The poor prognosis of patients with soft-tissue sarcoma as not changed in the past several decades, highlighting the necessity for new therapeutic approaches. T-cell based immunotherapies are a promising alternative to traditional cancer treatments due to their ability to target only malignant cells, leaving benign cells unharmed. The development of successful immunotherapy requires the identification and characterization of targetable immunogenic tumor antigens. Cancer-testis antigens (CTA) are a group of highly immunogenic tumor-associated proteins that have emerged as potential targets for CD8+ T-cell recognition. In addition to identifying a targetable antigen, it is crucial to understand the tumor immune microenvironment. The level of immune infiltration and mechanisms of immune suppression within the tumor play important roles in the outcome of immunotherapy. The goal of this study is to identify targetable immunogenic antigens for T-cell based immunotherapy and to characterize the tumor immune microenvironment in human dedifferentiated liposarcoma (DDLS) by Nanostring and IHC. To assess the complexity of the human DDLS tumor immune microenvironment and to identify target antigens we used the nCounter NanoString platform to generate a gene expression profile for hundreds of genes from RNA obtained from 29 DDLS and 10 control fat FFPE samples. To classify inflammatory status of DDLS tumors, we performed hierarchical clustering based on expression levels of selected tumor inflammatory signature genes (CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-E, IDO1, LAG3, PDCDILG2, PSMB10, STAT1, TIGIT). To confirm protein expression and distribution of identified antigens, we performed immunohistochemistry on human tissue micro-arrays encompassing DDLPS tumor tissues and matched normal control tissue from 63 patients. IHC for the cancer testis antigens PBK, SPA17, MAGE-A3, NY-ESO-1 and SSX2 was performed, and the staining results were scored by two authors based on maximal staining intensity on a scale of zero to three (absent=0, weak=1, moderate=2, or strong=3) and the percentage of tumor cells that stained. Hierarchical clustering of DDLS tumors based on expression of tumor inflammation signature genes revealed two distinct groups, consisting of 15 inflamed tumor and 14 non-inflamed tumors, demonstrating tumor heterogeneity within the DDLS sarcoma subtype. All antigens were found to be expressed in DDLS at an mRNA level. SPA17 was expressed at the highest levels in DDLS, however, this antigen was expressed at high levels in normal fat. Notably, antigens PBK and TTK had the largest fold change increase in expression in DDLS compared to normal fat controls. Immunohistochemical analysis of selected antigens revealed that PBK was found to be expressed in 96% (52/54) of DDLS samples at high levels. Other antigens were absent or expressed at low levels in DDLS; MAGEA3 in 15.87% (10/63) NY-ESO-1 in 6.35% (4/62) and SSX2 in 12.7% (8/63) and SPA17 in 5.5% (3/54). This data shows considerable inter-tumoral heterogeneity of inflammation, which should be taken into consideration when designing an immunotherapy for DDLS. To date, these results show promising expression of PBK antigen in DDLS, which may be used as a target in the future development of an immunotherapy for sarcoma

