Elizabeth C. Stahl

Polypropylene Surgical Mesh Coated with Extracellular Matrix Mitigates the Host Foreign Body Response

Surgical mesh devices composed of synthetic materials are commonly used for ventral hernia repair. These materials provide robust mechanical strength and are quickly incorporated into host tissue; factors that contribute to reduced hernia recurrence rates. However, such mesh devices cause a foreign body response with the associated complications of fibrosis and patient discomfort. In contrast, surgical mesh devices composed of naturally occurring extracellular matrix (ECM) are associated with constructive tissue remodeling, but lack the mechanical strength of synthetic materials. A method for applying a porcine dermal ECM hydrogel coating to a polypropylene mesh is described herein with the associated effects upon the host tissue response and biaxial mechanical behavior. Uncoated and ECM coated heavy-weight BARD™ Mesh were compared to the light-weight ULTRAPRO™ and BARD™ Soft Mesh devices in a rat partial thickness abdominal defect overlay model. The ECM coated mesh attenuated the pro-inflammatory response compared to all other devices, with a reduced cell accumulation and fewer foreign body giant cells. The ECM coating degraded by 35 days, and was replaced with loose connective tissue compared to the dense collagenous tissue associated with the uncoated polypropylene mesh device. Biaxial mechanical characterization showed that all of the mesh devices were of similar isotropic stiffness. Upon explanation, the light-weight mesh devices were more compliant than the coated or uncoated heavy-weight devices. This study shows that an ECM coating alters the default host response to a polypropylene mesh, but not the mechanical properties in an acute in vivo abdominal repair model.

Cell Therapy Strategies to Combat Immunosenescence

abstract Declining function of the immune system, termed “immunosenescence,” leads to a higher incidence of infection, cancer, and autoimmune disease related mortalities in the elderly population.1 Increasing interest in the field of immunosenescence is well-timed, as 20% of the United States population is expected to surpass the age of 65 by the year 2030.2 Our current understanding of immunosenescence involves a shift in function of both adaptive and innate immune cells, leading to a reduced capacity to recognize new antigens and widespread chronic inflammation. The present review focuses on changes that occur in haematopoietic stem cells, macrophages, and T-cells using knowledge gained from both rodent and human studies. The review will discuss emerging strategies to combat immunosenescence, focusing on cellular and genetic therapies, including bone marrow transplantation and genetic reprogramming. A better understanding of the mechanisms and implications of immunosenescence will be necessary to combat age-related mortalities in the future.

Effects of aging upon the host response to implants

Macrophage polarization during the host response is now a well-accepted predictor of outcomes following material implantation. Immunosenescence, dysregulation of macrophage function, and delayed resolution of immune responses in aged individuals have all been demonstrated, suggesting that host responses to materials in aged individuals should differ from those in younger individuals. However, few studies examining the effects of aging upon the host response have been performed. The present work sought to elucidate the impacts of aging upon the host response to polypropylene mesh implanted into 8-week-old and 18-month-old mice. The results showed that there are significant differences in macrophage surface marker expression, migration, and polarization during the early host macrophage response and delayed resolution of the host response in 18-month-old versus 8-week-old mice. These differences could not be attributed to cell-intrinsic defects alone, suggesting that the host macrophage response to implants is likely also dictated to a significant degree by the local tissue microenvironment. These results raise important questions about the design and testing of materials and devices often intended to treat aged individuals and suggest that an improved understanding of patient- and context-dependent macrophage responses has the potential to improve outcomes in aged individuals.

