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Purpose The purpose of this paper is to investigate how family members co-create value and improve the well-being of patients with chronic developmental disorders, such as Asperger syndrome (AS) that undertake permanent therapy services. Design/methodology/approach Qualitative methodology is used to identify family value co-creation activities and well-being outcomes. Extensive interviews with family members and professional therapists of AS patients were conducted as the main data collection method. Findings Drawing from previous conceptualizations of value co-creation activities in health contexts, the findings of this study identify the specific value co-creation activities held by family members that influence the different dimensions of well-being for AS patients and their families: co-learning, combining therapies, changing ways of doing things, connecting, co-operation and co-production, managing daily life, motivating, protecting, regulating and establishing roles. The findings also reveal improvements in the following dimensions of patient well-being: autonomy, self-acceptance, purpose in life, positive relationships with others, control of the environment and personal growth. In addition, value co-creation activities also improve family relationships at home and the well-being of patient family members. Originality/value This study contributes to the services literature and addresses a gap in transformative service research by exploring the value co-creation activities of family members for improving well-being outcomes of patients with chronic developmental disorders. People with chronic developmental disorders engage in permanent therapy services and tend to have below-average well-being scores, which also extends to their family members.

The immune system is tasked to keep our body unharmed and healthy. In the immune system, B- and T-lymphocytes are the two main components working together to stop and eliminate invading threats like virus particles, bacteria, fungi and parasite from attacking our healthy cells. The function of antibodies is relatively more direct in target recognition as compared to T-cell receptors (TCR) which recognizes antigenic peptides being presented on the major histocompatibility complex (MHC). Although phage display has been widely applied for antibody presentation, this is the opposite in the case of TCR. The cell surface TCR is a relatively large and complex molecule, making presentation on phage surfaces challenging. Even so, recombinant versions and modifications have been introduced to allow the growing development of TCR in phage display. In addition, the increasing application of TCR for immunotherapy has made it an important binding motif to be developed by phage display. This review will emphasize on the application of phage display for TCR discovery as well as the engineering aspect of TCR for improved characteristics.

The stabilized R-spine of oncogenic BRAF mutants impairs RAF inhibitor association and confers drug resistance., Although targeting BRAF mutants with RAF inhibitors has achieved promising outcomes in cancer therapy, drug resistance remains a remarkable challenge, and underlying molecular mechanisms are not fully understood. Here, we characterized a previously unknown group of oncogenic BRAF mutants with in-frame insertions (LLRins506 or VLRins506) of αC-β4 loop. Using structure modeling and molecular dynamics simulation, we found that these insertions formed a large hydrophobic network that stabilizes R-spine and thus triggers the catalytic activity of BRAF. Furthermore, these insertions disrupted BRAF dimer interface and impaired dimerization. Unlike BRAF(V600E), these BRAF mutants with low dimer affinity were strongly resistant to all RAF inhibitors in clinic or clinical trials, which arises from their stabilized R-spines. As predicted by molecular docking, the stabilized R-spines in other BRAF mutants also conferred drug resistance. Together, our data indicated that the stability of R-spine but not dimer affinity determines the RAF inhibitor resistance of oncogenic BRAF mutants.

Mesenchymal stem cells (MSCs) possess some unique features (inherent tumor tropism, anti-inflammatory and immunosuppressive properties) that are not commonly found in conventional anti-cancer agents. These cells are known to secrete a vast array of proteins including growth factors, cytokines, chemokines, extracellular matrix metalloproteinases, and their corresponding inhibitors which exhibit profound effects on the microenvironment. However, the lack of a uniform method for culturing MSCs and their paracrine factors has hindered our understanding of MSC biology. In this chapter, we describe methods for the isolation, in vivo expansion, and phenotypic characterization of MSCs. In addition, methods for the collection and concentration of conditioned medium from these MSCs are described. Using tumor cells that constitutively express fluorescence reporter proteins, the effect of conditioned medium on tumor cell viability can be easily tested in vitro.

