Oncotic Necrosis Research Articles

Drug-induced oxidative stress actively prevents caspase activation and hepatocyte apoptosis

Cell death is a fundamental process in health and disease. Emerging research shows the existence of numerous distinct cell death modalities with similar and intertwined signaling pathways, but resulting in different cellular outcomes, raising the need to understand the decision-making steps during cell death signaling. Paracetamol (Acetaminophen, APAP)-induced hepatocyte death includes several apoptotic processes but eventually is executed by oncotic necrosis without any caspase activation. Here, we studied this paradoxical form of cell death and revealed that APAP not only fails to activate caspases but also strongly impedes their activation upon classical apoptosis induction, thereby shifting apoptosis to necrosis. While APAP intoxication results in massive drop in mitochondrial respiration, low cellular ATP levels could be excluded as an underlying cause of missing apoptosome formation and caspase activation. In contrast, we identified oxidative stress as a key factor in APAP-induced caspase inhibition. Importantly, caspase inhibition and the associated switch from apoptotic to necrotic cell death was reversible through the administration of antioxidants. Thus, exemplified by APAP-induced cell death, our study stresses that cellular redox status is a critical component in the decision-making between apoptotic and necrotic cell death, as it directly affects caspase activity.

Acetaminophen Hepatotoxicity: Paradigm for Understanding Mechanisms of Drug-Induced Liver Injury.

Acetaminophen (APAP) overdose is the clinically most relevant drug hepatotoxicity in western countries, and, because of translational relevance of animal models, APAP is mechanistically the most studied drug. This review covers intracellular signaling events starting with drug metabolism and the central role of mitochondrial dysfunction involving oxidant stress and peroxynitrite. Mitochondria-derived endonucleases trigger nuclear DNA fragmentation, the point of no return for cell death. In addition, adaptive mechanisms that limit cell death are discussed including autophagy, mitochondrial morphology changes, and biogenesis. Extensive evidence supports oncotic necrosis as the mode of cell death; however, a partial overlap with signaling events of apoptosis, ferroptosis, and pyroptosis is the basis for controversial discussions. Furthermore, an update on sterile inflammation in injury and repair with activation of Kupffer cells, monocyte-derived macrophages, and neutrophils is provided. Understanding these mechanisms of cell death led to discovery of N-acetylcysteine and recently fomepizole as effective antidotes against APAP toxicity.

A Photoactivated Ru (II) Polypyridine Complex Induced Oncotic Necrosis of A549 Cells by Activating Oxidative Phosphorylation and Inhibiting DNA Synthesis as Revealed by Quantitative Proteomics.

The ruthenium polypyridine complex [Ru(dppa)2(pytp)] (PF6)2 (termed as ZQX-1), where dppa = 4,7-diphenyl-1,10-phenanthroline and pytp = 4'-pyrene-2,2':6',2''-terpyridine, has been shown a high and selective cytotoxicity to hypoxic and cisplatin-resistant cancer cells either under irradiation with blue light or upon two-photon excitation. The IC50 values of ZQX-1 towards A549 cancer cells and HEK293 health cells are 0.16 ± 0.09 µM and >100 µM under irradiation at 420 nm, respectively. However, the mechanism of action of ZQX-1 remains unclear. In this work, using the quantitative proteomics method we identified 84 differentially expressed proteins (DEPs) with |fold-change| ≥ 1.2 in A549 cancer cells exposed to ZQX-1 under irradiation at 420 nm. Bioinformatics analysis of the DEPs revealed that photoactivated ZQX-1 generated reactive oxygen species (ROS) to activate oxidative phosphorylation signaling to overproduce ATP; it also released ROS and pyrene derivative to damage DNA and arrest A549 cells at S-phase, which synergistically led to oncotic necrosis and apoptosis of A549 cells to deplete excess ATP, evidenced by the elevated level of PRAP1 and cleaved capase-3. Moreover, the DNA damage inhibited the expression of DNA repair-related proteins, such as RBX1 and GPS1, enhancing photocytotoxicity of ZQX-1, which was reflected in the inhibition of integrin signaling and disruption of ribosome assembly. Importantly, the photoactivated ZQX-1 was shown to activate hypoxia-inducible factor 1A (HIF1A) survival signaling, implying that combining use of ZQX-1 with HIF1A signaling inhibitors may further promote the photocytotoxicity of the prodrug.

