Reported effects of anti-CD154 treatment on autoimmunity, alloreactivity, and inf lammatory events mediated by macrophages and endothelial cells indicated that it might be an ideal agent for the prevention of intrahepatic islet allograft failure. This hypothesis was tested in MHCmismatched rhesus monkeys. Transplantation of an adequate number of viable islets resulted in engraftment and insulin independence in six of six recipients treated with anti-CD154 (hu5c8) induction plus monthly maintenance therapy (postoperative day >125, >246, >266, >405, >419, >476). Anti-CD154 (hu5c8) displayed no inhibitory effect on islet cell function. For monkeys followed for >100 days, continued improvement in graft function, as determined by first phase insulin release in response to intravenous glucose, was observed after the first 100 days post-transplant. No evidence of toxicity or infectious complications has been observed. All recipients treated with anti-CD154 became specifically nonresponsive to donor cells in mixed lymphocyte reactions. Furthermore, three monkeys are now off therapy (>113, >67, and >54 days off anti-CD154), with continued insulin independence and donor-specific mixed lymphocyte reaction hyporeactivity. In striking contrast to all previously tested strategies, transplantation of an adequate number of functional islets under the cover of anti-CD154 (hu5c8) monotherapy consistently allows for allogeneic islet engraftment and long-term insulin independence in this highly relevant preclinical model.Islet cell transplantation for patients with type 1 diabetes can result in the reversal of hyperglycemia and normalization of metabolic control (1-7). Broad-based application of curative islet cell transplantation has been limited, however, by the inability of current, generalized immunosuppressive reagents to reliably support long-term islet graft survival and function. The CD40-CD154 costimulation pathway has proven to be a critical interaction in the generation of a T-dependent immune response (8-10), and blockade of this pathway has prevented allograft rejection (11-16), graft versus host disease (17-19), and autoimmunity (20-29) in rodent models. Humanized anti-CD154 (30) (hu5c8, Biogen) has been shown to prevent renal allograft rejection in a rigorous non-human primate model (31). Additionally, blockade of the CD40-CD154 costimulation pathway can prevent production of proinflammatory mediators by activated macrophages (32-34) and endothelial cells (35)(36). Blockade of this pathway, therefore, has the potential to prevent allograft rejection, recurrent autoimmunity, and the nonspecific inflammatory events that occur on transplantation of islets into the liver, without the adverse effects of conventional, generalized immunosuppressive drugs on islet function (37). This study was undertaken to determine whether anti-CD154 (hu5c8) monotherapy would prevent the rejection of allogeneic islets in a preclinical, non-human primate model of pancreatectomy-induced diabetes.

No abstract

Clinical islet cell transplantation has resulted in insulin independence in a limited number of cases. Rejection, recurrence of autoimmunity, and impairment of normal islet function by conventional immunosuppressive drugs, e.g., steroids, tacrolimus, and cyclosporin A, may all contribute to islet allograft loss. Furthermore, intraportal infusion of allogeneic islets results in the activation of intrahepatic macrophages and endothelial cells, followed by production of proinflammatory mediators that can contribute to islet primary nonfunction. We reasoned that the beneficial effects of anti-CD154 treatment on autoimmunity, alloreactivity, and proinflammatory events mediated by macrophages and endothelial cells made it an ideal agent for the prevention of islet allograft failure. In this study, a nonhuman primate model (Papio hamadryas) was used to assess the effect of humanized anti-CD154 (hu5c8) on allogeneic islet engraftment and function. Nonimmunosuppressed and tacrolimus-treated recipients were insulin independent posttransplant, but rejected their islet allografts in 8 days. Engraftment and insulin independence were achieved in seven of seven baboon recipients of anti-CD154 induction therapy administered on days -1, 3, and 10 relative to the islet transplant. Three of three baboons treated with 20 mg/kg anti-CD154 induction therapy experienced delayed rejection episodes, first detected by elevations in postprandial blood glucose levels, on postoperative day (POD) 31 for one and on POD 58 for the other two. Re-treatment with three doses of anti-CD154 resulted in reversal of rejection in all three animals and in a return to normoglycemia and insulin independence in two of three baboons. It was possible to reverse multiple episodes of rejection with this approach. A loss of functional islet mass, as detected by reduced first-phase insulin release in response to intravenous glucose tolerance testing, was observed after each episode of rejection. One of two baboons treated with 10 mg/kg induction therapy became insulin independent post-transplant but rejected the islet graft on POD 10; the other animal experienced a reversible rejection episode on POD 58 and remained insulin independent and normoglycemic until POD 264. Two additional baboon recipients of allogeneic islets and donor bone marrow (infused on PODs 5 and 11) were treated with induction therapy (PODs -1, 3, 10), followed by initiation of monthly maintenance therapy (for a period of 6 months) on POD 28. Rejection-free graft survival and insulin independence was maintained for 114 and 238 days, with preservation of functional islet mass observed in the absence of rejection. Prevention and reversal of rejection, in the absence of the deleterious effects associated with the use of conventional immunosuppressive drugs, make anti-CD154 a unique agent for further study in islet cell transplantation.