The polyploid state restricts hepatocyte proliferation and liver regeneration

The liver contains a mixture of hepatocytes with diploid or polyploid (tetraploid, octaploid, etc.) nuclear content. Polyploid hepatocytes are commonly found in adult mammals, representing ~90% of the entire hepatic pool in rodents. The cellular and molecular mechanisms that regulate polyploidization have been well characterized; however, it is unclear whether diploid and polyploid hepatocytes function similarly in multiple contexts. Answering this question has been challenging because proliferating hepatocytes can increase or decrease ploidy, and animal models with healthy diploid‐only livers have not been available. Mice lacking E2f7 and E2f8 in the liver (liver‐specific E2f7/E2f8 knockout; LKO) were recently reported to have a polyploidization defect, but were otherwise healthy. Herein, livers from LKO mice were rigorously characterized, demonstrating a 20‐fold increase in diploid hepatocytes and maintenance of the diploid state even after extensive proliferation. Livers from LKO mice maintained normal function, but became highly tumorigenic when challenged with tumor‐promoting stimuli, suggesting that tumors in LKO mice were driven, at least in part, by diploid hepatocytes capable of rapid proliferation. Indeed, hepatocytes from LKO mice proliferate faster and out‐compete control hepatocytes, especially in competitive repopulation studies. In addition, diploid or polyploid hepatocytes from wild‐type (WT) mice were examined to eliminate potentially confounding effects associated with E2f7/E2f8 deficiency. WT diploid cells also showed a proliferative advantage, entering and progressing through the cell cycle faster than polyploid cells, both in vitro and during liver regeneration (LR). Diploid and polyploid hepatocytes responded similarly to hepatic mitogens, indicating that proliferation kinetics are unrelated to differential response to growth stimuli. Conclusion: Diploid hepatocytes proliferate faster than polyploids, suggesting that the polyploid state functions as a growth suppressor to restrict proliferation by the majority of hepatocytes.

Evaluation of the host immune response to decellularized lung scaffolds derived from α-Gal knockout pigs in a non-human primate model

Whole organ tissue engineering is a promising approach to address organ shortages in many applications, including lung transplantation for patients with chronic pulmonary disease. Engineered lungs may be derived from animal sources after removing cellular content, exposing the extracellular matrix to serve as a scaffold for recellularization with human cells. However, the use of xenogeneic tissue sources in human transplantation raises concerns due to the presence of the antigenic Gal epitope. In the present study, lungs from wild type or α-Gal knockout pigs were harvested, decellularized, and implanted subcutaneously in a non-human primate model to evaluate the host immune response. The decellularized porcine implants were compared to a sham surgery control, as well as native porcine and decellularized macaque lung implants. The results demonstrated differential profiles of circulating and infiltrating immune cell subsets and histological outcomes depending on the implanted tissue source. Upon implantation, the decellularized α-Gal knockout lung constructs performed similarly to the decellularized wild type lung constructs. However, upon re-implantation into a chronic exposure model, the decellularized wild type lung constructs resulted in a greater proportion of infiltrating CD45+ cells, including CD3+ and CD8+ cytotoxic T-cells, likely mediated by an increase in production of Gal-specific antibodies. The results suggest that removal of the Gal epitope can potentially reduce adverse inflammatory reactions associated with chronic exposure to engineered organs containing xenogeneic components.

Tumor-Derived α-Fetoprotein Directly Drives Human Natural Killer–Cell Activation and Subsequent Cell Death

Low NK cell numbers, function, and infiltration into tumors predict poor outcomes for patients with hepatocellular carcinoma (HCC). Tumor-derived α-fetoprotein (AFP) from HCCs directly impacted NK cell function and viability, through both the AFP protein and AFPs low-molecular-mass cargo. Hepatocellular carcinoma (HCC) patients with reduced natural killer (NK)–cell numbers and function have been shown to have a poor disease outcome. Mechanisms underlying NK-cell deficiency and dysfunction in HCC patients remain largely unresolved. α-Fetoprotein (AFP) is an oncofetal antigen produced by HCC. Previous studies demonstrated that tumor-derived AFP (tAFP) can indirectly impair NK-cell activity by suppressing dendritic cell function. However, a direct tAFP effect on NK cells remains unexplored. The purpose of this study was to examine the ability of cord blood-derived AFP (nAFP) and that of tAFP to directly modulate human NK-cell activity and longevity in vitro. Short-term exposure to tAFP and, especially, nAFP proteins induced a unique proinflammatory, IL2-hyperresponsive phenotype in NK cells as measured by IL1β, IL6, and TNF secretion, CD69 upregulation, and enhanced tumor cell killing. In contrast, extended coculture with tAFP, but not nAFP, negatively affected long-term NK-cell viability. NK-cell activation was directly mediated by the AFP protein itself, whereas their viability was affected by hydrophilic components within the low molecular mass cargo that copurified with tAFP. Identification of the distinct impact of circulating tAFP on NK-cell function and viability may be crucial to developing a strategy to ameliorate HCC patient NK-cell functional deficits. Cancer Immunol Res; 5(6); 493–502. ©2017 AACR.