Role of Pyroptosis in Acetaminophen-Induced Hepatotoxicity.

Acetaminophen (APAP) is a widely used pain reliever that can cause liver injury or liver failure in response to an overdose. Understanding the mechanisms of APAP-induced cell death is critical for identifying new therapeutic targets. In this respect it was hypothesized that hepatocytes die by oncotic necrosis, apoptosis, necroptosis, ferroptosis and more recently pyroptosis. The latter cell death is characterized by caspase-dependent gasdermin cleavage into a C-terminal and an N-terminal fragment, which forms pores in the plasma membrane. The gasdermin pores can release potassium, interleukin-1β (IL-1β), IL-18, and other small molecules in a sublytic phase, which can be the main function of the pores in certain cell types such as inflammatory cells. Alternatively, the process can progress to full lysis of the cell (pyroptosis) with extensive cell contents release. This review discusses the experimental evidence for the involvement of pyroptosis in APAP hepatotoxicity as well as the arguments against pyroptosis as a relevant mechanism of APAP-induced cell death in hepatocytes. Based on the critical evaluation of the currently available literature and understanding of the pathophysiology, it can be concluded that pyroptotic cell death is unlikely to be a relevant contributor to APAP-induced liver injury.

Pirfenidone attenuates acetaminophen-induced liver injury via suppressing c-Jun N-terminal kinase phosphorylation

Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in Western countries. Pirfenidone (PFD), an orally bioavailable pyridone derivative, is clinically used for idiopathic pulmonary fibrosis treatment and has antifibrotic, anti-inflammatory, and antioxidant effects. Here we examined the PFD effect on APAP-induced liver injury.In a murine model, APAP caused serum alanine aminotransferase elevation attenuated by PFD treatment. We performed terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and vital propidium iodide (PI) stainings simultaneously. APAP induced TUNEL-positive/PI-negative necrosis around the central vein and subsequent TUNEL-negative/PI-positive oncotic necrosis with hemorrhage and caused the upregulation of hypercoagulation- and hypoxia-associated gene expressions. PFD treatment suppressed these findings. Western blotting revealed PFD suppressed APAP-induced c-Jun N-terminal kinase (JNK) phosphorylation despite no effect on JNK phosphatase expressions.In conclusion, simultaneous TUNEL and vital PI staining is useful for discriminating APAP-induced necrosis from typical oncotic necrosis. Our results indicated that PFD attenuated APAP-induced liver injury by suppressing TUNEL-positive necrosis by directly blocking JNK phosphorylation. PFD is promising as a new option to prevent APAP-induced liver injury.

Post-treatment with glycyrrhizin can attenuate hepatic mitochondrial damage induced by acetaminophen in mice.

