Synthesis and Cell Adhesive Properties of Linear and Cyclic RGD Functionalized Polynorbornene Thin Films

Described herein is the efficient synthesis and evaluation of bioactive arginine-glycine-aspartic acid (RGD) functionalized polynorbornene-based materials for cell adhesion and spreading. Polynorbornenes containing either linear or cyclic RGD peptides were synthesized by ring-opening metathesis polymerization (ROMP) using the well-defined ruthenium initiator [(H_(2)IMes)(pyr)_(2)(Cl)_(2)Ru═CHPh]. The random copolymerization of three separate norbornene monomers allowed for the incorporation of water-soluble polyethylene glycol (PEG) moieties, RGD cell recognition motifs, and primary amines for postpolymerization cross-linking. Following polymer synthesis, thin-film hydrogels were formed by cross-linking with bis(sulfosuccinimidyl) suberate (BS^3), and the ability of these materials to support human umbilical vein endothelial cell (HUVEC) adhesion and spreading was evaluated and quantified. When compared to control polymers containing either no peptide or a scrambled RDG peptide, polymers with linear or cyclic RGD at varying concentrations displayed excellent cell adhesive properties in both serum-supplemented and serum-free media. Polymers with cyclic RGD side chains maintained cell adhesion and exhibited comparable integrin binding at a 100-fold lower concentration than those carrying linear RGD peptides. The precise control of monomer incorporation enabled by ROMP allows for quantification of the impact of RGD structure and concentration on cell adhesion and spreading. The results presented here will serve to guide future efforts for the design of RGD functionalized materials with applications in surgery, tissue engineering, and regenerative medicine

Collective Cell Migration on Artificial Extracellular Matrix Proteins Containing Full-Length Fibronectin Domains

Protein-based biomaterials that promote rapid wound healing are prepared by expression of artificial genes in bacterial cells. Artificial extracellular matrix proteins containing full-length fibronectin domains 9 and 10 exhibit strong α_5β_1 integrin binding and support rapid spreading, proliferation, and collective migration of fibroblasts

Foam and Antifoam Behavior of PDMS in MDEA-PZ Solution in the Presence of Different Degradation Products for CO2 Absorption Process

Absorption is one of the most established techniques to capture CO2 from natural gas and post-combustion processes. Nevertheless, the absorption process frequently suffers from various operational issues, including foaming. The main objective of the current work is to elucidate the effect of degradation product on the foaming behavior in methyldiethanolamine (MDEA) and piperazine (PZ) solution and evaluate the antifoaming performance of polydimethylsiloxane (PDMS) antifoam. The foaming behavior was investigated based on types of degradation product, temperature, and gas flow rate. The presence of glycine, heptanoic acid, hexadecane, and bicine in MDEA-PZ solution cause significant foaming. The presence of hexadecane produced the highest amount of foam, followed by heptanoic acid, glycine and lastly bicine. It was found that increasing the gas flow rate increases foaming tendency and foam stability. Furthermore, increasing temperature increases foaming tendency, but reduces foam stability. Moreover, PDMS antifoam was able to reduce foam formation in the presence of different degradation products and at various temperatures and gas flow rates. It was found that PDMS antifoam works best in the presence of hexadecane with the highest average foam height reduction of 19%. Hence, this work will demonstrate the cause of foaming and the importance of antifoam in reducing its effect

Failure patterns in resected pancreas adenocarcinoma: lack of predicted benefit to SMAD4 expression.

OBJECTIVE: To determine whether SMAD4 expression is associated with recurrence pattern after resection for pancreatic ductal adenocarcinoma (PDA). BACKGROUND: SMAD4 expression status has been reported to be associated with patterns of failure in PDA, but studies have not examined recurrence patterns after resection. METHODS: A tissue microarray was constructed including 127 patients with resected PDA and either short-term (\u3c12 \u3emonths) or long-term (\u3e30 months) survival. SMAD4 expression was evaluated by immunohistochemistry and categorized as present or lost in tumor cells. Conventional pathologic features (lymph node metastases, positive resection margin, poor grade, and tumor size) were recorded, and disease-specific outcomes (eg, recurrence pattern and early cancer-specific mortality) were determined. RESULTS: Loss of SMAD4 expression in pancreatic adenocarcinoma was identified in 40 of 127 patients (32%). SMAD4 loss occurred in 27% of patients who experienced isolated local recurrence, 33% of patients with a distant recurrence, 33% of patients who experienced local and distant site recurrences, and 25% of patients who were without evidence of recurrence (Fisher exact, P = 0.9). In a multivariate analysis, the presence of regional lymph node metastases was the only factor associated with the development of distant metastases (odds ratio = 4.7, P = 0.02). SMAD4 was neither associated with recurrence pattern (odds ratio = 0.9, P = 0.9) nor associated with early death (odds ratio = 0.5, P = 0.15). CONCLUSIONS: Primary tumor SMAD4 expression status was not a predictor of recurrence pattern in a large cohort of patients with resected PDA

Evaluating the feasibility and acceptability of an exercise and behaviour change intervention in socioeconomically deprived patients with peripheral arterial disease: The textpad study protocol

