Elevated ratio of MMP2/MMP9 activity is associated with poor response to chemotherapy in osteosarcoma

Background: Matrix metalloproteinases (MMPs) are crucially involved in the regulation of multiple stages of cancer progression. Elevated MMP levels have been associated with the development of metastases and poor prognosis in several types of cancer. However, the role of MMPs in osteosarcoma and their prognostic value is still unclear. Available data are conflicting, most likely due to different technical approaches. We hypothesized that in contrast to total mRNA or protein levels frequently analyzed in previous studies the enzymatic activities of MMPs and their inhibitors the tissue inhibitors of matrix metalloproteinases (TIMPs) are closer related to their biological functions. We therefore aimed to evaluate the reliability of different zymography techniques for the quantification of MMP and TIMP activities in osteosarcoma biopsies in order to investigate their distribution, possible regulation and prognostic value. Methods: All analyses were done using cryo-conserved osteosarcoma pretreatment biopsies (n = 18). Gene and protein expression of MMPs and TIMPs were analyzed by RT-qPCR and western blot analysis, respectively. Overall MMP activity was analyzed by in situ zymography, individual MMP activities were analyzed by gelatin zymography. Reverse zymography was used to detect and quantify TIMP activities. Results: Strong overall MMP activities could be detected in osteosarcoma pretreatment biopsies with MMP2 and MMP9 as predominant active MMPs. In contrast to total RNA or protein expression MMP2 and MMP9 activities showed significant quantitative differences between good and poor responders. While MMP9 activity was high in the good responder group and significantly decreased in the poor responder group, MMP2 activity showed a reverse distribution. Likewise, significant differences were detected concerning the activity of TIMPs resulting in a negative correlation of TIMP1 activity with MMP2 activity (p = 0.044) and negative correlations of TIMP2 and TIMP3 with MMP9 activity (p = 0.007 and p = 0.006). Conclusion: In contrast to mRNA or protein levels MMP and TIMP activities showed significant differences between the analyzed good and poor responder groups. A shift from MMP9 to predominant MMP2 activity is associated with poor response to chemotherapy suggesting that the ratio of MMP2/MMP9 activity might be a valuable and easily accessible marker to predict the response to chemotherapy in osteosarcoma

Staphylococcus aureus planktonic but not biofilm environment induces an IFN-β macrophage immune response via the STING/IRF3 pathway

ABSTRACTChronic implant-related bone infections are a severe complication in orthopaedic surgery. Biofilm formation on the implant impairs the immune response, leading to bacterial persistence. In a previous study, we found that Staphylococcus aureus (SA) induced interferon regulatory factor 3 (IRF3) activation and Ifnb expression only in its planktonic form but not in the biofilm. The aim of this study was to clarify the role of the stimulator of interferon genes (STING) in this process. We treated RAW 264.7 macrophages with conditioned media (CM) generated from planktonic or biofilm cultured SA in combination with agonists or inhibitors of the cyclic GMP-AMP synthase (cGAS)/STING pathway. We further evaluated bacterial gene expression of planktonic and biofilm SA to identify potential mediators. STING inhibition resulted in the loss of IRF3 activation and Ifnb induction in SA planktonic CM, whereas STING activation induced an IRF3 dependent IFN-β response in SA biofilm CM. The expression levels of virulence-associated genes decreased during biofilm formation, but genes associated with cyclic dinucleotide (CDN) synthesis did not correlate with Ifnb induction. We further observed that cGAS contributed to Ifnb induction by SA planktonic CM, although cGAS activation was not sufficient to induce Ifnb expression in SA biofilm CM. Our data indicate that the different degrees of virulence associated with SA planktonic and biofilm environments result in an altered induction of the IRF3 mediated IFN-β response via the STING pathway. This finding suggests that the STING/IRF3/IFN-β axis is a potential candidate as an immunotherapeutic target for implant-related bone infections

<i>Staphylococcus aureus</i> planktonic but not biofilm environment induces an IFN-β macrophage immune response via the STING/IRF3 pathway

Chronic implant-related bone infections are a severe complication in orthopaedic surgery. Biofilm formation on the implant impairs the immune response, leading to bacterial persistence. In a previous study, we found that Staphylococcus aureus (SA) induced interferon regulatory factor 3 (IRF3) activation and Ifnb expression only in its planktonic form but not in the biofilm. The aim of this study was to clarify the role of the stimulator of interferon genes (STING) in this process. We treated RAW 264.7 macrophages with conditioned media (CM) generated from planktonic or biofilm cultured SA in combination with agonists or inhibitors of the cyclic GMP-AMP synthase (cGAS)/STING pathway. We further evaluated bacterial gene expression of planktonic and biofilm SA to identify potential mediators. STING inhibition resulted in the loss of IRF3 activation and Ifnb induction in SA planktonic CM, whereas STING activation induced an IRF3 dependent IFN-β response in SA biofilm CM. The expression levels of virulence-associated genes decreased during biofilm formation, but genes associated with cyclic dinucleotide (CDN) synthesis did not correlate with Ifnb induction. We further observed that cGAS contributed to Ifnb induction by SA planktonic CM, although cGAS activation was not sufficient to induce Ifnb expression in SA biofilm CM. Our data indicate that the different degrees of virulence associated with SA planktonic and biofilm environments result in an altered induction of the IRF3 mediated IFN-β response via the STING pathway. This finding suggests that the STING/IRF3/IFN-β axis is a potential candidate as an immunotherapeutic target for implant-related bone infections.</p

Reference architectures for trustworthy energy management, desktop grid computing applications, and ubiquitous display environments

