LSD1 drives intestinal epithelial maturation and controls small intestinal immune cell composition independent of microbiota in a murine model.

Postnatal development of the gastrointestinal tract involves the establishment of the commensal microbiota, the acquisition of immune tolerance via a balanced immune cell composition, and maturation of the intestinal epithelium. While studies have uncovered an interplay between the first two, less is known about the role of the maturing epithelium. Here we show that intestinal-epithelial intrinsic expression of lysine-specific demethylase 1A (LSD1) is necessary for the postnatal maturation of intestinal epithelium and maintenance of this developed state during adulthood. Using microbiota-depleted mice, we find plasma cells, innate lymphoid cells (ILCs), and a specific myeloid population to depend on LSD1-controlled epithelial maturation. We propose that LSD1 controls the expression of epithelial-derived chemokines, such as Cxcl16, and that this is a mode of action for this epithelial-immune cell interplay in local ILC2s but not ILC3s. Together, our findings suggest that the maturing epithelium plays a dominant role in regulating the local immune cell composition, thereby contributing to gut homeostasis.

Mmp17-deficient mice exhibit heightened goblet cell effector expression in the colon and increased resistance to chronic Trichuris muris infection.

Intestinal epithelial homeostasis is maintained by intrinsic and extrinsic signals. The extrinsic signals include those provided by mesenchymal cell populations that surround intestinal crypts and is further facilitated by the extracellular matrix (ECM), which is modulated by proteases such as matrix metalloproteinases (MMPs). Extrinsic signals ensure an appropriate balance between intestinal epithelial proliferation and differentiation. This study explores the role of MMP17, which is preferentially expressed by smooth muscle cells in the intestine, in intestinal homeostasis and during immunity to infection. Mice lacking MMP17 expressed high levels of goblet-cell associated genes and proteins, such as CLCA1 and RELM-ß, which are normally associated with immune responses to infection. Nevertheless, Mmp17 KO mice did not have altered resistance during a bacterial Citrobacter rodentium infection. However, when challenged with a low dose of the helminth Trichuris muris, Mmp17 KO mice had increased resistance, without a clear role for an altered immune response during infection. Mechanistically, we did not find changes in traditional modulators of goblet cell effectors such as the NOTCH pathway or specific cytokines. We found MMP17 expression in smooth muscle cells as well as lamina propria cells such as macrophages. Together, our data suggest that MMP17 extrinsically alters goblet cell maturation which is sufficient to alter clearance in a helminth infection model.

Olfactory Stimulation Successfully Modulates the Neurochemical, Biochemical and Behavioral Phenotypes of the Visceral Pain.

Visceral pain (VP) is the organ-derived nociception in which increased inflammatory reaction and exaggerated activation of the central nucleus of the amygdala (CeA) may contribute to this deficiency. Considering the amygdala also serves as the integration center for olfaction, the present study aimed to determine whether olfactory stimulation (OS) would effectively depress over-activation and inflammatory reaction in CeA, and successfully relieve VP-induced abnormalities. Adult rats subjected to intraperitoneal injection of acetic acid inhaled lavender essential oil for 2 or 4 h. The potential benefits of OS were determined by measuring the pro-inflammatory cytokine level, intracellular potassium and the upstream small-conductance calcium-activated potassium (SK) channel expression, together with detecting the stress transmitters that participated in the modulation of CeA activity. Results indicated that in VP rats, strong potassium intensity, reduced SK channel protein level, and increased corticotropin-releasing factor, c-fos, and substance P immuno-reactivities were detected in CeA. Enhanced CeA activation corresponded well with increased inflammatory reaction and decreased locomotion, respectively. However, in rats subjected to VP and received OS, all above parameters were significantly returned to normal levels with higher change detected in treating OS of 4h. As OS successfully depresses inflammation and CeA over-activation, application of OS may serve as an alternative and effective strategy to efficiently relieve VP-induced deficiency.

Mono- and combinational drug therapies for global viral pandemic preparedness.

Broadly effective antiviral therapies must be developed to be ready for clinical trials, which should begin soon after the emergence of new life-threatening viruses. Here, we pave the way towards this goal by reviewing conserved druggable virus-host interactions, mechanisms of action, immunomodulatory properties of available broad-spectrum antivirals (BSAs), routes of BSA delivery, and interactions of BSAs with other antivirals. Based on the review, we concluded that the range of indications of BSAs can be expanded, and new pan- and cross-viral mono- and combinational therapies can be developed. We have also developed a new scoring algorithm that can help identify the most promising few of the thousands of potential BSAs and BSA-containing drug cocktails (BCCs) to prioritize their development during the critical period between the identification of a new virus and the development of virus-specific vaccines, drugs, and therapeutic antibodies.

