Pulsed Electric Fields Induce STING Palmitoylation and Polymerization Independently of Plasmid DNA Electrotransfer.

Gene therapy approaches may target skeletal muscle due to its high protein-expressing nature and vascularization. Intramuscular plasmid DNA (pDNA) delivery via pulsed electric fields (PEFs) can be termed electroporation or electrotransfer. Nonviral delivery of plasmids to cells and tissues activates DNA-sensing pathways. The central signaling complex in cytosolic DNA sensing is the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING). The effects of pDNA electrotransfer on the signaling of STING, a key adapter protein, remain incompletely characterized. STING undergoes several post-translational modifications which modulate its function, including palmitoylation. This study demonstrated that in mouse skeletal muscle, STING was constitutively palmitoylated at two sites, while an additional site was modified following electroporation independent of the presence of pDNA. This third palmitoylation site correlated with STING polymerization but not with STING activation. Expression of several palmitoyl acyltransferases, including zinc finger and DHHC motif containing 1 (zDHHC1), coincided with STING activation. Expression of several depalmitoylases, including palmitoyl protein thioesterase 2 (PPT2), was diminished in all PEF application groups. Therefore, STING may not be regulated by active modification by palmitate after electroporation but inversely by the downregulation of palmitate removal. These findings unveil intricate molecular changes induced by PEF application.

Modification of the tumor microenvironment enhances immunity with plasmid gene therapy.

Local intratumor delivery with electroporation of low levels of plasmids encoding molecules, induces an antitumor effect without causing systemic toxicity. However, previous studies have predominately focused on the function of the delivered molecule encoded within the plasmid, and ignored the plasmid vector. In this study, we found vectors pUMVC3 and pVax1 induced upregulation of MHC class I (MHC-I) and PD-L1 on tumor cell surface. These molecules participate in a considerable number of immunoregulatory functions through their interactions with and activating inhibitory immune cell receptors. MHC molecules are well-known for their role in antigen (cross-) presentation, thereby functioning as key players in the communication between immune cells and tumor cells. Increased PD-L1 expression on tumor cells is an important monitor of tumor growth and the effectiveness of immune inhibitor therapy. Results from flow cytometry confirmed increased expression of MHC-I and PDL-1 on B16F10, 4T1, and KPC tumor cell lines. Preliminary animal data from tumor-bearing models, B16F10 melanoma, 4T1 breast cancer and KPC pancreatic cancer mouse models showed that tumor growth was attenuated after pUMVC3 intratumoral electroporation. Our data also documented that pSTAT1 signaling pathway might not be associated with plasmid vectors' function of upregulating MHC-I, PD-L1 on tumor cells.

Synergistic effects of nanosecond pulsed plasma and electric field on inactivation of pancreatic cancer cells in vitro.

Nanosecond pulsed atmospheric pressure plasma jets (ns-APPJs) produce reactive plasma species, including charged particles and reactive oxygen and nitrogen species (RONS), which can induce oxidative stress in biological cells. Nanosecond pulsed electric field (nsPEF) has also been found to cause permeabilization of cell membranes and induce apoptosis or cell death. Combining the treatment of ns-APPJ and nsPEF may enhance the effectiveness of cancer cell inactivation with only moderate doses of both treatments. Employing ns-APPJ powered by 9 kV, 200 ns pulses at 2 kHz and 60-nsPEF of 50 kV/cm at 1 Hz, the synergistic effects on pancreatic cancer cells (Pan02) in vitro were evaluated on the metabolic activities of cells and transcellular electrical resistance (TER). It was observed that treatment with ns-APPJ for > 2 min disrupts Pan02 cell stability and resulted in over 30% cell death. Similarly, applying nsPEF alone, > 20 pulses resulted in over 15% cell death. While the inactivation activity from the individual treatment is moderate, combined treatments resulted in 80% cell death, approximately 3-to-fivefold increase compared to the individual treatment. In addition, reactive oxygen species such as OH and O were identified at the plasma-liquid interface. The gas temperature of the plasma and the temperature of the cell solution during treatments were determined to be near room temperature.

Synergistic effects of nanosecond pulsed plasma and electric field on inactivation of pancreatic cancer cells in vitro.

Nanosecond pulsed atmospheric pressure plasma jets (ns-APPJs) produce reactive plasma species, including charged particles and reactive oxygen and nitrogen species (RONS), which can induce oxidative stress in biological cells. Nanosecond pulsed electric field (nsPEF) has also been found to cause permeabilization of cell membranes and induce apoptosis or cell death. Combining the treatment of ns-APPJ and nsPEF may enhance the effectiveness of cancer cell inactivation with only moderate doses of both treatments. Employing ns-APPJ powered by 9 kV, 200 ns pulses at 2 kHz and 60-nsPEF of 50 kV/cm at 1 Hz, the synergistic effects on pancreatic cancer cells (Pan02) in vitro were evaluated on cell viability and transcellular electrical resistance (TER). It was observed that treatment with ns-APPJ for >2 min disrupts Pan02 cell stability and resulted in over 30% cell death. Similarly, applying nsPEF alone, >20 pulses resulted in over 15% cell death. While the inactivation activity from the individual treatment is moderate, combined treatments resulted in 80% cell death, approximately 3-to-5-fold increase compared to the individual treatment. In addition, reactive oxygen species such as OH and O were identified at the plasma-liquid interface. The gas temperature of the plasma and the temperature of the cell solution during treatments were determined to be near room temperature.