Aim. In vivo biofilm models play major role to study biofilm development, morphology, and regulatory molecules involve in biofilm. Due to ethical restrictions, the use mammalian models are replaced with other alternative models in basic research. Recently, we have developed insect infection model G. mellonella larvae to study implant associated biofilm infections. This model organism is easy to handle, cheap and ethical restriction free and could be used for the high through put screening of antimicrobial compounds to treat biofilm. To promote the use of this model in basic research we aimed to validate this based on the typical biofilm features such as less susceptible to the antibiotics, complexity of the biofilm structure and gene expression profile of biofilms. Method. G. mellonella larvae are maintained at 30oC on artificial diet in an incubator. Titanium and Stainless steel K-wires were cut into small pieces with size of 4mm. After sterilization with 100% alcohol, these K-wires were pre-incubated in S. aureus bacterial suspension (5×10. 6. CFU/ml) for 30 min, washed in PBS and implanted inside the larva after with help of scalpel. The larvae were incubated at 37. o. C for two day for the survival analysis. To analyze the less susceptibility of the biofilms towards antibiotics, the larvae were treated with gentamicin and compared survival with planktonic infection in G. mellonella. To reveal the complex structure of biofilm, the implants were removed and processed for the MALDI analysis. Whole genome-based transcriptome of biofilm was performed to explore the changes in transcriptional landscapes. Results. The results are very promising to validate the use of G. mellonella as in vivo model to study the biofilm formation on implanted materials. The gentamicin treatment could rescue the larvae from the planktonic infection, but not from the biofilm infection on the implants. Further, the MALDI analysis could reveal the complex structure and components of S. aureus biofilm formed on the implant inside the larvae. Finally, the transcriptomic analysis revealed the gene expression changes that can be compared to normal biofilm expression profile. Conclusions. Further, comparison of these results with other in vivo models such as rat and mouse as well as acute and chronic clinical samples from patients with implant-associated bone infections could validate and relevant use of this model to study S. aureus biofilm infections

Introduction. Improvements in material properties of total joint prostheses and methods of fixation mean that arthroplasty is the most effective means of restoring mobility in osteoarthritic patients. Aseptic loosening is the major cause of long-term failure of prostheses. Cobalt particles may act directly on osteoblasts, decreasing bone formation and potentially playing a role in osteolysis and aseptic loosening. Objectives. To assess gene expression profiles of primary human osteoblasts exposed to cobalt ions in a temporal manner, and to identify gene clusters underpinning the osteoblast response to cobalt. Methods. Primary human osteoblasts were exposed to cobalt ions at a concentration of 10ppm. To determine gene expression profiles, comparisons were made between control and 12, 24, 48 and 72 hour time exposures. RNA isolation and cDNA synthesis were performed. Gene profiling was carried out using the Affymetrix Human Genome UI33 Plus 2.0 array. Data was normalised using RMA express and an average expression measure for each time point used to identify alterations in gene expression. Validation was achieved by performing quantitative real time RT-PCR on selected genes. Results. Oligonucleotide microarray profiling identified significant alterations in osteoblast gene expression in response to cobalt exposure. Distinct phase patterns were observed, with significant altered expression following 12, 24 and 48 hours cobalt ion exposure. Of the 22,233 gene sequences represented on the Affymetrix microarray, 4.8% (1077 genes), 4.1% (930 genes), and 2.13% (486 genes) were significantly altered. We identified dysregulation of key functional families in response to cobalt ions, including alterations in cellular proliferation, development and and inflammation. Conclusion. These data will provide novel avenues for exploration to further characterise the molecular mechanisms underpinning the initiation and progression of osteolysis

Osteoarthritis (OA) is a chronic degenerative joint disorder that affects millions of people. There are currently no therapies that reverse or repair cartilage degradation in OA patients. Link N (DHLSDNYTLDHDRAIH) is a naturally occurring peptide that has been shown to increase both collagen and proteoglycan synthesis in chondrocytes and intervertebral disc cells [1,2]. Recent evidence indicates that Link N activates Smad1/5 signaling in cultured rabbit IVD cells presumably by interacting with the bone morphogenetic protein (BMP) type II receptor [3], however, whether a similar mechanism exists in chondrocytes remains unknown. In this study we determined whether Link N can stimulate matrix production and reverse degradation of human OA cartilage under inflammatory conditions. OA cartilage was obtained from donors undergoing total knee arthroplasty with informed consent. OA cartilage/bone explants and OA chondrocytes were prepared from each donor. Cells were prepared in alginate beads (2×106 cells/mL) for gene expression analysis using qPCR. Cells and cartilage explants were exposed to IL-1β (10ng/ml), human Link N (hLN) (1μg/ml) or co-incubated with IL-1β+hLN for 7 and 21 days, respectively. Media was supplemented every three days. Cartilage/bone explants were measured for total glycosaminoglycan (GAG) content (retained and released) using the dimethylmethylene blue (DMMB) assay. Western blotting was performed to determine aggrecan and collagen expression in cartilage tissue. To determine NFκB activation, Western blotting was performed for detection of P-p65 in chondrocytes cultured in 2D following 10 min exposure of IL-1β in the presence of 10, 100, or 1000 ng/mL hLN. Link N significantly decreased in a dose-dependent manner IL-1β-induced NFκB activation in chondrocytes. Gene expression profiling of matrix proteins indicated that there was a trend towards increased aggrecan and decreased collagen type I expression following hLN and IL-1β co-incubation. HLN significantly decreased the IL-1β-induced expression of catabolic enzymes MMP3 and MMP13, and the neuronal growth factor NGF (p < 0 .0001, n=3). In OA cartilage/bone explants, hLN reversed the loss of proteoglycan in cartilage tissue and significantly increased its synthesis whilst in the presence of IL-1β. Link N stimulated proteoglycan synthesis and decreased MMP expression in OA chondrocytes under inflammatory conditions. One mechanism for Link N in preserving matrix protein synthesis may, in part, be due to its ability in rapidly suppressing IL-1β-induced activation of NF-κB. Further work is needed to determine whether Link N directly inhibits the IL-1β receptor or interferes with NFκB activation through an independent pathway(s)

