Cell Death Markers Research Articles

Patient-derived explant model of appendiceal cancer.

4160 Background: Appendiceal cancers (AC) are rare with 5-year survival of 15% for high grade (HG) adenocarcinomas and 75% for low grade (LG) mucinous neoplasms. There is limited literature on the AC tumour microenvironment (TME) in disease progression and drug resistance. Ex vivo cultures, such as patient derived explants (PDEs), have been used for other solid tumours to test anticancer agents, explore the TME, and are being developed as personalised models. The aim of our study was to develop a PDE model of AC, preserve the TME, study the biological profile of AC and test novel therapies. Methods: Fresh tissue was collected during cytoreductive surgery (CRS) from consenting patients with AC with peritoneal disease, Jul 2020-Mar 2021. Tissues from 10 patients were dissected and cultured as PDEs under varying conditions of tissue size, media, matrix support and duration (Table). Uncultured Day 0 tissues and PDEs were fixed in formalin prior to paraffin embedding (FFPE). Immunohistochemical staining was performed on sections of FFPE tissue to assess viability with antibodies against the tumour marker, Cytokeratin-20 (CK20), and cell death marker, cleaved caspase 3 (CC3). Tissue architecture was rated on a 4-point scale (MS, J-SS). Cancer cells were counted in 6 Day 0 samples and PDEs from 3 patients using QuPath v0.3.2. Apoptotic index (AI) was calculated as the proportion of cells positive for CC3 divided by the number of total CK20 positive cells. Results: The mean proportion of tumour and mucin in the tissues was 3% (0-60%) and 39% (0-95%) respectively. Day 0 samples were viable with mean AI of cancer cells 7% (0-4%). Tissue architecture was maintained, as compared to Day 0 control, for varying sizes of PDE and culture durations up to 4d. More small or medium-sized PDEs had improved architecture compared to large sized PDEs (Table). PDEs at 4d had poorer architecture compared to at 1-3d. There was improved architecture in PDEs using enriched media with support factors compared to base media, and in specimens with matrix support compared to none. The mean AI of PDE cancer cells was 21% (0-92%) and 51% of PDEs had no cancer cell death. Conclusions: This is the first study demonstrating that AC tissue is amenable to ex vivo culture as PDEs. The optimal PDE model was <10mm tissue placed on a gelatine sponge in enriched media for 1-3d. PDEs had preserved tissue architecture and viability compared to uncultured tissue. Protein expression was in keeping with the original tumour. We plan to use this model to test anticancer agents and explore the TME of AC.[Table: see text]

993-P: Cell-Free DNA Methylation as a Biomarker of Islet-Cell Death in Youth with Type 2 Diabetes

Type 2 diabetes (T2D) in youth is diagnosed after irreparable islet damage. Cell-free DNA methylation can be used as a marker of islet cell death after islet transplantation. We assessed the performance of cell-free (cf) DNA in the discrimination of T2D and as a circulating biomarker of islet cell death. Methods: We recruited 72 youth 10-20 years old through provider networks in Atlanta, Georgia. Participant groups were (a) new onset T2D (n=21, within 16 weeks of diagnosis) ; (b) healthy, normal weight (n=23, no family history of diabetes) ; and (c) High Risk (HR, n=31, BMI ≥ 95th %ile or acanthosis nigricans; with family history) . All participants had an oral glucose tolerance test. Based on genes uniquely unmethylated in islets, plasma was analyzed at 9 CpG sites across 3 genes (INS, GCK, IADD) using bisulfite conversion and next generation sequencing. T2D and HR were grouped and compared to Healthy by (a) linear regression of % methylation on group, and (b) area under the curve (AUC) ROC analysis, adjusted for age and BMI %ile. Results: Mean age was higher among healthy participants (17yr SD±3) vs. T2D+HR (14±2) . Age- & BMI %ile- adjusted methylation of INS GRCh38 CpG Chr11:2160843 was highest in T2D+HR (94.8%, 95%CI: 93.5, 96.1) and lowest in Healthy (90.0%, 95%CI: 87.7, 92.2) , p=0.0051. Secondly, at CpG Chr11:2160806, methylation was highest in T2D+HR (85.6%, 95%CI: 83.3, 87.8) vs. Healthy (83.1%, 95%CI: 79.1, 87.1) , p=0.0409. Cell-free INS methylation improved the AUC for T2D or severe prediabetes (i.e., combined impaired fasting glucose + impaired glucose tolerance, per ADA cutpoints) from 0.72 in models of age and BMI %ile to 0.81 in models also including cfINS methylation. Conclusions: Higher % methylated cfINS was found in T2D+HR youth, rather than hypothesized lower levels. It is possible that during T2D onset, non-islet apoptosis occurs in larger concentration than islet cell apoptosis. Methylated cfINS improved discrimination of T2D and severe prediabetes by nearly 10% beyond age and BMI. Disclosure L.R.Staimez: None. J.Ham: None. T.Daley: None. P.Vellanki: n/a. A.K.Smith: None. K.Narayan: n/a. A.Baccarelli: None. K.Conneely: None. Funding Georgia CTSA UL1 (UL1TR002378) and KL2 (KL2TR002381) ; Emory University Woodruff Health Sciences Center Foundation

