Antagonism of regulatory ISGs enhances the anti-melanoma efficacy of STING agonists.

Background: Stimulator of Interferon Genes (STING) is a dsDNA sensor that triggers type I inflammatory responses. Recent data from our group and others support the therapeutic efficacy of STING agonists applied intratumorally or systemically in a range of murine tumor models, with treatment benefits associated with tumor vascular normalization and improved immune cell recruitment and function within the tumor microenvironment (TME). However, such interventions are rarely curative and STING agonism coordinately upregulates expression of immunoregulatory interferon-stimulated genes (ISGs) including , and that may limit treatment benefits.

Immunohistochemistry.

Immunohistochemistry is an essential technique for the localization and measurement of proteins in cells and tissues. This article describes methods for labeling proteins in adherent and suspension cell cultures and in tissue sections. Choices of antibodies and detection methods are discussed, and detailed troubleshooting guidelines are provided.

Co-expression of TNF receptors 1 and 2 on melanomas facilitates soluble TNF-induced resistance to MAPK pathway inhibitors.

Background: The effectiveness of MAPK pathway inhibitors (MAPKi) used to treat patients with BRAF-mutant melanoma is limited by a range of resistance mechanisms, including soluble TNF (solTNF)-mediated NF-kB signaling. solTNF preferentially signals through type-1 TNF receptor (TNFR1), however, it can also bind to TNFR2, a receptor that is primarily expressed on leukocytes. Here, we investigate the TNFR2 expression pattern on human BRAF melanomas and its role in solTNF-driven resistance reprogramming to MAPKi.

Increased LCN2 (lipocalin 2) in the RPE decreases autophagy and activates inflammasome-ferroptosis processes in a mouse model of dry AMD.

In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis.

Fatty acid nitroalkene reversal of established lung fibrosis.

Tissue fibrosis occurs in response to dysregulated metabolism, pro-inflammatory signaling and tissue repair reactions. For example, lungs exposed to environmental toxins, cancer therapies, chronic inflammation and other stimuli manifest a phenotypic shift to activated myofibroblasts and progressive and often irreversible lung tissue scarring. There are no therapies that stop or reverse fibrosis.

Cryosectioning.

The protocols presented here describe steps for cryosectioning tissue samples to be used in light microscopy methodologies including histochemistry, enzyme immunohistochemistry, and immunofluorescence. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cryosectioning.

A Tissue Perfusion Harvest Model for Optimal Multisystem Comparisons of Pathobiology.

Gravity flow whole-body perfusion maintains effective and reproducible preservation of tissue architecture critical to investigate pathobiology of multiple organs from the same specimen. The purpose of the protocols described within this article is to help the researcher optimize tissue harvest for multisystem pathobiology comparison. The protocols presented here describe tissue harvest for processing and cryopreservation to generate optimal samples for microscopy and parallel biochemical and molecular biology analysis.

Single-cell analyses of human pancreas: characteristics of two populations of acinar cells in chronic pancreatitis.

Chronic pancreatitis (CP) is a complex inflammatory disorder with numerous associated genetic and environmental risk factors. The most distressing characteristic of CP is recalcitrant pain, often requiring surgical resection including total pancreatectomy with islet autotransplantation (TPIAT). We studied five consented subjects undergoing pancreatic resection and processed isolated cells for single-cell RNA sequencing (scRNA-Seq).

CaMKII exacerbates heart failure progression by activating class I HDACs.

Background: Persistent cardiac Ca/calmodulin dependent Kinase II (CaMKII) activation plays an essential role in heart failure development. However, the molecular mechanisms underlying CaMKII induced heart failure progression remains incompletely understood. Histone deacetylases (HDACs) are critical for transcriptional responses to stress, and contribute to expression of pathological genes causing adverse ventricular remodeling.

CD47 Promotes Age-Associated Deterioration in Angiogenesis, Blood Flow and Glucose Homeostasis.

The aged population is currently at its highest level in human history and is expected to increase further in the coming years. In humans, aging is accompanied by impaired angiogenesis, diminished blood flow and altered metabolism, among others. A cellular mechanism that impinges upon these manifestations of aging can be a suitable target for therapeutic intervention.

Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice.