Overdose of acetaminophen (APAP) is responsible for the most cases of acute liver failure worldwide. Hepatic mitochondrial damage mediated by neuronal nitric oxide synthase- (nNOS) induced liver protein tyrosine nitration plays a critical role in the pathophysiology of APAP hepatotoxicity. It has been reported that pre-treatment or co-treatment with glycyrrhizin can protect against hepatotoxicity through prevention of hepatocellular apoptosis. However, the majority of APAP-induced acute liver failure cases are people intentionally taking the drug to commit suicide. Any preventive treatment is of little value in practice. In addition, the hepatocellular damage induced by APAP is considered to be oncotic necrosis rather than apoptosis. In the present study, our aim is to investigate if glycyrrhizin can be used therapeutically and the underlying mechanisms of APAP hepatotoxicity protection. Hepatic damage was induced by 300 mg/kg APAP in balb/c mice, followed with administration of 40, 80, or 160 mg/kg glycyrrhizin 90 min later. Mice were euthanized and harvested at 6 h post-APAP. Compared with model controls, glycyrrhizin post-treatment attenuated hepatic mitochondrial and hepatocellular damages, as indicated by decreased serum glutamate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities as well as ameliorated mitochondrial swollen, distortion, and hepatocellular necrosis. Notably, 80 mg/kg glycyrrhizin inhibited hepatic nNOS activity and its mRNA and protein expression levels by 16.9, 14.9, and 28.3%, respectively. These results were consistent with the decreased liver nitric oxide content and liver protein tyrosine nitration indicated by 3-nitrotyrosine staining. Moreover, glycyrrhizin did not affect the APAP metabolic activation, and the survival rate of ALF mice was increased by glycyrrhizin. The present study indicates that post-treatment with glycyrrhizin can dose-dependently attenuate hepatic mitochondrial damage and inhibit the up-regulation of hepatic nNOS induced by APAP. Glycyrrhizin shows promise as drug for the treatment of APAP hepatotoxicity.

Engineering the Human Fc Region Enables Direct Cell Killing by Cancer Glycan-Targeting Antibodies without the Need for Immune Effector Cells or Complement.

Murine IgG3 glycan-targeting mAb often induces direct cell killing in the absence of immune effector cells or complement via a proinflammatory mechanism resembling oncotic necrosis. This cancer cell killing is due to noncovalent association between Fc regions of neighboring antibodies, resulting in enhanced avidity. Human isotypes do not contain the residues underlying this cooperative binding mode; consequently, the direct cell killing of mouse IgG3 mAb is lost upon chimerization or humanization. Using the Lewisa/c/x -targeting 88mAb, we identified the murine IgG3 residues underlying the direct cell killing and increased avidity via a series of constant region shuffling and subdomain swapping approaches to create improved ("i") chimeric mAb with enhanced tumor killing in vitro and in vivo. Constant region shuffling identified a major CH3 and a minor CH2 contribution, which was further mapped to discontinuous regions among residues 286-306 and 339-378 that, when introduced in 88hIgG1, recapitulated the direct cell killing and avidity of 88mIgG3. Of greater interest was the creation of a sialyl-di-Lewisa-targeting i129G1 mAb via introduction of these selected residues into 129hIgG1, converting it into a direct cell killing mAb with enhanced avidity and significant in vivo tumor control. The human iG1 mAb, termed Avidimabs, retained effector functions, paving the way for the proinflammatory direct cell killing to promote antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity through relief of immunosuppression. Ultimately, Fc engineering of human glycan-targeting IgG1 mAb confers proinflammatory direct cell killing and enhanced avidity, an approach that could be used to improve the avidity of other mAb with therapeutic potential. SIGNIFICANCE: Fc engineering enhances avidity and direct cell killing of cancer-targeting anti-glycan antibodies to create superior clinical candidates for cancer immunotherapy.

Ultrastructural Features of Mast Cell Death in Autotransplanted Lymph Heart of Adult Frog Rana temporaria