WP1066 induces cell death in a schwannomatosis patient–derived schwannoma cell line

Schwannomatosis is a rare genetic disorder that predisposes individuals to development of multiple schwannomas mainly in spinal and peripheral nerves and to debilitating chronic pain often unrelated to any schwannoma. Pathogenic variants of two genes, SMARCB1 and LZTR1, are causal in familial cases. However, many schwannomatosis patients lack mutations in these genes. Surgery is the standard treatment for schwannomas but leaves patients with increasing neurological deficits. Pain management is a daily struggle controlled by the use of multiple analgesic and anti-inflammatory drugs. There is a need for both nonsurgical treatment to manage tumor growth and nonaddictive, nonsedative pain control. Because standard clinical trials are exceedingly difficult for patients with rare disorders, precision medicine approaches offer the possibility of bespoke therapeutic regimens to control tumor growth. As a proof of principle, we obtained a bio-specimen of paraspinal schwannoma from a schwannomatosis patient with a germline point mutation in the SMARCB1/INI gene. We created an hTERT immortalized cell line and tested the ability of targeted small molecules with efficacy in neurofibromatosis type 2–related schwannomas to reduce cell viability and induce cell death. We identified WP1066, a STAT3 inhibitor, currently in phase 2 clinical trials for pediatric and adult brain tumors as a lead compound. It reduced cell viability and STAT-3 phosphorylation and induced expression of markers for both necroptosis and caspase-dependent cell death. The results demonstrate feasibility in creating patient-derived cell lines for use in precision medicine studies.

Cell death in lung transplantation. The roles of apoptosis, necroptois, and pyroptosis

Abstract Objective For patients with end stage lung disease, lung transplantation (Ltx) is the unique treatment option. Nevertheless, a large number of lung allografts fail, because of acute or chronic rejection. Innate immune responses following Ltx play a critical role in the development of primary graft dysfunction, and ultimately promote the development of chronic lung allograft dysfunction, leading to increased mortality after lung transplantation. Here, we focus on the mechanisms responsible for disruption of the cells membranes integrity, resulting in the release of Damage Associated Molecular Patterns (DAMPs) and activation of cell death pathways triggering cell injury in lungs with different preservation status. Methods Rat lungs were exposed to cold ischemia (CI group, n=9) or warm ischemia (WI group, n=9). In CI group, after Perfadex flushing, and hypothermic preservation (4 h) left lung was transplanted. In WI group, after 1 h of in situ WI, flashing and 3 h cold preservation the left lung was transplanted. Lung mechanics were determined during reperfusion. Recipients were sacrificed at 30, 60 and 120 min after Ltx. Bronchoalveolar lavage (BAL) was performed on the grafts to measure markers of cellular injury, and cytokines. Cell death markers were determined in graft tissue. Biological samples were kept at -80°C for additional analysis. Results Compared to CI, WI group displayed significantly decreased graft function associated with increased activation of pyroptotic cell death (NLRP3, caspase1, IL-1β) pathway, whereas apoptotic (caspas3 and caspase7) and necroptotic (RIP1, RIP3, MLKL) cell death pathways activation were similar in both groups. WI group showed more important cellular damage with DAMPs release (LDH, sRAGE, HMGB) and inflammation (CXCL1, IL1-β, IL-33). Conclusion Our results suggest that pyroptosis could play a critical role in the development of early graft dysfunction in lungs subjected to warm ischemia. Inhibition of pyroptotic pathway could be a strategy to improve damaged donor lungs.

High anesthetic (isoflurane) indoor pollution is associated with genetic instability, cytotoxicity, and proliferative alterations in professionals working in a veterinary hospital.

