Rho-mediated Contractility Exposes a Cryptic Site in Fibronectin and Induces Fibronectin Matrix Assembly

Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447–1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987. FEBS (Fed. Eur. Biochem. Soc.) Lett. 217:124–128). When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. We have investigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30–35% stretch to immobilized fibronectin induced binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension

TAT-Mediated Transduction of MafA Protein In Utero Results in Enhanced Pancreatic Insulin Expression and Changes in Islet Morphology

Alongside Pdx1 and Beta2/NeuroD, the transcription factor MafA has been shown to be instrumental in the maintenance of the beta cell phenotype. Indeed, a combination of MafA, Pdx1 and Ngn3 (an upstream regulator of Beta2/NeuroD) was recently reported to lead to the effective reprogramming of acinar cells into insulin-producing beta cells. These experiments set the stage for the development of new strategies to address the impairment of glycemic control in diabetic patients. However, the clinical applicability of reprogramming in this context is deemed to be poor due to the need to use viral vehicles for the delivery of the above factors. Here we describe a recombinant transducible version of the MafA protein (TAT-MafA) that penetrates across cell membranes with an efficiency of 100% and binds to the insulin promoter in vitro. When injected in utero into living mouse embryos, TAT-MafA significantly up-regulates target genes and induces enhanced insulin production as well as cytoarchitectural changes consistent with faster islet maturation. As the latest addition to our armamentarium of transducible proteins (which already includes Pdx1 and Ngn3), the purification and characterization of a functional TAT-MafA protein opens the door to prospective therapeutic uses that circumvent the use of viral delivery. To our knowledge, this is also the first report on the use of protein transduction in utero

2012 ACCF/AHA/ACP/AATS/PCNA/SCAI/STS guideline for the diagnosis and management of patients with stable ischemic heart disease

The recommendations listed in this document are, whenever possible, evidence based. An extensive evidence review was conducted as the document was compiled through December 2008. Repeated literature searches were performed by the guideline development staff and writing committee members as new issues were considered. New clinical trials published in peer-reviewed journals and articles through December 2011 were also reviewed and incorporated when relevant. Furthermore, because of the extended development time period for this guideline, peer review comments indicated that the sections focused on imaging technologies required additional updating, which occurred during 2011. Therefore, the evidence review for the imaging sections includes published literature through December 2011

The Role of Oxygen During In Vitro Culture and Immunoisolation of Islets of Langerhans

While clinical transplantation of islets of Langerhans for the treatment of insulin dependent Diabetes Mellitus has shown significant promise in recent years, there remains a need for procedural optimizations to improve cell viability, functionality and ultimately, graft longevity. One of the most critical factors to islet cell survival is the proper oxygenation of these highly metabolic cellular aggregates. In culture, islets experience suboptimal oxygen profiles delimited by steep gradients across culture media. When retransplanted, they are subjected to extremes of hypoxia and anoxia, resulting in pronounced graft dysfunction and cell loss, which is further exacerbated when these cells are immunoisolated in polymer matrices. This study examined the effects of improving both in-vitro culture and immunoisolation of islet cells by optimizing oxygen mass transfer via oxygen carriers in the form of perfluorocarbons. Specifically, new systems for these applications were developed utilizing perfluoromoeities and conventional culture (polydimethylsiloxane) and immunoisolation (sodium alginate) matrices. During in vitro culture of islet cells, the use of perfluoro-impregnated PDMS culture platforms enhanced cell recovery, viability and function over the culture period. Additionally, marginal mass transplants of the islets cultured in these novel platforms functioned better in recipients than relevant controls. In immunoisolation, the optimization of perfluorocarbon emulsions was performed investigating the effects of combinations of surfactants and perfluorocarbons on oxygen mass transfer and cell viability. Emulsions were well characterized using particle size analysis by dynamic light scattering, perfluorocarbon inclusion by gravimetry and oxygen diffusivity measurements utilizing fluorescent optodes. A novel method was developed for the assessment of dissolved oxygen content of these emulsions. Optimal emulsions, as determined by predicted/measured oxygen transfer enhancement over relevant controls, were utilized in alginate matrices for microencapsulation of cell lines, initially, and then, islets of Langehans. The effects of these potential improvements were assessed by in-vitro potency assays, including a novel method for assessing glucose stimulated insulin release, and in transplantation efficacy in rodent marginal mass models. While the improvements in culture were promising in cell line studies, the observed benefit did not translate in islet culture. The cause was found to be related to permeability impediments generated from the surfactant components utilized in emulsion manufacture. In addition to the development of several new methods for the characterization of oxygen containing solutions and the potency assessment of isolated islets of Langerhans, the impact of these studies is important in the field of polymer engineering. We observed that the use of Polyethylene glycol (PEG) based materials may limit transport of nutrients and oxygen critical to cells. Additionally, we developed cell culture platforms that enhance the viability, number and function of cultured islet cells, potentially impacting the clinical realm where cell preservation is critical to transplant outcome.</p