Abstract—Lymph heart of frog are hollow pulsating organs 1–2 mm in diameter that pump lymph into the venous system. They were used in this work as an experimental model to study the processes of initiation and development of aseptic inflammation in damaged tissues and necrosis-like death of mast cells (MCs). The ultrastructure of damaged and dying MCs was studied in striated muscle tissue of the posterior lymph heart of the adult frog in conditions of temporary ischemia (hypoxia) caused by autotransplantation of this organ into its own bed. It was found that damaged MCs die via oncotic necrosis after autotransplantation of the lymph heart within the period of time from 3 days to 6 weeks. Swelling of the cells and their membrane organelles (except for cytoplasmic secretory granules), dilatation of perinuclear space, budding of vesicles strewn with ribosomes from the outer nuclear membrane, and enrichment of nuclei of some MCs with heterochromatin occur at the early stages of oncosis. For late stages of oncotic necrosis, disintegration of the plasma membrane, release of intact and structurally little modified secretory granules into extracellular space, and karyolysis are typical. The presence of presumably nuclear material was found in extended perinuclear spaces of some damaged and dying MCs. The release of vesicles of rough endoplasmic reticulum (RER) into extracellular space was detected in places of local violation of the plasma membrane integrity of still undestroyed MCs. Immunocytochemical labeling of MCs using antibodies to histamine, substance P (SP), and atrial natriuretic peptide (ANP) (conducted at the ultrastructural level) detected the localization of aurum particles both over secretory granules located in the cytoplasm of damaged and dying cells and over the granules released into extracellular space after disintegration of MCs. Data obtained in the present study suggest that MCs dying via oncotic necrosis may be involved in the initiation and development of aseptic inflammatory process in autotransplanted lymph heart of frog and be a source of alarmins that contribute to the development of inflammation.

Emerging and established modes of cell death during acetaminophen-induced liver injury.

Acetaminophen (APAP)-induced liver injury is an important clinical and toxicological problem. Understanding the mechanisms and modes of cell death are vital for the development of therapeutic interventions. The histological and clinical features of APAP hepatotoxicity including cell and organelle swelling, karyolysis, and extensive cell contents release lead to the characterization of the cell death as oncotic necrosis. However, the more recent identification of detailed signaling mechanisms of mitochondrial dysfunction, the amplification mechanisms of mitochondrial oxidant stress and peroxynitrite formation by a mitogen-activated protein kinase cascade, mechanisms of the mitochondrial permeability transition pore opening and nuclear DNA fragmentation as well as the characterization of the sterile inflammatory response suggested that the mode of cell death is better termed programmed necrosis. Additional features like mitochondrial Bax translocation and cytochrome c release, mobilization of lysosomal iron and the activation of receptor-interacting protein kinases and the inflammasome raised the question whether other emerging modes of cell death such as apoptosis, necroptosis, ferroptosis and pyroptosis could also play a role. The current review summarizes the key mechanisms of APAP-induced liver injury and compares these with key features of the newly described modes of cell death. Based on the preponderance of experimental and clinical evidence, the mode of APAP-induced cell death should be termed programmed necrosis; despite some overlap with other modes of cell death, APAP hepatotoxicity does not fulfill the characteristics of either apoptosis, necroptosis, ferroptosis, pyroptosis or autophagic cell death.

Prevention of acetaminophen-induced liver injury by alginate

IntroductionAcetaminophen (APAP) intoxication is a major cause of acute liver failure. Alginate, an anionic polysaccharide, was previously shown as a macroporous scaffold, to reduce liver inflammation and sustain hepatic synthetic function, when implanted on liver remnant after extended partial hepatectomy. In the recent study we wanted to examine in a model of APAP intoxication the potential of a specially formulated alginate solution to prevent APAP toxicity. MethodsThree alginate solutions from low (30–50 kDa, VLVG), medium (100 kDa, LVG54) and high (150 kDa, LVG150) molecular weights were examined. Mice were orally administered with the alginate solution before, with and after APAP administration and were compared to control mice which received vehicle only. All mice were euthanized 24 h after APAP administration. Liver enzyme, blood APAP, IL-6 and liver histology including Ki-67 proliferation, IgG necrosis and nitrotyrosine staining were studied. ResultsVLVG- treated mice presented low ALT levels while 20–40 fold increase was demonstrated in control mice. The effect of LVG solutions was marginal. Accordingly, liver histology was normal with no hepatocytes proliferation in the VLVG group while massive centrilobular necrosis, increased nitrotyrosine staining and high proliferation appeared in livers of control mice. APAP blood levels were comparable in the two groups. Treatment with VLVG was associated with prevention of increase of IL-6 serum levels. ConclusionVLVG, a novel alginate solution, alleviated the liver toxicity and inhibited oncotic necrosis and related immune-mediated damage. VLVG may serve as a novel hepato-protector and prevent drug induced liver injury.