This is the first study to monitor anesthetic pollution in veterinary operating rooms (VOR) and assess the toxicological impact of the inhalational anesthetic isoflurane (exposed group) compared to matched volunteers (control group). DNA damage was evaluated in mononuclear cells by the comet assay while genetic instability (including micronucleus-MN), cell proliferation, and cell death markers were assessed by the buccal MN cytome assay. Residual isoflurane concentrations in VOR (air monitoring) lacking the scavenging system were assessed by infrared spectrophotometry; the mean concentration was 11ppm (≥ 5 times above the international recommended threshold). Comet assay results did not differ between groups; however, both younger exposed professionals (with higher week workload) compared to older individuals exposed for the same period and older professionals with greater time of exposure (years) compared to those in the same age group with fewer years of exposure presented higher DNA damage. The exposed group had a higher frequency of MN, nuclear buds, binucleated cells, karyorrhexis, and karyolysis and a lower frequency of basal cells than the control group. Exposed women were more vulnerable to genetic instability and proliferative index; exposed men presented more cytotoxicity. High WAG exposure has deleterious effects on exposed professionals.

Theragnostic Efficacy of K18 Response in Alcohol Use Disorder with Clinically Significant Fibrosis Using Gut-Liver Axis.

(1) Background: Fibrosis in early-stage alcohol-associated liver disease (ALD) is commonly under-diagnosed in routine clinical practice. This study characterized the liver-injury and cell death response in alcohol use disorder (AUD) patients with ALD who also exhibited fibrosis and assessed the efficacy of standard of care (SOC) treatment in the improvement in liver injury. (2) Methods: Forty-eight heavy-drinking AUD patients aged 21–65 yrs. without clinical manifestations of liver injury were grouped by Fibrosis-4 (FIB-4) score, as negative (Gr.1 < 1.45, n = 21) or positive (Gr.2 ≥ 1.45, n = 27). Patients received 2-weeks (2 w) inpatient SOC. Data on demographics, drinking patterns, liver-injury, immune markers, and liver cell death (K18s) markers were analyzed at baseline (BL) and after 2 w SOC. (3) Results: Lifetime drinking (LTDH, yrs.) and acute heavy drinking (Heavy Drinking Days Past 90 Days [HDD90]) markers were significantly higher in Gr.2 vs. Gr.1. BL ALT, AST, AST:ALT and K18M65 were considerably higher in Gr.2. Dysregulated gut dysfunction and elevated immune activity were evident in Gr.2 characterized by TNF-α, IL-8 and LPS levels. After SOC, Gr.2 showed improvement in AST, ALT, AST/ALT ratio; and in the K18M65, K18M30 and K18M65/M30 ratio vs. Gr.1. The true positivity of BL IL-8 response to predict the improvement in K18M65 to normal levels among Gr.2 patients against those who did not have improvement after 2 w SOC was very high (AUROC = 0.830, p = 0.042). (4) Conclusions: Gut dysfunction, elevated cytokine response and necrotic liver cell death were elevated in AUD patients with early-stage ALD. K18 showed promise as a predictive theragnostic factor to differentiate among the AUD patients with early-stage ALD and baseline fibrosis who had improvement in liver injury against those who did not, by the levels of baseline IL-8.

The Rysto immune receptor recognises a broadly conserved feature of potyviral coat proteins.

Summary Knowledge of the immune mechanisms responsible for viral recognition is critical for understanding durable disease resistance and successful crop protection. We determined how potato virus Y (PVY) coat protein (CP) is recognised by Rysto, a TNL immune receptor.We applied structural modelling, site‐directed mutagenesis, transient overexpression, co‐immunoprecipitation, infection assays and physiological cell death marker measurements to investigate the mechanism of Rysto–CP interaction.Rysto associates directly with PVY CP in planta that is conditioned by the presence of a CP central 149 amino acids domain. Each deletion that affects the CP core region impairs the ability of Rysto to trigger defence. Point mutations in the amino acid residues Ser125, Arg157, and Asp201 of the conserved RNA‐binding pocket of potyviral CP reduce or abolish Rysto binding and Rysto‐dependent responses, demonstrating that appropriate folding of the CP core is crucial for Rysto‐mediated recognition.Rysto recognises the CPs of at least 10 crop‐damaging viruses that share a similar core region. It confers immunity to plum pox virus and turnip mosaic virus in both Solanaceae and Brassicaceae systems, demonstrating potential utility in engineering virus resistance in various crops. Our findings shed new light on how R proteins detect different viruses by sensing conserved structural patterns.