Pulmonary hypertension (PH) is a leading cause of death in sickle cell disease (SCD) patients. Hemolysis and oxidative stress contribute to SCD-associated PH. We have reported that the protein thrombospondin-1 (TSP1) is elevated in the plasma of patients with SCD and, by interacting with its receptor CD47, limits vasodilation of distal pulmonary arteries ex vivo.

Enhanced Neutrophil Extracellular Trap Formation in Acute Pancreatitis Contributes to Disease Severity and Is Reduced by Chloroquine.

Neutrophil extracellular traps (NETs) are generated when activated neutrophils, driven by PAD4, release their DNA, histones, HMGB1, and other intracellular granule components. NETs play a role in acute pancreatitis, worsening pancreatic inflammation, and promoting pancreatic duct obstruction. The autophagy inhibitor chloroquine (CQ) inhibits NET formation; therefore, we investigated the impact of CQ mediated NET inhibition in murine models of pancreatitis and human correlative studies.

Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps.

Background: The hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis.

Methods: Utilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine.

Spontaneous DNA damage to the nuclear genome promotes senescence, redox imbalance and aging.

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals.

Graft-infiltrating PD-L1 cross-dressed dendritic cells regulate antidonor T cell responses in mouse liver transplant tolerance.

Unlabelled: Although a key role of cross-dressing has been established in immunity to viral infection and more recently in the instigation of transplant rejection, its role in tolerance is unclear. We investigated the role of intragraft dendritic cells (DCs) and cross-dressing in mouse major histocompatibility complex (MHC)-mismatched liver transplant tolerance that occurs without therapeutic immunosuppression. Although donor interstitial DCs diminished rapidly after transplantation, they were replaced in the liver by host DCs that peaked on postoperative day (POD) 7 and persisted indefinitely.

Influence of the Novel ATP-Competitive Dual mTORC1/2 Inhibitor AZD2014 on Immune Cell Populations and Heart Allograft Rejection.

Background: Little is known about how new-generation adenosine triphosphate-competitive mechanistic target of rapamycin (mTOR) kinase inhibitors affect immunity and allograft rejection.

Methods: mTOR complex (C) 1 and 2 signaling in dendritic cells and T cells was analyzed by Western blotting, whereas immune cell populations in normal and heart allograft recipient mice were analyzed by flow cytometry. Alloreactive T cell proliferation was quantified in mixed leukocyte reaction; intracellular cytokine production and serum antidonor IgG levels were determined by flow analysis and immunofluorescence staining used to detect IgG in allografts.

IRF-1 promotes liver transplant ischemia/reperfusion injury via hepatocyte IL-15/IL-15Rα production.

Ischemia and reperfusion (I/R) injury following liver transplantation (LTx) is an important problem that significantly impacts clinical outcomes. IFN regulatory factor-1 (IRF-1) is a nuclear transcription factor that plays a critical role in liver injury. Our objective was to determine the immunomodulatory role of IRF-1 during I/R injury following allogeneic LTx.

Air medical retrieval of acute psychiatric patients.

Background: The aim of this study is to review the characteristics of acute psychiatric patients requiring air medical retrieval across the Northern Territory (NT) of Australia, to assess the sedation requirements and incidence of in-flight complications when retrieving such patients, and to review the optimal flight crew composition required for safe retrieval.

Methods: Retrospective data were collected for all psychiatric patients retrieved by an air medical retrieval service of the NT of Australia over a 12-month period between February 1, 2012, and January 31, 2013.

Results: Two hundred sixty-two patients were retrieved using fixed wing transport; 90% were indigenous.

Thrombospondin-1 activation of signal-regulatory protein-α stimulates reactive oxygen species production and promotes renal ischemia reperfusion injury.

Ischemia reperfusion injury (IRI) causes tissue and organ injury, in part, through alterations in tissue blood flow and the production of reactive oxygen species. The cell surface receptor signal-regulatory protein-α (SIRP-α) is expressed on inflammatory cells and suppresses phagocytosis, but the function of SIRP-α in IRI has not been determined. We reported previously that the matricellular protein thrombospondin-1 is upregulated in IRI.

West nile virus infection and myasthenia gravis.

Introduction: Viruses are commonly cited as triggers for autoimmune disease. It is unclear if West Nile virus (WNV) initiates autoimmunity.

Methods: We describe 6 cases of myasthenia gravis (MG) that developed several months after WNV infection.