Characterization of Triptolide-Induced Hepatotoxicity by Imaging and Transcriptomics in a Novel Zebrafish Model.

Triptolide is a vine extract used in traditional Chinese medicines and associated with hepatotoxicity. In vitro data suggest that inhibition of RNA synthesis may be the mechanism of toxicity. For studying drug-induced liver injury the zebrafish has experimental, practical and financial advantages compared with rodents. The aim of this study was to explore the mechanism of triptolide toxicity using zebrafish as the model system. The effect of triptolide exposure on zebrafish larvae was determined with regard to mortality, histology, expression of liver specific microRNA-122 and liver volume. Fluorescent microscopy was used to track toxicity in the Tg(-2.8lfabp:GFP)as3 zebrafish line. Informed by microscopy, RNA-sequencing was used to explore the mechanism of toxicity. Triptolide exposure resulted in dose-dependent mortality, a reduction in the number of copies of microRNA-122 per larva, hepatocyte vacuolation, disarray and oncotic necrosis, and a reduction in liver volume. These findings were consistent across replicate experiments. Time-lapse imaging indicated the onset of injury was 6 h after the start of exposure, at which point, RNA-sequencing revealed that 88% of genes were down-regulated. Immune response associated genes were up-regulated in the triptolide-treated larvae including nitric oxide synthase. Inhibition of nitric oxide synthase increased mortality. Triptolide induces hepatotoxicity in zebrafish larvae. This represents a new model of drug-induced liver injury that complements rodents. RNA sequencing, guided by time-lapse microscopy, revealed early down-regulation of genes consistent with previous invitro studies, and facilitated the discovery of mechanistic inflammatory pathways.

Microcystin-LR induced liver injury in mice and in primary human hepatocytes is caused by oncotic necrosis

Microcystins are a group of toxins produced by freshwater cyanobacteria. Uptake of microcystin-leucine arginine (MC-LR) by organic anion transporting polypeptide 1B2 in hepatocytes results in inhibition of protein phosphatase 1A and 2A, and subsequent cell death. Studies performed in primary rat hepatocytes demonstrate prototypical apoptosis after MC-LR exposure; however, no study has directly tested whether apoptosis is critically involved in vivo in the mouse, or in human hepatocytes. MC-LR (120 μg/kg) was administered to C57BL/6J mice and cell death was evaluated by alanine aminotransferase (ALT) release, caspase-3 activity in the liver, and histology. Mice exposed to MC-LR had increases in plasma ALT values, and hemorrhage in the liver, but no increase in capase-3 activity in the liver. Pre-treatment with the pan-caspase inhibitor z-VAD-fmk failed to protect against cell death measured by ALT, glutathione depletion, or hemorrhage. Administration of MC-LR to primary human hepatocytes resulted in significant toxicity at concentrations between 5 nM and 1 μM. There were no elevated caspase-3 activities and pretreatment with z-VAD-fmk failed to protect against cell death in human hepatocytes. MC-LR treated human hepatocytes stained positive for propidium iodide, indicating membrane instability, a marker of necrosis. Of note, both increases in PI positive cells, and increases in lactate dehydrogenase release, occurred before the onset of complete actin filament collapse. In conclusion, apoptosis does not contribute to MC-LR-induced cell death in the in vivo mouse model or in primary human hepatocytes in vitro. Thus, targeting necrotic cell death mechanisms will be critical for preventing microcystin-induced liver injury.