Stem Cell Transplantation for Degenerative Muscle Diseases

Under normal physiological conditions, skeletal muscle has a robust potential for tissue regeneration, which requires the activation of muscle progenitor cells (MPCs). However, degenerative muscle disease triggers recurrent muscle fiber destruction and repair, requiring persistent activation of MPCs, and ultimately leading to depletion of the progenitor cell pool. Duchenne muscular dystrophy (DMD) is a classic example of degenerative muscle disease which results in a reduced and dysfunctional progenitor cell pool, a phenomenon known as MPC depletion. Although DMD is a congenital disease, patients do not exhibit symptoms until 3–5 years of age, which coincides with MPC depletion. Replenishment of the MPC pool through cell therapy has therefore been explored as a potential treatment for DMD and other related degenerative muscle diseases. Several preclinical and clinical studies are ongoing to evaluate the efficacy of stem cell transplantation in patients with DMD. Notably, these studies are investigating various cell sources, genetic manipulations, and administration routes to achieve optimal treatment. In the past, studies have been hindered by cell availability, poor engraftment, and limited myogenic differentiation ability. Recent findings have revealed the many complexities of the degenerative muscle microenvironment, which include chronic inflammation, ectopic ossification, fatty infiltration, and pathological fibrosis. This hostile environment likely influences progenitor cell survival, engraftment, and differentiation after stem cell transplantation. This chapter aims to describe several clinical and preclinical examples of cell transplantation, highlighting the advantages and disadvantages of various approaches, while emphasizing the importance of the cellular and molecular disease environment in order to afford successful cell therapy.

Tumor-derived alpha fetoprotein directly impacts human natural killer cell activity and viability

Alpha-fetoprotein (AFP) is an oncofetal antigen produced by hepatocellular carcinomas (HCC). Previous studies demonstrated that tumor-derived AFP (tAFP) is a glycoprotein that has an immunosuppressive role on natural killer (NK), T, B, and dendritic (DC) cells which may play a role in HCC pathogenesis. Defects in NK cells have been attributed to tAFP-mediated immunosuppression of DC. However, a direct tAFP effect on NK cells remains unexplored. Here we compared the ability of cord blood-derived AFP (nAFP) to that of tAFP to modulate human NK cell activity and longevity in vitro. Short-term exposure to tAFP and, especially, nAFP proteins induced a unique pro-inflammatory, IL-2 hyperresponsive phenotype in healthy donor NK cells as measured by CD69 upregulation, IL-1β, IL-6 and TNF secretion, and enhanced tumor cell killing. In contrast, extended co-culture with tAFP, but not nAFP, inhibited NK cell proliferation and viability. NK cell activation was directly mediated by the AFP protein itself, while their viability was affected by the low molecular mass cargo that co-purified with tAFP. Overall, these data show that nAFP and tAFP induce similar yet distinct changes in NK cell function and viability, respectively. Defining the impact of circulating AFP on NK cells may be crucial to understand the NK cell functional deficits described in HCC patients.

Alpha fetoprotein directly induces a unique pro-inflammatory, IL-2 hyperresponsive phenotype in human natural killer cells

Alpha fetoprotein (AFP) is an oncofetal antigen commonly produced by hepatocellular carcinomas (HCC). Previous studies have shown that tumor-derived AFP (tAFP) has an immunosuppressive role on natural killer (NK), T, B, and dendritic (DC) cells which may play a role in HCC pathogenesis. Defects in NK cell frequency and function have partially been attributed to tAFP-mediated immunosuppression of DC function. However, a direct tAFP effect on NK cells remains unclear. Here we examine the ability of cord blood-derived AFP (nAFP) and tAFP to modulate human NK cell activity in vitro. We show that exposure to tAFP or, especially, nAFP proteins induces a unique pro-inflammatory NK cell activation profile as measured by CD69 upregulation, IL-1β and IL-6 secretion, and enhanced tumor cell killing. Interestingly, AFP-treated plus interleukin-2 (IL-2) stimulated cultures promote a degree of NK cell activation that is higher than that of NK cells activated with IL-2 alone by both phenotypic and functional measures, including elevated IFN-γ and GM-CSF secretion. To confirm that the observed effects are directly mediated by AFP protein, we confirmed that NK cells can readily bind to and take up nAFP and tAFP. The observed synergism between AFP and IL-2 may be mediated by the ability of AFP to modulate IL-2 receptor signaling, as shown by the ability of AFP to upregulate CD25, and downregulate CD122 and CD132 on NK cells. Overall, these data show that nAFP and tAFP induce a unique pro-inflammatory, IL-2 hyperresponive phenotype in NK cells. Defining the impact of circulating AFP on NK cells may be crucial to understand the NK cell functional deficits described in HCC patients, and for the development of an effective HCC-targeting immunotherapy.