Evaluation of the healing properties of Garcinia brasiliensis extracts in a cutaneous wound model

Wound healing is a complex process that can leave pathological scars, especially in case of infections from opportunistic microorganisms. In this context, herbal medicines open up great possibilities for investigation. One of the species of interest native to Brazil is Garcinia brasiliensis ("bacupari"). Traditionally known for treating wounds and ulcers, G. brasiliensis presents anti-inflammatory, antioxidant and antimicrobials properties. But, its wound healing profile in experimental models, in order to validate its efficacy, is still litle studied. Thus, the objective of this work was to evaluate, in an infected cutanous wound model, the potential of formulations incorporated with G. brasiliensis leaves extracts. Crude extract (CE), Ethyl Acetate Fraction (EAF) and Hexanic Fraction (HF) were submitted to phytochemical assays, high performance thin layer chromatography (HTPLC) and cytotoxicity studies. CE and EAF were also tested for microbicidal properties and incorporated in cream and gel formulations at 10% concentration. After stability testing, the gel formulations with CE or EAF at 10% were selected and applied to skin wounds infected or not with Staphylococcus aureus in Wistar rats. The healing potenttial of the extracts was verified by the expression of the protein Annexin A1 (AnxA1), related to the processes of inflammation and antifibrotic function, the cells immunostaining for Gasdermin-D (GSDM-D), a marker of pyroptotic cell death, and the dosage of interleukin-10 (IL-10) and monocyte chemotactic protein (MCP)-1 inflammatory mediators. Phytochemical studies indicated the presence of compounds of pharmacological interest, including Catechin, Quercetin and Berberine in addition to low cytotoxicity of CE and EAF at 10%. After the 6-day topical treatments, CE and EAF gel formulations demonstrated to control the pruritus formation process. The treatments decreased AnxA1 expression and the amount of cells immunostained for GSDM-D, and increased the expression of MCP-1 in infected wounds. Together, the results show important anti-inflammatory profile and skin healing potential of CE and EAF from G. brasiliensis leaves, even in infected lesions, with therapeutic perspectives.

Neuregulin-1/ErbB4 signaling modulates Plasmodium falciparum HRP2-induced damage to brain cortical organoids

SummaryHuman cerebral malaria (HCM) is a severe complication of Plasmodium falciparum (P.f.) infection that is characterized by capillary occlusions, rupture of the blood-brain barrier (BBB), perivascular cellular injury, and brain swelling. P.f.histidine-rich protein 2 (HRP2), a byproduct of parasitized red blood cell (pRBC) lysis, crosses the BBB when compromised to cause brain injury. We hypothesized that HRP2-induced neuronal damage can be attenuated by Neuregulin-1 (NRG1), an anti-inflammatory neuroprotective factor. Using brain cortical organoids, we determined that HRP2 upregulated cell death and inflammatory markers and disorganized brain organoid tissue. We identified toll-like receptors (TLR1 and 2) as potential mediators of HRP2-induced cellular damage and inflammation. Exogenous acute treatment of organoids with NRG1 attenuated HRP2 effects. The results indicate that HRP2 mediates malaria-associated HRP2-induced brain injury and inflammation and that NRG1 may be an effective therapy against HRP2 effects in the brain.

Rationale for the Use of Cord Blood in Hypoxic-Ischaemic Encephalopathy.

Hypoxic-ischaemic encephalopathy (HIE) is a severe complication of asphyxia at birth. Therapeutic hypothermia, the standard method for HIE prevention, is effective in only 50% of the cases. As the understanding of the immunological basis of these changes increases, experiments have begun with the use of cord blood (CB) because of its neuroprotective properties. Mechanisms for the neuroprotective effects of CB stem cells include antiapoptotic and anti-inflammatory actions, stimulation of angiogenesis, production of trophic factors, and mitochondrial donation. In several animal models of HIE, CB decreased oxidative stress, cell death markers, CD4+ T cell infiltration, and microglial activation; restored normal brain metabolic activity; promoted neurogenesis; improved myelination; and increased the proportion of mature oligodendrocytes, neuron numbers in the motor cortex and somatosensory cortex, and brain weight. These observations translate into motor strength, limb function, gait, and cognitive function and behaviour. In humans, the efficacy and safety of CB administration were reported in a few early clinical studies which confirmed the feasibility and safety of this intervention for up to 10 years. The results of these studies showed an improvement in the developmental outcomes over hypothermia. Two phase-2 clinical studies are ongoing under the United States regulations, namely one controlled study and one blinded study.

Indirect and Direct Effects of SARS-CoV-2 on Human Pancreatic Islets.