Impact of Mercury on Liver and Ovary of Yellowfin Sea bream (Acanthopagrus latus) in the Persian Gulf

This study reports the incidence of histological alterations in morphometric changes in the liver and ovary of Acanthopagrus latus collected from an urban stream impaired by anthropogenic activities and from a clean site (reference). Mercury concentration was determined using a standard cold vapor atomic absorption. The results showed that mercury concentration at the creek waters, and specially sediments along Mahshahr coast, was higher than in the other marine environment, and in Zangi was lower than in the other sites of sampling. Several liver lesions were identified, including enlarged and lateral nuclei, nuclear degeneration and vacuolation, oncotic, apoptic, focal, massive, centrilobular and periportal necrosis, atrophy, lipidosis, hydropic and cloudy swelling, oval cell proliferation, cirrhosis, hemorrhage, macrophage aggregates, bile stagnation, dilation of sinusoid, intracellular edema, and dark granules. Ovary histology revealed some changes in higher concentrations such as increase in the number of pre-vitellogenic oocytes, oocyte atresia, and adhesion in the ovaries of females that were sampled from the sites with high concentration of mercury. In this study, existence of mercury in Mahshahr coast is demonstrated and high incidence of histological alterations in the liver and ovary of A. latus is an evidence of the poor environmental quality of creek waters. Hence, histopathological changes were induced by the mercury adversely affected the proper functioning of these organs in these fish.

Molecular responses to ischemia and reperfusion in the liver.

Ischemia/reperfusion (IR) injury occurs when oxygen is rapidly reintroduced into ischemic tissue, resulting in cell death and necrotic tissue damage. This is a major concern during liver transplantation procedures since there is an inevitable interruption and subsequent restoration of circulation. IR injury in liver tissue is initiated through reactive oxygen species (ROS), which are generated by hepatocytes during IR insult. Although these ROS are thought to play a protective roll since they are known to activate several pathways involved in the hypoxic response, they also trigger a localized sterile immune response that results in the recruitment of Kupffer cells and neutrophils to the site of IR insult. These immune cells generate larger quantities of ROS that trigger apoptosis and oncotic necrosis in liver tissue. In this review, we will summarize what is currently known about the response of liver tissue to IR insult at the molecular level.

Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice.

Drug-induced liver injury (DILI) is an important cause of acute liver failure, with limited therapeutic options. During DILI, oncotic necrosis with concomitant release and recognition of intracellular content amplifies liver inflammation and injury. Among these molecules, self-DNA has been widely shown to trigger inflammatory and autoimmune diseases; however, whether DNA released from damaged hepatocytes accumulates into necrotic liver and the impact of its recognition by the immune system remains elusive. Here we show that treatment with two different hepatotoxic compounds (acetaminophen and thioacetamide) caused DNA release into the hepatocyte cytoplasm, which occurred in parallel with cell death in vitro. Administration of these compounds in vivo caused massive DNA deposition within liver necrotic areas, together with an intravascular DNA coating. Using confocal intravital microscopy, we revealed that liver injury due to acetaminophen overdose led to a directional migration of neutrophils to DNA-rich areas, where they exhibit an active patrolling behavior. DNA removal by intravenous DNASE1 injection or ablation of Toll-like receptor 9 (TLR9)-mediated sensing significantly reduced systemic inflammation, liver neutrophil recruitment, and hepatotoxicity. Analysis of liver leukocytes by flow cytometry revealed that emigrated neutrophils up-regulated TLR9 expression during acetaminophen-mediated necrosis, and these cells sensed and reacted to extracellular DNA by activating the TLR9/NF-κB pathway. Likewise, adoptive transfer of wild-type neutrophils to TLR9(-/-) mice reversed the hepatoprotective phenotype otherwise observed in TLR9 absence. Hepatic DNA accumulation is a novel feature of DILI pathogenesis. Blockage of DNA recognition by the innate immune system may constitute a promising therapeutic venue.