This report presents three reference architectures that can be used as architectural blueprints for applications of three different system classes. The first system class comprises applications in the field of energy management; the second one contains applications in the domain of desktop grid computing; the third system class contains multi-user multi-display applications. Because applications in the scope of energy management are safety-critical and desktop grid computing applications have to cope with a variety of self-interested participants, applications of these domains have in common that they can increase their robustness and efficiency by considering the trustworthiness of participants. Multi-user multi-display applications have to assess the social relationships between its users and adapt based on trustworthiness facets such as transparency and controllability. Therefore, the reference architectures given here are based on a multi-agent system that provides functionality for the utilization of trust. Experiences with participants can be stored and evaluated so that trust can be derived and incorporated into the applications. Additionally, the platform provides the basis for open systems in which agents enter and leave dynamically

Melanocortin 1 receptor-signaling deficiency results in an articular cartilage phenotype and accelerates pathogenesis of surgically induced murine osteoarthritis

Proopiomelanocortin-derived peptides exert pleiotropic effects via binding to melanocortin receptors (MCR). MCR-subtypes have been detected in cartilage and bone and mediate an increasing number of effects in diathrodial joints. This study aims to determine the role of MC1-receptors (MC1) in joint physiology and pathogenesis of osteoarthritis (OA) using MC1-signaling deficient mice (Mc1re/e). OA was surgically induced in Mc1re/e and wild-type (WT) mice by transection of the medial meniscotibial ligament. Histomorphometry of Safranin O stained articular cartilage was performed with non-operated controls (11 weeks and 6 months) and 4/8 weeks past surgery. µCT–analysis for assessing epiphyseal bone architecture was performed as a longitudinal study at 4/8 weeks after OA-induction. Collagen II, ICAM-1 and MC1 expression was analysed by immunohistochemistry. Mc1re/e mice display less Safranin O and collagen II stained articular cartilage area compared to WT prior to OA-induction without signs of spontaneous cartilage surface erosion. This MC1-signaling deficiency related cartilage phenotype persisted in 6 month animals. At 4/8 weeks after OA-induction cartilage erosions were increased in Mc1re/e knees paralleled by weaker collagen II staining. Prior to OA-induction, Mc1re/e mice do not differ from WT with respect to bone parameters. During OA, Mc1re/e mice developed more osteophytes and had higher epiphyseal bone density and mass. Trabecular thickness was increased while concomitantly trabecular separation was decreased in Mc1re/e mice. Numbers of ICAM-positive chondrocytes were equal in non-operated 11 weeks Mc1re/e and WT whereas number of positive chondrocytes decreased during OA-progression. Unchallenged Mc1re/e mice display smaller articular cartilage covered area without OA-related surface erosions indicating that MC1-signaling is critical for proper cartilage matrix integrity and formation. When challenged with OA, Mc1re/e mice develop a more severe OA-pathology. Our data suggest that MC1-signaling protects against cartilage degradation and subchondral bone sclerosis in OA indicating a beneficial role of the POMC system in joint pathophysiology

TAFRO Syndrome in Caucasians: A Case Report and Review of the Literature

TAFRO syndrome has been reported in Japan among human herpesvirus 8 (HHV-8)-negative/idiopathic multicentric Castleman's disease (iMCD) patients. To date, the majority of iMCD patients with TAFRO syndrome originate from Japan

HLA-E/human beta2-microglobulin transgenic pigs: protection against xenogeneic human anti-pig natural killer cell cytotoxicity

BACKGROUND: Natural killer (NK) cells participate in pig-to-primate xenograft rejection both by antibody-dependent and -independent mechanisms. A majority of human NK cells express the inhibitory receptor CD94/NKG2A, which binds specifically to human leukocyte antigen (HLA)-E, a trimeric complex consisting of the HLA-E heavy chain, beta2-microglobulin (beta2m), and a peptide derived from the leader sequence of some major histocompatibility complex class I molecules. METHODS: To use this mechanism for protection of pig tissues against human NK cell-mediated cytotoxicity, we generated transgenic pigs by pronuclear microinjection of genomic fragments of HLA-E with an HLA-B7 signal sequence and of human beta2-microglobulin (hubeta2m) into zygotes. RESULTS: Three transgenic founder pigs were generated. Northern blot analysis of RNA from peripheral blood mononuclear cells revealed the presence of the expected transcript sizes for both transgenes in two of the three founders. The founder with the highest expression and his offspring were characterized in detail. Fluorescence-activated cell sorting (FACS) and Western blot analyses demonstrated consistent expression of HLA-E and hubeta2m in peripheral blood mononuclear cells. Immunohistochemistry revealed the presence of HLA-E and hubeta2m on endothelial cells of many organs, including heart and kidney. In vitro studies showed that lymphoblasts and endothelial cells derived from HLA-E/hubeta2m transgenic pigs are effectively protected against human NK cell-mediated cytotoxicity, depending on the level of CD94/NKG2A expression on the NK cells. Further, HLA-E/hubeta2m expression on porcine endothelial cells inhibited the secretion of interferon (IFN)-gamma by co-cultured human NK cells. CONCLUSIONS: This novel approach against cell-mediated xenogeneic responses has important implications for the generation of multitransgenic pigs as organ donors for clinical xenotransplantation

Radiographic evaluation of osteophytes at 4 and 8 weeks past OA induction.

<p>A–C) Knees were oriented as described in material and methods section and were analyzed for osteophytes (white arrow) in transaxial (A), sagittal (B) and coronal (C) plane. F: femur, T: tibia, Fib: fibula, L: lateral and M: medial. D) At 4 and 8 weeks the number and size of osteophytes was calculated for the right and the left knee of each animal, respectively. 4 of 5 Mc1re/e mice developed osteophytes whereas only 2 from 7 WT mice developed one osteophyte after OA-induction.</p