This pilot randomised controlled trial aims to assess the feasibility and acceptability of a 12-week home-based telehealth exercise and behavioural intervention delivered in socioeconomically deprived patients with peripheral artery disease (PAD). The study will also determine the preliminary effectiveness of the intervention for improving clinical and health outcomes. Sixty patients with PAD who meet the inclusion criteria will be recruited from outpatient clinic at the Freeman Hospital, United Kingdom. The intervention group will undergo telehealth behaviour intervention performed 3 times per week over 3 months. This program will comprise a home-based exercise (twice a week) and an individual lifestyle program (once per week). The control group will receive general health recommendations and advice to perform unsupervised walking training. The primary outcome will be feasibility and acceptability outcomes. The secondary outcomes will be objective and subjective function capacity, quality of life, dietary quality, physical activity levels, sleep pattern, alcohol and tobacco use, mental wellbeing, and patients’ activation. This pilot study will provide preliminary evidence of the feasibility, acceptability and effectiveness of home-based telehealth exercise and behavioural intervention delivered in socioeconomically deprived patients with PAD. In addition, the variance of the key health outcomes of this pilot study will be used to inform the sample size calculation for a future fully powered, multicentre randomized clinical trial

Association between pre-biologic T2-biomaker combinations and response to biologics in patients with severe asthma

Funding This study was conducted by the Observational and Pragmatic Research Institute (OPRI) Pte Ltd and was partially funded by Optimum Patient Care Global (OPCG) and AstraZeneca Ltd. No funding was received by the OPRI for its contribution. The International Severe Asthma Registry (ISAR) is operated by OPCG and co-funded by OPCG and AstraZenecaPeer reviewe

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Wound Healing on Artificial Extracellular Matrix Proteins

Collective cell migration is a key process in tissue repair, and in drawing parallels from complex multi-cellular events such as tumor morphogenesis and embryogenesis. Mechanisms of wound healing have been studied extensively in vitro. Extracellular matrix (ECM) is required to support cell migration and ensure rapid coverage of the wound area. The main challenge in designing biomaterials for tissue repair is to provide cells with the appropriate biological and mechanical cues. Hence, understanding key cell-ECM interactions during wound healing is necessary for effective biomaterial design. Genetic engineering provides a convenient avenue to customize materials for any given application. The artificial protein-based biomaterials discussed in this work were derived from fibronectin and elastin. These proteins have a modular design, and have material properties that can be fine-tuned according to specific applications. The artificial extracellular matrix (aECM) proteins prepared by previous members of our laboratory have been shown to promote attachment of endothelial cells. In this work, we studied extensively epithelial and fibroblast wound healing behavior on these aECM biomaterials. Crosslinked aECM protein films of varying RGD densities have been prepared by mixing aECM proteins with the RGD cell binding domain with aECM proteins containing the scrambled RDG sequence. Corneal epithelial wound healing was observed on aECM films with 100% RGD but not on aECM films with 2.5% RGD. Surprisingly, we found a five fold difference between the wound closure rates between these surfaces, but individual cell speeds did not increase significantly. We proposed that the five fold increase in wound closure rate was determined by the rate of crossing the boundary between the wound area and the area underneath the cell sheet. Both simulation and experimental data verified that the rate of boundary-crossing was sufficient to account for five-fold difference in wound closure rates between 100% RGD and 2.5% RGD surfaces. Full-length fibronectin domains have also been incorporated to improve the overall cell binding properties of the aECM proteins. The aECM proteins containing full-length fibronectin domains were shown to facilitate rapid spreading of Rat-1 fibroblasts. The aECM protein containing both fibronectin domains 9 and 10 exhibited an increased binding affinity to the α5β1 integrin. More importantly, these aECM proteins also promoted rapid wound closure, which was comparable to that on fibronectin. We showed that aECM proteins containing full-length fibronectin domains also promoted higher phosphorylated levels of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), consistent with the faster cell migration and proliferation observed. To try to understand how cells select wound healing mechanisms, wound healing of Madin-Darby Canine Kidney (MDCK) epithelial cells were examined in vitro. On surfaces containing the aECM protein bearing the fibronectin domain 10, characteristic healing patterns were observed in MDCK wound healing. These patterns are defined by the formation of leader cells at regular intervals of actomyosin purse strings. The spacing between consecutive leader cell groups was also found to be independent of the wound diameter. This spacing however, was found to decrease with increasing myosin II inhibition. These observations could be explained using a simple force transmission mechanical model. Consistent with the model predictions, we demonstrated that wounds with a zigzag geometry biased the selection of the wound healing mechanism along the wound edge. These zigzag wounds also healed nearly eight fold faster than wounds with straight edges.</p

Collective cell migration on artificial extracellular matrix proteins containing full-length fibronectin domains

The discovery of the cell-adhesive properties of the Arg-Gly-Asp (RGD) sequence located in the tenth type III domain of fi bronectin triggered widespread use of RGD-functionalized materials for directing cell behavior. [ 1,2 ] In studies of cell adhesion and migration, however, cell responses on RGD surfaces are never identical to those observed on fi bronectin. [ 3 ] For example, we recently examined the attachment and patterning of Rat-1 fi broblasts on elastin-based artifi cial extracellular matrix (aECM) proteins bearing a fi bronectin-derived RGD sequence and found the average projected area of such cells to be approximately 60% of those spread on fi bronectin.Accepted versio