Recent studies have shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may induce metabolic distress, leading to hyperglycemia in patients affected by coronavirus disease 19 (COVID-19). We investigated the potential indirect and direct effects of SARS-CoV-2 on human pancreatic islets in 10 patients who became hyperglycemic after COVID-19. Although there was no evidence of peripheral anti-islet autoimmunity, the serum of these patients displayed toxicity on human pancreatic islets, which could be abrogated by the use of anti-interleukin-1β (IL-1β), anti-IL-6, and anti-tumor necrosis factor α, cytokines known to be highly upregulated during COVID-19. Interestingly, the receptors of those aforementioned cytokines were highly expressed on human pancreatic islets. An increase in peripheral unmethylated INS DNA, a marker of cell death, was evident in several patients with COVID-19. Pathology of the pancreas from deceased hyperglycemic patients who had COVID-19 revealed mild lymphocytic infiltration of pancreatic islets and pancreatic lymph nodes. Moreover, SARS-CoV-2-specific viral RNA, along with the presence of several immature insulin granules or proinsulin, was detected in postmortem pancreatic tissues, suggestive of β-cell-altered proinsulin processing, as well as β-cell degeneration and hyperstimulation. These data demonstrate that SARS-CoV-2 may negatively affect human pancreatic islet function and survival by creating inflammatory conditions, possibly with a direct tropism, which may in turn lead to metabolic abnormalities observed in patients with COVID-19.

The role of IR‐induced swelling in OPA1‐MICOS interaction in cardiac mitochondria

Mitochondria are membrane‐bound organelles composed of two membranes, the inner mitochondrial membrane (IMM) and the outer mitochondrial membrane (OMM), with the intermembrane space (IMS) localized between them. The IMM is divided into two subcompartments, the inner boundary membrane (IBM) that lies parallel to the OMM, and the cristae membrane (CM), which are IMM invaginations folded into cristae. The IBM and CM are connected at terminals forming circular‐like openings known as cristae junctions (CJs). The structural integrity of the mitochondrial CJs is maintained by the mitochondrial contact site and cristae organizing system (MICOS), a specific protein complex containing several structural and regulatory proteins. The MICOS proteins control the IMM architecture through direct membrane shaping, formation of contact sites, and biogenesis of proteins and lipids. In addition, the optic atrophy 1 (OPA1), a mitochondrial fusion protein, localized also in the CM, has been shown to participate in IMM remodeling which mediates the fusion of the IMM. The structural organization of the IMM is regulated by changes in the matrix volume of mitochondria; excessive matrix swelling in response to energetic and oxidative stress induced by pathological stimuli such as cardiac ischemia‐reperfusion (IR) impairs the integrity of CJs and thereby, alters mitochondrial function. The main goal of this study is to investigate the effects of cardiac IR‐induced swelling on OPA1 and MICOS proteins. Hearts were isolated from male Sprague Dawley rats and perfused with Krebs‐Henseleit solution (KHS) using the Langendorff‐mode technique at a constant flow rate (10‐12 ml/min). The animals were randomly assigned to the following groups: i) perfusion (no ischemia) for 55 min (C‐55 group), ii) ischemia (I group) for 25 min, iii) ischemia in the presence of sanglifehrin A (SfA), an inhibitor of the permeability transition pore (IS), iv) perfusion (no ischemia) for 95 min (C‐95), v) ischemia for 25‐min followed by 40‐min reperfusion (IR group), and vi) IR in the presence of SfA (IRS). SfA (0.5 µM) was present 10 min before ischemia (IS group) and throughout the entire period of reperfusion (IRS group). LDH activity was determined in the coronary effluent as a marker of cell death. At the end of reperfusion, mitochondria were isolated from the hearts by differential centrifugation for analysis of oxygen consumption rates, mitochondrial swelling, and protein levels of long and short forms of OPA1, and MICOS proteins. Our results show that ischemia diminished post‐ischemic recovery of the hearts as evidenced by impaired cardiac contractility, increased LDH activity in the coronary effluent, and reduced mitochondrial respiration. Ischemia alone and IR differently affected the expression of both, OPA1 and mitofilin (Mic‐60, a core MICOS protein) as well as other MICOS components. In conclusion, our data suggest that OPA1 and MICOS proteins are affected differently by ischemia alone and IR.

Anti‐Epileptic Drug and Teratogen Valproic Acid Induces Microglial Cell Death in a Valproic Acid Murine Model of Autism

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects approximately 1 in 44 children in North America, and its association with neuronal connectivity is an area of intense research. Valproic acid (VPA) is a multi‐target drug widely used to treat epilepsy. It is classified as a teratogen and as a histone deacetylase inhibitor (HDACi) and fetal exposure to VPA increases the risk of ASD. The VPA model has been well characterized for behavioral and neuronal deficits, including hyperconnectivity. The cause of hyperconnectivity is poorly understood however, it is speculated that lack of pruning may be partly responsible for this anomaly. Microglia, the principal immune cells of CNS regulate dendrite and synapse formation during early brain development. The effect of VPA on microglia during early development has not been well characterized and may provide potential hints regarding the etiology of this disorder. Preliminary studies from our lab indicate that VPA reduces microglial number during peak periods of postnatal synaptogenesis in the mouse brain primary cortex. Nevertheless, the cause of this reduction is not known. Therefore, in this study, we determined the mechanism for reduced microglial numbers in the VPA model of autism.Pregnant BALBc mice were administered 600 mg/kg of VPA by subcutaneous injection on day 13.5 of gestation. We harvested the mice brains at embryonic (E) day 14, E17, and postnatal day 1 (PD1). Brain tissue was double immunolabeled for microglia (IBA1) and cell death marker (TUNEL). We found a gradual increase in TUNEL staining beginning E14 to PD1 compared to age‐matched controls. The mechanism for reduced microglial number may be due to cell death. Thus, our findings possibly have pathophysiological/pathogenic implications in autism and may, ultimately, lead to the discovery of new therapeutic target(s) for autism.