Natural cytotoxic antibodies against tumor N-glycolilated ganglioside decrease with age and are absent in NSCLC patients (TUM4P.906)

Abstract N-glycolylated (NeuGc) gangliosides are not naturally expressed in normal human tissues but are overexpressed in several tumors and have immunosuppressive capacity, contributing to cancer progression. The present work shows the existence of a natural antibody response against NeuGcGM3 in healthy donors. These antibodies specifically recognized NeuGcGM3 and killed tumor cells expressing the antigen by a complement mediated mechanism. Interestingly, even after complement inactivation 17% of the positive sera showed a direct cytotoxic effect over the tumor cells. This cytotoxicity was dependent on the presence of the antigen on the tumor cell and resembles an oncotic necrosis kind of cell death. The levels of anti-NeuGcGM3 antibodies in the sera of healthy donors and the percentage of donors with this natural immunity decreases with age. Furthermore we could not detect this reactivity in the sera of 51 non-small cell lung cancer (NSCLC) patients that matched in sex and age the assessed healthy donors. Interestingly the level of sera IgM, but not IgG, was significantly lower in cancer patients in comparison with healthy donors. Also, the capacity of patients B1 cells to spontaneously secrete IgM and to respond to DAMPs, like CpG, seems to be affected. Defects in the natural surveillance against tumor antigens could increase the risk of elderly donors to develop cancer and affect the capacity of cancer patients to effectively fight against them.

PKCs: Pernicious kinase culprits in acetaminophen pathogenesis

PKCs: Pernicious kinase culprits in acetaminophen pathogenesis

Role Of Heme Oxygenase 1 In TNF/TNF Receptor-Mediated Apoptosis After Hepatic Ischemia/Reperfusion In Rats. Shock 39

In a recent study in SHOCK on the role of heme oxygenase-1 (HO-1) in hepatic ischemia-reperfusion injury, Kim et al. concluded that “the cytoprotective effects of HO-1 are mediated by suppression of TNF/TNFR1-mediated apoptotic signaling, ….” (1). Due to the word limitation, we will focus only on one fundamental concern with this study: the assumed role of apoptotic cell death in this rat model. The evidence for apoptosis provided by the authors is based on an increase of caspase-3 and -8 activities, cytochrome c release and TUNEL-positive cells (1). A major contradiction is already quite obvious from the authors’ own data: The authors report a 2-fold increase in caspase activity compared to a massive increase in TUNEL-positive cells (60% of all hepatocytes) (1). In untreated controls the number of TUNEL-positive cells is generally <0.3% (2), which amounts to a more than 200-fold increase over baseline after ischemia-reperfusion (1). It is well recognized that the TUNEL assay detects DNA strand breaks independent of the mode of cell death (3) and in the case of hepatic ischemia-reperfusion injury, TUNEL-positive cells are almost exclusively necrotic (2,4). This is consistent with morphological assessment by professional pathologists who were unable to detect any relevant increases in the number of cells that showed cell shrinkage, chromatin condensation and apoptotic body formation during hepatic ischemia and up to 24h of reperfusion in a rat model (2). In addition, other features like mitochondrial cytochrome c release can also be found during necrotic cell death either after bax translocation or mitochondrial swelling after the membrane permeability transition pore opening (5). Thus, none of these parameters provides any support for apoptotic cell death being important in the pathophysiology. The only reliable parameter for apoptotic signaling is activation of caspases. However, quantitative changes need to be considered. A modest 2-fold increase in caspase activation is insufficient to trigger relevant apoptotic cell death (6). In a true model of TNF-mediated apoptosis (galactosamine/endotoxin), generally increases in caspase-3 activity of 20–50-fold of baseline can be observed when 20–30% of all hepatocytes undergoing apoptosis (7). Thus, if the massive cell death (60% of hepatocytes) during hepatic ischemia-reperfusion would include a relevant contribution of apoptosis, the caspase activities would have to be 2 orders of magnitude higher than reported here (1). Most importantly, a pancaspase inhibitor, even when administered after TNF formation, completely prevents apoptotic cell death and any related liver injury (7). Caspase inhibitors generally do not protect against ischemia-reperfusion injury (2,4) and even if there is some protection, it is likely due to an off-target effect on other proteases (8). Why should we care if the mode of cell death is apoptosis or oncotic necrosis? First, in contrast to apoptosis, necrosis causes much more inflammation that aggravates the injury (9) but is also necessary for the repair (10). Second, for apoptosis we already have highly effective therapeutics (pancaspase inhibitors). However, because apoptosis is not a relevant process during hepatic ischemia-reperfusion injury, it is still necessary to evaluate therapeutic strategies such as HO-1 induction.