Mesenchymal stem cell (MSC) based therapies are currently being evaluated as a putative therapeutic in numerous human clinical trials. Recent reports have established that exosomes mediate much of the therapeutic properties of MSCs. Exosomes are nanovesicles which mediate intercellular communication, transmitting signals between cells which regulate a diverse range of biological processes. MSC-derived exosomes are packaged with numerous types of proteins and RNAs, however, their metabolomic and lipidomic profiles to date have not been well characterized. We previously reported that MSCs, in response to priming culture conditions that mimic the in vivo microenvironmental niche, substantially modulate cellular signaling and significantly increase the secretion of exosomes. Here we report that MSCs exposed to such priming conditions undergo glycolytic reprogramming, which homogenizes MSCs’ metabolomic profile. In addition, we establish that exosomes derive from primed MSCs are packaged with numerous metabolites that have been directly associated with immunomodulation, including M2 macrophage polarization and regulatory T lymphocyte induction.

Directing stem cells to the heart is critical in producing an effective cell therapy for myocardial infarction (MI). Mesenchymal stem cells (MSCs) offer an exquisite drug delivery platform with environment-sensing cytokine release and MSCs have shown therapeutic potential in MI. Peptide-based targeting offers a novel method to increase cell homing, wherein MI-specific peptides, identified by phage display, are synthesized with a palmitic acid tail to facilitate cell membrane integration. Phage-peptides were screened in a mouse MI model and four peptides (CRPPR, CRKDKC, KSTRKS, and CARSKNKDC) were selected and synthesized as palmitated derivatives for further investigation. Cell coating was optimized and coating persistence and cytotoxicity were evaluated. MSCs were coated with peptides, injected into mice with MI, and MSCs in the heart quantified. Greater numbers of MSCs were found in heart of animals treated with the peptide-coated MSCs compared to uncoated controls. MSC numbers had positive correlation with MI severity in peptide-coated cells but a negative correlation in MSCs alone. A transient cell coating ("painting") method has been developed that labels cells efficiently, non-toxically and increases cell localization in MI hearts.

Mesenchymal stem cells (MSCs) facilitate functional recovery in numerous animal models of inflammatory and ischemic tissue-related diseases with a growing body of research suggesting that exosomes mediate many of these therapeutic effects. It remains unclear, however, which types of proteins are packaged into exosomes compared with the cells from which they are derived. In this study, using comprehensive proteomic analysis, we demonstrated that human primed MSCs secrete exosomes (pMEX) that are packaged with markedly higher fractions of specific protein subclasses compared with their cells of origin, indicating regulation of their contents. Notably, we found that pMEX are also packaged with substantially elevated levels of extracellular-associated proteins. Fibronectin was the most abundant protein detected, and data established that fibronectin mediates the mitogenic properties of pMEX. In addition, treatment of SHSY5Y cells with pMEX induced the secretion of growth factors known to possess mitogenic and neurotrophic properties. Taken together, our comprehensive analysis indicates that pMEX are packaged with specific protein subtypes, which may provide a molecular basis for their distinct functional properties.

COL10A1 was identified as a gene with restricted expression in most normal tissues and elevated expression in many diverse tumor types. By contrast, COL10A1 expression was undetectable in the 68 tumor cell lines surveyed in this study. Immunofluorescence studies localized collagen, type X, α-1 (collagen X) staining to tumor vasculature in breast tumors, whereas the vasculature of normal breast tissue was either collagen X-negative or had markedly lower levels. The tumor microenvironment-specific expression of collagen X, together with its localization in the vasculature, may facilitate its use as a novel target for the diagnosis and treatment of diverse solid tumor types.

In prior work, Friends of Cancer Research convened multiple data partners to establish standardized definitions for oncology real-world end points derived from electronic health records (EHRs) and claims data. Here, we assessed the performance of real-world overall survival (rwOS) from data sets sourced from EHRs by evaluating the ability of the end point to reflect expected differences from a previous randomized controlled trial across five data sources, after applying inclusion/exclusion criteria. The KEYNOTE-189 clinical trial protocol of platinum doublet chemotherapy (chemotherapy) vs. programmed cell death protein 1 (PD-1) in combination with platinum doublet chemotherapy (PD-1 combination) in first-line nonsquamous metastatic non-small cell lung cancer guided retrospective cohort selection. The Kaplan-Meier product limit estimator was used to calculate 12-month rwOS with 95% confidence intervals (CIs) in each data source. Cox proportional hazards models estimated hazard ratios (HRs) and associated 95% CIs, controlled for prognostic factors. Once the inclusion/exclusion criteria were applied, the five resulting data sets included 155 to 1,501 patients in the chemotherapy cohort and 36 to 405 patients in the PD-1 combination cohort. Twelve-month rwOS ranged from 45% to 58% in the chemotherapy cohort and 44% to 68% in the PD-1 combination cohort. The adjusted HR for death ranged from 0.80 (95% CI: 0.69, 0.93) to 1.15 (95% CI: 0.71, 1.85), controlling for age, gender, performance status, and smoking status. This study yielded insights regarding data capture, including ability of real-world data to precisely identify patient populations and the impact of criteria on end points. Sensitivity analyses could elucidate data set-specific factors that drive results.