Glioblastoma multiforme (GBM) is the most aggressive human brain tumor. Standard of care includes surgical resection, radiation therapy, and concomitant adjuvant chemotherapy with temozolomide (TMZ) which can only modestly improve median survival. Resistance to treatment is often associated with a heterogeneous population of cells, with various genetic aberrations coexisting within the same cancer. In recent years, improvements have been targeted at improving the precision of tumor debulking during surgery and using thermal and electric fields to increase tumor cell kill. In this chapter however, we discuss innovative technologies for targeted GBM therapies under preclinical and clinical evaluation. These include modulating the MGMT activities to overcome TMZ resistance, the use of virotherapies, microRNAs, antiangiogenic and anti-EGFR therapies.

Glial cell invasion is a multistep cellular process that involves a complex system of tightly regulated proteases (matrix metalloproteinases; MMPs) and their endogenous inhibitors (tissue inhibitors of metalloproteinases; TIMPs) to mediate the degradation of the basement membrane and extracellular matrix. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a matrix-bound inhibitor of MMPs. In the present study, we have overexpressed the TIMP3 gene in human glioma cells with a herpes simplex virus type 1 amplicon-based vector. Oligonucleotide DNA arrays were employed to identify genes that were differentially modulated by the overexpression of TIMP-3. Consistent with the function of TIMP-3, genes associated with angiogenesis, growth factors, cytokines, death receptors, and substrates of the various MMPs were found to be up-regulated. Furthermore, caspases are important in the signaling pathway of cellular apoptosis, and the overexpression of TIMP-3 in glioma cells is tightly associated with the activation of caspases, including caspase-1, at both the mRNA level (P=0.0371) and the protein level. Moreover, the activation of an apoptotic pathway via the overexpression of TIMP-3 induced apoptosis of transduced human glioma cells in vitro and the growth inhibition of human glioma tumor xenografts in immunodeficient mice.

Pax3 is an evolutionarily conserved transcription factor expressed in the lateral dermomyotome, a region that gives rise to limb muscle progenitors. Mutations in Pax-3 account for the mouse mutant Splotch which develops without limb musculature. We demonstrate that Pax3 can inhibit myogenic differentiation of C2C12 myoblasts normally induced by exposure to low serum. Specific missense mutations that affect the DNA binding characteristics of the two distinct DNA binding domains of Pax3 abolish this effect. Furthermore, we show that Pax3 can inhibit myogenic differentiation of 10T1/2 fibroblasts transfected with MyoD, but not of 10T1/2 cells transfected with myogenin. This anti-myogenic property is shared by a PAX3-forkhead fusion protein resulting from a t(2;13) chromosomal translocation found in pediatric alveolar rhabdomyosarcomas. These results suggest that Pax3 may suppress the terminal differentiation of migrating limb myoblasts and that the PAX3-forkhead fusion may contribute to the phenotype of alveolar rhabdomyosarcoma by preventing terminal differentiation.

Celastrol, a plant triterpene has attracted great interest recently, especially for its potential anti-inflammatory and anti-cancer activities. In the present report, we investigated the effect of celastrol on proliferation of various cancer cell lines. The mechanism, by which this triterpene exerts its apoptotic effects, was also examined in detail. We found that celastrol inhibited the proliferation of wide variety of human tumor cell types including multiple myeloma, hepatocellular carcinoma, gastric cancer, prostate cancer, renal cell carcinoma, head and neck carcinoma, non-small cell lung carcinoma, melanoma, glioma, and breast cancer with concentrations as low as 1 μM. Growth inhibitory effects of celastrol correlated with a decrease in the levels of cyclin D1 and cyclin E, but concomitant increase in the levels of p21 and p27. The apoptosis induced by celastrol was indicated by the activation of caspase-8, bid cleavage, caspase-9 activation, caspase-3 activation, PARP cleavage and through the down regulation of anti-apoptototic proteins. The apoptotic effects of celastrol were preceded by activation of JNK and down-regulation of Akt activation. JNK was needed for celastrol-induced apoptosis, and inhibition of JNK by pharmacological inhibitor abolished the apoptotic effects. Overall, our results indicate that celastrol can inhibit cell proliferation and induce apoptosis through the activation of JNK, suppression of Akt, and down-regulation of anti-apoptotic protein expression.