In addition to mechanical stresses, an inflammatory mediated association between obesity and knee osteoarthritis (OA) is increasingly being recognised. Adipokines, such as adiponectin and leptin, have been postulated as likely mediators. Clinical and epidemiological differences in OA by race have been reported. What contributes to these differences is not well understood. In this study, we examined the profile of adipokines in knee synovial fluid (SF) and the gene expression profile of the infra-patellar fat pad (IFP) by race among patients with end-stage knee OA scheduled for knee arthroplasty. Age, sex, weight and height (used to derive body mass index (BMI)) and race (White, Asian and Black) were elicited through self-report questionnaire prior to surgery. SF and IFP samples were collected at the time of surgery. Adipokines (adiponectin and leptin) were examined in the SF using MAGPIX Multiplex platform. IFP was profiled using Human Adipogenesis PCRArray and genes of interest were further validated via quantitative relative RT-PCR using Student's t-test. Overall differences in adiponectin and leptin concentrations were tested across race. Linear regression modeling was used to investigate the association between adiponectin and leptin concentrations (outcomes) and race (predictor; referent group: White), adjusting for age, sex and BMI. 67 patients (18 White, 33 Asian, 16 Black) were included. Mean SF adiponectin concentration was greatest in Whites (1175.05 ng/mL), followed by Blacks (868.53 ng/mL) and Asians (702.23 ng/mL) (p=0.034). The mean SF leptin concentration was highest in Blacks (44.88 ng/mL), followed by Whites (29.86 ng/mL) and Asians (20.18 ng/mL) (p=0.021). Regression analysis showed Asians had significantly lower adiponectin concentrations compared to Whites (p<0.05). However, leptin concentrations did not differ significantly by race after adjusting for covariates. Testing of the IFP, using the Adipogenesis PCRArray, showed significant higher expression of LEP gene (leptin, p=0.03) in Asians (n=4) compared to Whites (n=4). There appears to be important racial differences in the SF adiponectin profile among individuals with end-stage knee OA. Differential gene expression in the IFP across racial groups could be a potential contributory source for the noted SF variations. Further work to determine the source and function of adipokines in knee OA pathophysiology across racial groups is warranted

Tendinopathy is a debilitating musculoskeletal condition which can cause significant pain and lead to complete rupture of the tendon, which often requires surgical repair. Due in part to the large spectrum of tendon pathologies, these disorders continue to be a clinical challenge. Animal models are often used in this field of research as they offer an attractive framework to examine the cascade of processes that occur throughout both tendon pathology and repair. This review discusses the structural, mechanical, and biological changes that occur throughout tendon pathology in animal models, as well as strategies for the improvement of tendon healing.

Cite this article: Bone Joint Res 2014;3:193–202.