Neurological restorative effects of the flavanol (‐)‐epicatechin in a rodent model of Gulf War Illness

Gulf War Illness (GWI) afflicts ~30% of the US military personnel who served in the 1990‐91 Persian Gulf War. Symptoms include cognitive deficits, muscle pain, weakness, exercise intolerance and fatigue which are still present after 30 years in war Veterans. Suspect causal agents include exposure to the compounds pyridostigmine (PB), permetrim (PM) and N,N‐diethyl‐m‐toluamide (DEET) which were used as protectants against nerve gases (PB) and insects. We previously reported a study to model GWI in which young male rats were provided PB, PM and DEET at equivalent human doses and physical restraint (to induce stress) for 3 weeks. In GWI animals, skeletal muscle weight was 35% lower vs. controls, which correlated with decreases in myofiber area, limb strength and treadmill time/distance. In a follow up study, we reported on the beneficial effects of the cacao flavanol (‐)‐epicatechin (epi) provided for 2 weeks by oral gavage on skeletal muscle atrophy and loss of function. In the current study, we wished to explore the potential of Epi to beneficially impact neurological measures of memory including multiple markers of hippocampus structure and function. Male Wistar rats underwent 3 weeks of exposure to either vehicles or DEET, PM, PB and stress. Subgroups of GWI rats (n=7/subgroup) received 15 days of either water (vehicle) or 1 mg/kg/day of Epi treatment by oral gavage and were compared to normal control (n=7). Object recognition tasks were performed to assess effects on memory and hippocampus samples were collected for histological and biochemical analysis. Results demonstrate that Epi treatment yields significant improvements in short and long‐term memory vs. GWI rats yielding values similar to those of controls. The assessment of hippocampus oxidative stress (protein carbonylation) and pro‐inflammatory cytokines levels (ELISA) of IL‐1β, TNF‐α and IFN‐γ (figure) yielded significant increases with GWI that were fully normalized with Epi to become comparable to controls. Significant increases in markers of hippocampus neuroinflammation (Iba1 and GFAP) and cell death (caspase activity) were noted with GWI and were significantly reduced with Epi. The neuronal survival signaling pathways (TREM2, PI3K and AKT) were adversely impacted by GWI and were partially or fully restored by Epi. Multiple markers of mitochondrial function (citrate synthase activity and ATP levels) adversely impacted by GWI and were fully restored by Epi. Thus, in an animal model of GWI, Epi beneficially impacted markers of hippocampus neuroinflammation, oxidative stress, cell survival, neurotoxicity and mitochondrial function. Such effects translated into improvements in functional endpoints associated with memory centers. As per the recognized safety profile of Epi clinical studies are warranted to explore the effects of treatment in Gulf War Veterans.

IL-23 signaling limits ferroptosis-driven immunopathology during systemic fungal infection

Abstract Pattern recognition receptors initiate antifungal immune responses during infection. Recently, we identified that the ephrin type-A receptor 2 (EphA2) is a fungal β-glucan receptor which orchestrates innate immune responses and prevents oral Candida infection. However, the role of EphA2 during invasive candidiasis (IC) remains unknown. Here we found that EphA2 induces renal inflammation and injury; thus, promoting immunopathology during IC. In Epha2−/− mice, dendritic cells migrated more efficient in infected tissues, while secreting more IL-23. Furthermore, Epha2−/− mice had reduced lipid peroxidation in kidneys, a marker of ferroptotic cell death. Pharmacological inhibtion of ferroptosis increased macrophage fungal killing, reduced inflammation, and increased disease outcomes during IC. Previously, IL-23 signaling has been associated with myeloid cell survival during candidiasis. Treatment of macrophages with IL-23 reduced ferroptotic macrophage cell death and excessive inflammation, while increasing killing capacity. Furthermore, IL-23 treatment of infected mice increased survival and reduced ferroptosis during IC. Collectively, our study demonstrates that ferroptotic host cell death is linked to immunopathology and can be targeted by recombinant cytokine therapy during fungal infection. This work was supported in part by NIH grant R00DE026856, an American Association of Immunologists Careers in Immunology Fellowship, and a TLI Seed grant to MS