Previously, we have demonstrated that not all adult human bone marrow-derived mesenchymal stem cells can migrate efficiently towards tumor cells. In the current study, we attempt to address whether different samples of human fetal bone marrow-derived mesenchymal stem cells (hfMSC) also exhibit different migratory capacities toward tumors as was found to be the case with adult human MSC. Further, we are keen to explore the underlying mechanism of how hfMSC home to tumor cells. Using modified Boyden chamber assay, we demonstrated that hfMSC migrate at an efficiency comparable or better than the highly migratory adult MSC. Unlike the adult MSC, the extent of migration was not correlated to the expression and activity of MMP1. Rather, it appeared to be dependent in part on the PAR1 expression which may in turn be modulated by GPCR signal pathways. Additional evaluation will need to be done to further confirm the exact migratory mechanism. Nevertheless, hfMSC may potentially serve as equally efficient carriers of therapeutic genes to tumors.

One of the challenges in gene therapy is to ensure stable transgene expression at the site of disease with a high degree of accuracy and safety. In this paper, we examine both viral and cellular elements that may affect the level of transgene expression mediated by herpes simplex virus type 1 (HSV-1) adeno-associated virus (AAV) amplicon vectors. These elements include the AAV inverted terminal repeats (ITRs), the AAV Rep proteins, and the allelic status of 19q in human glioma cell lines. The latter is of particular interest because the AAV integration site (AAVS1) is located on the long arm of chromosome 19 and 30-40% of human glioblastoma tumors are reported to have loss of heterozygosity in this region of chromosome 19q. Fluorescence-activated cell-sorting analysis results indicate that inclusion of minimal or full-length AAV ITRs in HSV-1 amplicon vectors markedly increases the efficiency of transgene expression. On the other hand, insertion of the AAV rep gene decreases the level of transgene expression, apparently because of the cytotoxic effects of Rep proteins. Further, the levels of transgene expression appear to be independent of 19q allelic status or the number of endogenous AAVS1 sequences in the various glioma cell lines studied. Taken together, these data support employing AAV ITRs, in the context of HSV-1 amplicon vectors, to enhance short-term levels of transgene expression.

2066 Background: The human epidermal growth factor receptor (EGFR) is an ideal therapeutic target for inhibiting glioblastoma (GBM) growth as the signaling pathway is highly dysregulated in GBM. However, trials so far with EGFR inhibitors have not shown clinically meaningful improvement in response rates and survival. One of the postulated mechanisms of resistance is the constitutive activation of the downstream, PI3K/AKT/mTOR pathway, independent of the upstream EGFR activation status. Hence, the dual blockage of the pathways with an anti-EGFR agent and mTOR inhibitor is postulated to have synergistic anti-tumour effects. We aim to investigate the anti-tumour effect of combined nimotuzumab (anti-EGFR monoclonal antibody) and sirolimus (mTOR inhibitor) in a GBM preclinical model. Methods: Primary human GBM cells were derived from surgical GBM specimens. The endogenous expressions of glial-fibrillary acidic proteins and EGFR were determined by IHC staining and Western Blot analysis. Cell viability assays were then carried out in these GBM cells and immortalized human normal astrocytes (iHNA) using a range of concentrations of nimotuzumab alone, sirolimus alone or combined treatment. Results: GBM cells treated with nimotuzumab showed a dose- dependent cell kill and the optimal concentration was determined to be 2 µg/ml. Treatment of the GBM cells with sirolimus showed that the drug was capable of inducing cell death at varying levels and the optimal level was determined to be 0.1 mM. Combined treatment with nimotuzumab (2µg/ml) and rapamycin (0.1 mM) showed a dose dependent cell kill in GBM cells which was not observed in iNHA cells. The combined treatment resulted in only 10% of residual gliomas at 24 h post treatment. Single treatment with rapamycin has no cytotoxic effect whereas treatment with nimotuzumab alone exerts a cytotoxicity effect of 33%. Taken together, we observed an additive effect of cell kill when rapamycin is used together with nimotuzumab in human glioma cells. Conclusions: In this study, combined treatment of nimotuzumab and sirolimus resulted in a greater cytocidal effects in GBM cells than either agent alone. We will further examine this combination regimen in a subsequent phase I clinical trial.