Effect of human Decellularized Skeletal Muscle on Recovery from Volumetric Muscle Loss Injury

Volumetric muscle loss (VML) overwhelms muscle’s robust capacity for regeneration. A key event in the etiology of VML injury is the bulk loss of structural cues provided by the underlying extracellular matrix (ECM). While muscle is a highly structured tissue, with cell and ECM alignment in the direction of contractile force production, the impact of scaffold alignment on recovery remains unclear. Bulk human decellularized skeletal muscle (DSM) tissues were sectioned into 10 x 1-2 mm fibers. VML defects were repaired using multi-fiber implants consisting of approximately 8 fibers per defect arranged into two layers. Fibers were oriented 1) to the surrounding tibialis anterior (TA) muscle fibers (aligned), or 2) randomly (unaligned) within the defect and coated with minced muscle paste. At 8 weeks, peak tetanic force was not significantly different between the aligned and unaligned repair groups (73%±14% versus 70%±12%). TA muscle mass was also similar between the repair groups. Collagen and Laminin immuno-stains did not reveal structural differences between the groups. The results suggest it may not be necessary to alter the structure of the scaffold to match the surrounding native muscle. This could suggest that cellular and chemical cues provided by the scaffolds are the key design parameters to be considered. Short-term follow-up studies (1 and 2 weeks following repair) may reveal mechanistic insights

Effect of human Decellularized Skeletal Muscle on Recovery from Volumetric Muscle Loss Injury

Volumetric muscle loss (VML) overwhelms muscle’s robust capacity for regeneration. A key event in the etiology of VML injury is the bulk loss of structural cues provided by the underlying extracellular matrix (ECM). While muscle is a highly structured tissue, with cell and ECM alignment in the direction of contractile force production, the impact of scaffold alignment on recovery remains unclear. Bulk human decellularized skeletal muscle (DSM) tissues were sectioned into 10 x 1-2 mm fibers. VML defects were repaired using multi-fiber implants consisting of approximately 8 fibers per defect arranged into two layers. Fibers were oriented 1) to the surrounding tibialis anterior (TA) muscle fibers (aligned), or 2) randomly (unaligned) within the defect and coated with minced muscle paste. At 8 weeks, peak tetanic force was not significantly different between the aligned and unaligned repair groups (73%±14% versus 70%±12%). TA muscle mass was also similar between the repair groups. Collagen and Laminin immuno-stains did not reveal structural differences between the groups. The results suggest it may not be necessary to alter the structure of the scaffold to match the surrounding native muscle. This could suggest that cellular and chemical cues provided by the scaffolds are the key design parameters to be considered. Short-term follow-up studies (1 and 2 weeks following repair) may reveal mechanistic insights

Characterization of the Response of TRPV4 to Chemical Stimulation

Traumatic Brain Injury (TBI) is a source of acute and chronic health issues for many patients. One of the components of the brain’s response to injury is astrogliosis, in which astrocytes that normally function to repair the brain instead form scar tissue that halts repair processes. Transient Receptor Potential Vanilloid Type 4 (TRPV4) is a trans-membrane calcium channel involved in astrogliosis. Through Fura-2AM based calcium imaging, the base activity of this channel in mouse astrocyte cells was recorded. The cells were then subjected to TRPV4 agonist and antagonist stimulation and their subsequent activity levels were recorded. The data showed that increasing agonist levels garnered higher TRPV4 activity and increasing antagonist levels suppressed TRPV4 activity, though to a lesser extent. With further characterization of the exact nature of TRPV4’s role in TBI response, potential treatment plans for TBI could be developed

Engineering of extracellular matrix scaffolds via hollow fiber cell culture

Extracellular matrix (ECM) tissue scaffolds are seeing increased use for clinical applications, as they significantly decrease the time course of healing for injured tissues; however, the use of animal-sourced matrix for these scaffolds introduces xenogeneic epitopes into the patient toward which deleterious immune responses are directed, decreasing the effectiveness of the scaffold. ECM scaffolds produced in vitro have potential to minimize the foreign body response, as ECM can be cultured using human cell lines and decellularized to produce an allogeneic scaffold with high biocompatibility. The primary challenge of producing ECM-based therapeutics in vitro is fabricating such material in a manner which approximates the composition and architecture of native matrix while maintaining high yield and ease-of-handling. In previous work, we have demonstrated that sacrificial open-cell foams can be used for the production of ECM scaffolds with properties approximating those of native tissues.1 Herein we demonstrate a novel approach for the production of continuous threads of extracellular matrix by statically culturing ECM-secreting fibroblasts in the lumina of mesoporous hollow fiber membranes (HFMs). This approach exploits the fact that mesoporous HFMs prevent cross-membrane transport of high molecular weight proteins produced by cells in their lumina, while allowing for diffusion of low molecular weight cell medium components. Please click Additional Files below to see the full abstract

Inflammatory chemokine receptors regulate CD8+ T cell contraction and memory generation following infection

CD8+ T cells lacking CXCR3 and CCR5 expression have impaired contraction and generate an increased number of memory cells after virus infection

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

Transcriptome profiling of a synergistic volumetric muscle loss repair strategy

Abstract Volumetric muscle loss overwhelms skeletal muscle’s ordinarily capable regenerative machinery, resulting in severe functional deficits that have defied clinical repair strategies. In this manuscript we pair the early in vivo functional response induced by differing volumetric muscle loss tissue engineering repair strategies that are broadly representative of those explored by the field (scaffold alone, cells alone, or scaffold + cells) to the transcriptomic response induced by each intervention. We demonstrate that an implant strategy comprising allogeneic decellularized skeletal muscle scaffolds seeded with autologous minced muscle cellular paste (scaffold + cells) mediates a pattern of increased expression for several genes known to play roles in axon guidance and peripheral neuroregeneration, as well as several other key genes related to inflammation, phagocytosis, and extracellular matrix regulation. The upregulation of several key genes in the presence of both implant components suggests a unique synergy between scaffolding and cells in the early period following intervention that is not seen when either scaffolds or cells are used in isolation; a finding that invites further exploration of the interactions that could have a positive impact on the treatment of volumetric muscle loss

The molecular program induced in T cells undergoing homeostatic proliferation

Naïve T cells proliferate independently of cognate antigen when introduced into lymphopenic hosts. Lymphopenia-induced proliferation depends on low-affinity MHC/self-peptide complexes and on IL-7. To elucidate the intracellular signals mediating this proliferation, we analyzed changes in gene expression in naïve CD8(+) T cells at different times after their transfer into a lymphopenic environment. The genes induced in response to lymphopenia were largely an attenuated subset of those turned up by full antigenic stimulation, including genes related to cell cycling, whereas excluding genes specifically associated with effector activity. After the initial phase of proliferation in an empty compartment, the naïve T cells adopted a stable pattern of gene expression similar to that of antigen-experienced memory cells. Thus, T cells proliferating in lymphopenic hosts do not exhibit a unique gene-expression profile, instead relying on “traditional” signals for this antigen-independent proliferation; this process ultimately results in differentiation to “authentic” memory cells