Conventional amphotericin B elicits markers of immunogenic cell death on leukemic blasts, mediates immunostimulatory effects on phagocytic cells, and synergizes with PD-L1 blockade

ABSTRACT Immunostimulatory regimens are a game changer in the fight against cancer, but still only a minority of patients achieve clinical benefit. Combination with immunomodulatory drugs and agents converting otherwise non-immunogenic forms of cell death into bona fide “immunogenic cell death” (ICD) could improve the efficacy of these novel therapies. The aim of our study was to investigate conventional Amphotericin B (AmB) as an enhancer of antitumor immune responses. In tumor cell line models, AmB induced ICD with its typical hallmarks of calreticulin (CALR) expression and release of high mobility group box 1 (HMGB1) as well as Adenosine 5’-triphosphate (ATP). Interestingly, in contrast to non-ICD inducing treatments, ICD induction led to up-regulation of PD-L1-expression by ICD experiencing cells, resulting in decreased maturation of dendritic cells (DCs). Blocking this PD-L1 expression on tumor cells could unleash full ICD effects on antigen presenting cells. Even at sub-toxic concentrations, AmB was able to enhance CALR on leukemic blasts, particularly on phagocytic monoblastic THP-1 cells, which also showed features of “M1-like” differentiation after AmB exposure. The ability of AmB to increase the immunogenicity of tumor cells was confirmed in vivo in a mouse vaccination experiment. In conclusion, we demonstrate that AmB can promote antitumor immune responses in a dose-dependent manner by ICD induction, surface translocation of CALR on leukemic blasts even at sub-toxic concentrations, and “M1-like” polarization of phagocytic cells, making it noteworthy as potential booster for cancer immunotherapy. We additionally report for the first time that PD-L1 expression may be a feature of ICD, possibly as a negative feedback mechanism regulating the maturation status of DCs and thus indirectly affecting T-cell priming.

Anethole's effects against myocardial infarction: The role of TLR4/NFκB and Nrf2/HO1 pathways

BackgroundExploring new drugs for the management of myocardial infarction (MI) is crucial, as MI is a major contributor to mortality worldwide. Anethole, a naturally occurring essential oil component, has numerous medicinal, pharmaceutical, and cosmetic purposes. This study explored the potential action of anethole to protect myocytes against MI injure. MethodsWistar rats were divided into five groups: normal; anethole; and isoproterenol (ISO) groups in addition to two groups of ISO + anethole (125 and 250 mg/kg). All anethole groups were administered the oil component for 30 days, and all ISO groups were challenged with ISO on the 28th and 29th days. Parameters measured included infracted area, ECG, cardiac markers, the expression of Keap 1, nuclear Nrf2, and heme oxygenase-1, as well as the expression of TLR4 and MYD88 together with subsequent downstream oxidative stress, inflammatory, and apoptotic markers. ResultsAnethole reduced infarct region, degenerated cardiac indicators levels, amended ECG alterations, and diminished myocardial necrosis. Anethole reduced Keap-1, activated Nrf2/HO-1 pathway, increased mitochondrial antioxidant enzyme activities, declined the TLR4/MYD88 pathway, and ameliorated myocardial inflammation and cell death markers. ConclusionAnethole may retain a cardio-protective potential by controlling myocardial oxidative stress (through Nrf2 pathway) and diminishing inflammation and apoptosis via the TLR4/MYD88 pathway.

Sonosensitizer nanoplatform-mediated sonodynamic therapy induced immunogenic cell death and tumor immune microenvironment variation

Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic malignancies, and effective treatments are still lacking due to drug tolerance and tumor recurrence. In this study, we aimed to investigate the effects of sonodynamic therapy (SDT) on ovarian cancer and its potential mechanism. Folate receptor-targeted and ultrasound-responsive nanoparticles (NPs) were constructed using PLGA-PEG-FA (PLGA: poly (lactic-co-glycolic) acid, polyethylene glycol (PEG), FA: folate), the reactive oxygen species (ROS)-generating sonosensitizer IR780 and the oxygen-carrying material perfluorohexane (PFH), termed IRO@FA NPs. The antitumor effect of NPs triggered by ultrasound (US) was measured by an apoptosis assay in a C57/BL6 mouse model. Immunochemistry and flow cytometry were used to detect the proportion of CD3+ T, CD4+ T, CD8+ T cells and activated dendritic cells (DCs) in spleens and tumor tissues to assess variation in the immune response. Moreover, endoplasmic reticulum (ER) stress and immunogenic cell death (ICD) markers (high mobility group protein box-1, ATP and calreticulin) were detected to identify potential mechanisms. The results showed that IRO@FA NP-mediated SDT promoted ID8 cell apoptosis both in vitro and in vivo. The densities of CD3+ and CD8+ T lymphocytes and inflammatory markers were upregulated in tumor tissues. IRO@FA NP-mediated SDT prompted DC maturation and T lymphocyte infiltration by inducing ID8 cell ICD.

