunofluorescence and immunohistochemistry are efficient tools to detect labelled AT-MSCs after intralesional injection into surgically created equine SDFT lesions. Intralesional injection of 10 × 106 AT-MSCs leads to the presence of high numbers of AT-MSCs in and around surgically created tendon lesions for up to 9 weeks. Integration of injected AT-MSCs into healing tendon tissue is an essential pathway after intralesional administration. Injection techniques have to be chosen deliberately to avoid reflux of the cell substrate injected. In vivo low-field MRI may be used as a non-invasive tool to monitor homing and engraftment of AT-MSCs in horses with tendinopathy of the SDFT