Culture and Transportation of Human Islets Between Centers

Significant progress has been achieved in clinical islet transplantation in the past two decades (1-7). Islet transplantation continues to gain promise as a potential cure for patients with Type 1 diabetes mellitus (8). Numerous technological improvements have been introduced in pancreas procurement (9) and preservation (10-12), islet isolation (13-15), purification (16-18), assessment (19,20), and culture (21), as well as in the management of islet transplant recipients (1-6)

Natural Killer Cells as Key Mediators in Type I Diabetes Immunopathology

The immunopathology of type I diabetes (T1D) presents a complicated case in part because of the multifactorial origin of this disease. Typically, T1D is thought to occur as a result of autoimmunity toward islets of Langerhans, resulting in the destruction of insulin-producing cells (β cells) and thus lifelong reliance on exogenous insulin. However, that explanation obscures much of the underlying mechanism, and the actual precipitating events along with the associated actors (latent viral infection, diverse immune cell types and their roles) are not completely understood. Notably, there is a malfunctioning in the regulation of cytotoxic CD8+ T cells that target endocrine cells through antigen-mediated attack. Further examination has revealed the likelihood of an imbalance in distinct subpopulations of tolerogenic and cytotoxic natural killer (NK) cells that may be the catalyst of adaptive immune system malfunction. The contributions of components outside the immune system, including environmental factors such as chronic viral infection also need more consideration, and much of the recent literature investigating the origins of this disease have focused on these factors. In this review, the details of the immunopathology of T1D regarding NK cell disfunction is discussed, along with how those mechanisms stand within the context of general autoimmune disorders. Finally, the rarer cases of latent autoimmune, COVID-19 (viral), and immune checkpoint inhibitor (ICI) induced diabetes are discussed as their exceptional pathology offers insight into the evolution of the disease as a whole

The Folate Cycle As a Cause of Natural Killer Cell Dysfunction and Viral Etiology in Type 1 Diabetes

The folate pathway is critical to proper cellular function and metabolism. It is responsible for multiple functions, including energy (ATP) production, methylation reactions for DNA and protein synthesis and the production of immunomodulatory molecules, inosine and adenosine. These play an important role in immune signaling and cytotoxicity. Herein, we hypothesize that defects in the folate pathway in genetically susceptible individuals could lead to immune dysfunction, permissive environments for chronic cyclical latent/lytic viral infection, and, ultimately, the development of unchecked autoimmune responses to infected tissue, in this case islet beta cells. In the context of type 1 diabetes (T1D), there has been a recent increase in newly diagnosed cases of T1D in the past 20 years that has exceeded previous epidemiological predictions with yet unidentified factor(s). This speaks to a potential environmental trigger that adversely affects immune responses. Most research into the immune dysfunction of T1D has focused on downstream adaptive responses of T and B cells neglecting the role of the upstream innate players such as natural killer (NK) cells. Constantly, surveilling the blood and tissues for pathogens, NK cells remove threats through direct cytotoxic responses and recruitment of adaptive responses using cytokines, such as IL-1β and IFN-γ. One long-standing hypothesis suggests viral infection as a potential trigger for the autoimmune response in T1D. Recent data suggest multiple viruses as potential causal agents. Intertwined with this is an observed reduced NK cell enumeration, cytotoxicity, and cytokine signaling in T1D patients. Many of the viruses implicated in T1D are chronic latent/lysogenic infections with demonstrated capacity to reduce NK cell response and number through mechanisms that resemble those of pregnancy tolerance. Defects in the folate pathway in T1D patients could result in decreased immune response to viral infection or viral reactivation. Dampened NK responses to infections result in improper signaling, improper antigen presentation, and amplified CD8+ lymphocyte proliferation and cytotoxicity, a hallmark of beta cell infiltrates in patients with T1D onset. This would suggest a critical role for NK cells in T1D development linked to viral infection and the importance of the folate pathway in maintaining proper NK response

Complementary methods for the determination of dissolved oxygen content in perfluorocarbon emulsions and other solutions

Perfluorocarbons (PFCs) are compounds with increased oxygen solubility and effective diffusivity, making them ideal candidates for improving oxygen mass transfer in numerous biological applications. Historically, quantification of the mass transfer characteristics of these liquids has relied on the use of elaborate laboratory equipment and complicated methodologies, such as in-line gas chromatography coupled with temperature-controlled glass fritted diffusion cells. In this work, we present an alternative method for the determination of dissolved oxygen content in PFC emulsions and, by extrapolation, pure PFCs. We implemented a simple stirred oxygen consumption microchamber coupled with an enzymatic reaction for the quantitative determination of oxygen by optical density measurements. Chambers were also custom fitted with lifetime oxygen sensors to permit simultaneous measurement of internal chamber oxygen levels. Analyzing the consumption of oxygen during the enzymatic reaction via recorded oxygen depletion traces, we found a strong degree of correlation between the zero-order reaction rate and the total measured oxygen concentrations, relative to control solutions. The values obtained were in close agreement with published values in the literature, establishing the accuracy of this method. Overall, this method allows for easy, reliable, and reproducible measurements of oxygen content in aqueous solutions, including, but not limited to PFC emulsions