Human antibodies reactive to NeuGcGM3 ganglioside have cytotoxic antitumor properties

N-glycolylated gangliosides are not naturally expressed in healthy human tissues but are overexpressed in several tumors. We demonstrate the existence of antibodies that bind (N-glycolylneuraminyl)-lactosylceramide (NeuGcGM3) and are detectable in the sera of 65 from the 100 donors (65%) tested by ELISA. From those 65 NeuGcGM3 antibody-positive donors, 35 had antibodies that were able to recognize and kill NeuGcGM3-expressing tumor cells by a complement-mediated mechanism. After complement inactivation, 11 of the 35 positive sera showed a direct cytotoxic effect on the tumor cells. This complement-independent cytotoxicity was dependent on the presence of antigen on the membrane and resembles an oncotic necrosis cell death. Both the levels of anti-NeuGcGM3 antibodies in the sera as well as the percentage of healthy donors with this immunity decreased with the age of the donor. In contrast to age and gender-matched healthy donors, we could only detect low reactivity against NeuGcGM3 in the sera of six out of 53 non-small cell lung cancer patients. These results suggest the existence of antibodies against NeuGcGM3 with antitumor immune surveillance functions, reinforcing the importance of N-glycolylated gangliosides as antitumor targets.

Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure

Acetaminophen (APAP) is a safe analgesic and antipyretic drug. However, APAP overdose leads to massive hepatocyte death. Cell death during APAP toxicity occurs by oncotic necrosis, in which the release of intracellular contents can elicit a reactive inflammatory response. We have previously demonstrated that an intravascular gradient of chemokines and mitochondria-derived formyl peptides collaborate to guide neutrophils to sites of liver necrosis by CXC chemokine receptor 2 (CXCR2) and formyl peptide receptor 1 (FPR1), respectively. Here, we investigated the role of CXCR2 chemokines and mitochondrial products during APAP-induced liver injury and in liver neutrophil influx and hepatotoxicity. During APAP overdose, neutrophils accumulated into the liver, and blockage of neutrophil infiltration by anti-granulocyte receptor 1 depletion or combined CXCR2-FPR1 antagonism significantly prevented hepatotoxicity. In agreement with our in vivo data, isolated human neutrophils were cytotoxic to HepG2 cells when cocultured, and the mechanism of neutrophil killing was dependent on direct contact with HepG2 cells and the CXCR2-FPR1-signaling pathway. Also, in mice and humans, serum levels of both mitochondrial DNA (mitDNA) and CXCR2 chemokines were higher during acute liver injury, suggesting that necrosis products may reach remote organs through the circulation, leading to a systemic inflammatory response. Accordingly, APAP-treated mice exhibited marked systemic inflammation and lung injury, which was prevented by CXCR2-FPR1 blockage and Toll-like receptor 9 (TLR9) absence (TLR9(-/-) mice). Chemokines and mitochondrial products (e.g., formyl peptides and mitDNA) collaborate in neutrophil-mediated injury and systemic inflammation during acute liver failure. Hepatocyte death is amplified by liver neutrophil infiltration, and the release of necrotic products into the circulation may trigger a systemic inflammatory response and remote lung injury.