Medicaid and the Children's Health Insurance Program: an overview for the pediatric radiologist.

In terms of number of beneficiaries, Medicaid is the single largest health insurance program in the US. Along with the Children's Health Insurance Program (CHIP), Medicaid covers nearly half of all births and provides health insurance to nearly half of the children in the country. This article provides a broad introduction to Medicaid and CHIP for the pediatric radiologist with a special focus on topics relevant to pediatric imaging and population health. This includes an overview of Medicaid's structure and eligibility criteria and how it differs from Medicare. The paper examines the means-tested programs within the context of pediatric radiology, reviewing pertinent topics such as the rise of Medicaid managed care plans, Medicaid expansion, the effects of Medicaid on child health, and COVID-19. Beyond the basics of benefits coverage, pediatric radiologists should understand how Medicaid and CHIP financing and reimbursement affect the ability of pediatric practices, radiology groups, and hospitals to provide services for children in a sustainable manner. The paper concludes with an analysis of future opportunities for Medicaid and CHIP.

Modification of the Tumor Microenvironment Enhances Anti-PD-1 Immunotherapy in Metastatic Melanoma.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. Both primary and acquired resistance have become major obstacles, greatly limiting the long-lasting effects and wide application of blockade therapy. Many patients with metastatic melanoma eventually require further therapy. The absence of T-cell infiltration to the tumor site is a well-accepted contributor limiting immune checkpoint inhibitor efficacy. In this study, we combined intratumoral injection of plasmid IL-12 with electrotransfer and anti-PD-1 in metastatic B16F10 melanoma tumor model to increase tumor-infiltrating lymphocytes and improve therapeutic efficacy. We showed that effective anti-tumor responses required a subset of tumor-infiltrating CD8+ and CD4+ T cells. Additionally, the combination therapy induced higher MHC-I surface expression on tumor cells to hamper tumor cells escaping from immune recognition. Furthermore, we found that activating T cells by exposure to IL-12 resulted in tumors sensitized to anti-PD-1 treatment, suggesting a therapeutic strategy to improve responses to checkpoint blockade.

Current Controversies in Radiology on Cost, Reimbursement, and Price Transparency: AJR Expert Panel Narrative Review.

Many believe that fundamental reform of the U.S. health care system is overdue and necessary given rising national health care expenditures, poor performance on key population health metrics, meaningful health disparities, concerns about potential financial toxicity of care, inadequate price transparency, pending insolvency of Medicare Part A, increasing commercial insurance premiums, and large uninsured and underinsured populations. The Medicare Payment Advisory Commission, an independent congressional agency, believes that part of this reform includes redistribution of reimbursements away from specialties such as radiology. Thus, despite an increase in the Medicare population and spending, Medicare payments for medical imaging have been decreasing for years. Further, the No Surprises Act, a federal law intended to curb the problem of surprise medical billing, was repurposed in federal rulemaking to reduce reimbursement from commercial payers to certain specialties, including radiology. In this article, we examine challenges facing the U.S. health care system, focusing on cost, reimbursement, and price transparency and the role of radiology in addressing such challenges. Medical imaging is a minor contributor to national health care expenditures but has an outsized impact on patient care. The radiology community should work together to reinforce the value of medical imaging and reduce inappropriate utilization of low-value care.

Reduction of plasmid vector backbone length enhances reporter gene expression.

Gene therapy has a wide range of applications for various types of pathologies. Viral methods of gene delivery provide high levels of gene expression but have various safety concerns. Non-viral methods are largely known to provide lower levels of expression. We aim to address this issue by using plasmid DNA with smaller backbones to increase gene expression levels when delivered using non-viral methods. In this study we compare gene expression levels between two vectors with firefly luciferase encoding gene insert using liposome complexes and gene electrotransfer as delivery methods. A 2-fold reduction in plasmid vector backbone size, disproportionately enhanced gene expression levels more than 10-fold in rat tenocytes in vitro, and rat myocardium in vivo, while improvements in delivery to the skin were more moderate.

Synergistic effects of an atmospheric pressure plasma jet and pulsed electric field on cells and skin.

Nonthermal atmospheric pressure plasmas produce reactive plasma species including charged particles and reactive oxygen nitrogen species, which are known to induce oxidative stress in living cells in liquid or tissue. In the meantime, pulsed electric fields have been widely used in reversible or irreversible electropermeabilization for either the delivery of plasmid DNA or inactivation of cancer cells. This work discusses the synergistic effects of nanosecond pulsed plasma jets and pulsed electric field on inactivation of pancreatic cancer cells in vitro and enhancement of plasmid DNA delivery to guinea pig skin in vivo. Higher inactivation rates of the cancer cells in suspension were obtained with combined treatment of 300-ns 50 kV/cm pulsed electric field and a 1-min exposure of a nanosecond pulsed, 250-µm plasma jet. Increased efficiency of gene electrotransfer to skin was also observed after a 3-min treatment of a nanosecond pulsed, 1-mm plasma jet. Application of the plasma alone at the same dosage did not have significant effect on gene delivery. These findings signify the dosage-dependent cell-response to both the electric fields and plasma. Importantly, the use of cold plasma to increase the sensitization of the biological cells in response to pulsed electric fields could be an effective approach to enhance the desired effects in electroporation-based applications.