Novel Portable Hypothermic Cardiac Preservation Device Extends Cardiac Viability and Function Beyond the Standard of Care Using Isolated Porcine Hearts

<h3>Purpose</h3> The standard of care: cold static storage (CSS), limits the time a heart can be preserved to 4-6 hours. Machine perfusion preservation has been shown to improve organ viability. However, current machine perfusion technology is complex and requires the use of blood or blood products. The purpose of this study was to test the efficacy of a portable hypothermic oxygenated and acellular perfusion machine device, in preserving cardiac viability and function in comparison to CSS for 4 hours at different perfusion times using an isolated heart porcine model. <h3>Methods</h3> Isolated porcine hearts (N=17) were recovered under anesthesia and by inducing cardiac arrest through cardioplegia administration. Experimental hearts were perfused using a novel cardiac preservation device with an acellular perfusate at 4-8°C for 4 hours(n=3), 8 hours(n=4), and 24 hours(n=3). In addition, 3 hearts were perfused for 2 hours and then kept inside the perfusor at 4-8°C for an additional 4 hours without perfusion (extended mode), to determine efficacy during inoperable conditions. Controls (n=4) were hearts kept in CSS for 4 hours. Hourly flow, pressure, temperature, and blood gas measurements were taken from perfused hearts. After preservation, the hearts were placed in a Langendorff for functional recovery determined by +/- dP/dT and maximum pressure. Biopsy samples were taken before and at the end of either preservation method for histological and gene expression analyses. <h3>Results</h3> Control hearts weight gain (%) was -3.4 ± 4.9 and perfused hearts was 1.52 ± 7.6. Venous and arterial lactate concentrations were less than 2.5 mmol/L across all experiments. Perfused hearts at 4 hours, 8 hours, and extended mode had significantly increased contractility (p<0.05) than CSS demonstrated by +dP/dT and peak pressure. Gene expression showed a significant reduction (p<0.005) of inflammation (IL-6, IL-8, TNFα) and cell death markers (p53,cTnI) in perfused hearts compared to control hearts. Furthermore, histological assessment confirmed greater myocardium damage in CSS hearts. <h3>Conclusion</h3> This novel cardiac preservation device enhances cardiac viability and function compared to CSS. Furthermore, it allows for 4 hours of CSS after 2 hours of perfusion in case of cessation of perfusion. Animal transplant studies will be conducted to verify these results.

Circulating Proteomic Analysis Identifies Reduced Inflammation After Initiation of Hemodynamic Support with Either Veno-Arterial Extracorporal Membrane Oxygenation or Impella in Patients with Cardiogenic Shock

<h3>Purpose</h3> Use of acute mechanical circulatory support (AMCS) for cardiogenic shock (CS) is growing. We sought to determine whether plasma protein profiles are differentially regulated after initiation of veno-arterial extracorporeal membrane oxygenation (ECMO) or Impella in patients with CS. <h3>Methods</h3> Plasma samples were collected before device implantation and 72 hours after initiation of support in 11 CS patients receiving ECMO (n=5) or Impella (n=6). The aptamer-based SOMAscan assay was used to detect 1305 circulating proteins. Ingenuity Pathway Analysis identified cellular pathways and network interactions defined by proteins with a fold change >1.3 or <0.7 and p<0.05 after AMCS initiation. <h3>Results</h3> 67/1305 proteins were changed after ECMO (18 upregulated and 49 downregulated, p<0.05) and 38/1305 after Impella (10 upregulated and 28 downregulated, p<0.05). Only 8 proteins were commonly affected by ECMO and Impella. Despite minimal overlap in individual proteins, both devices were associated with markers of reduced inflammation including regulatory cytokines and increased apoptosis of leukocytes, phagocytes, and antigen-presenting cells (Figures A-B). <h3>Conclusion</h3> Using unbiased proteomic screening we identified that ECMO and Impella are associated with reduced expression of inflammatory markers and increased markers of inflammatory cell death. These circulating proteins may serve as novel targets of therapy or novel biomarkers to tailor AMCS device use and improve patient outcomes.