Immunoregulatory Intestinal Microbiota and COVID-19 in Patients with Type Two Diabetes: A Double-Edged Sword.

Coronavirus disease 2019, or COVID-19, is a major challenge facing scientists worldwide. Alongside the lungs, the system of organs comprising the GI tract is commonly targeted by COVID-19. The dysbiotic modulations in the intestine influence the disease severity, potentially due to the ability of the intestinal microbiota to modulate T lymphocyte functions, i.e., to suppress or activate T cell subpopulations. The interplay between the lungs and intestinal microbiota is named the gut-lung axis. One of the most usual comorbidities in COVID-19 patients is type 2 diabetes, which induces changes in intestinal microbiota, resulting in a pro-inflammatory immune response, and consequently, a more severe course of COVID-19. However, changes in the microbiota in this comorbid pathology remain unclear. Metformin is used as a medication to treat type 2 diabetes. The use of the type 2 diabetes drug metformin is a promising treatment for this comorbidity because, in addition to its hypoglycemic action, it can increase amount of intestinal bacteria that induce regulatory T cell response. This dual activity of metformin can reduce lung damage and improve the course of the COVID-19 disease.

Synergistic Interferon-Alpha-Based Combinations for Treatment of SARS-CoV-2 and Other Viral Infections.

BACKGROUND: There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. METHODS: Here, we tested the antiviral properties of interferons (IFNs), alone and with other drugs in vitro. RESULTS: While IFNs alone were insufficient to completely abolish replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), IFNα, in combination with remdesivir, EIDD-2801, camostat, cycloheximide, or convalescent serum, proved to be more effective. Transcriptome and metabolomic analyses revealed that the IFNα-remdesivir combination suppressed SARS-CoV-2-mediated changes in Calu-3 cells and lung organoids, although it altered the homeostasis of uninfected cells and organoids. We also demonstrated that IFNα combinations with sofosbuvir, telaprevir, NITD008, ribavirin, pimodivir, or lamivudine were effective against HCV, HEV, FLuAV, or HIV at lower concentrations, compared to monotherapies. CONCLUSIONS: Altogether, our results indicated that IFNα can be combined with drugs that affect viral RNA transcription, protein synthesis, and processing to make synergistic combinations that can be attractive targets for further pre-clinical and clinical development against emerging and re-emerging viral infections.

Nafamostat-Interferon-α Combination Suppresses SARS-CoV-2 Infection In Vitro and In Vivo by Cooperatively Targeting Host TMPRSS2.

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNα) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNα and that both Serpin E1 and nafamostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.

Active Components of Commonly Prescribed Medicines Affect Influenza A Virus-Host Cell Interaction: A Pilot Study.

Background: Every year, millions of people are hospitalized and thousands die from influenza A virus (FLUAV) infection. Most cases of hospitalizations and death occur among the elderly. Many of these elderly patients are reliant on medical treatment of underlying chronic diseases, such as arthritis, diabetes, and hypertension. We hypothesized that the commonly prescribed medicines for treatment of underlying chronic diseases can affect host responses to FLUAV infection and thus contribute to the morbidity and mortality associated with influenza. Therefore, the aim of this study was to examine whether commonly prescribed medicines could affect host responses to virus infection in vitro. Methods: We first identified 45 active compounds from a list of commonly prescribed medicines. Then, we constructed a drug-target interaction network and identified the potential implication of these interactions for FLUAV-host cell interplay. Finally, we tested the effect of 45 drugs on the viability, transcription, and metabolism of mock- and FLUAV-infected human retinal pigment epithelial (RPE) cells. Results: In silico drug-target interaction analysis revealed that drugs such as atorvastatin, candesartan, and hydroxocobalamin could target and modulate FLUAV-host cell interaction. In vitro experiments showed that at non-cytotoxic concentrations, these compounds affected the transcription and metabolism of FLUAV- and mock-infected cells. Conclusion: Many commonly prescribed drugs were found to modulate FLUAV-host cell interactions in silico and in vitro and could therefore affect their interplay in vivo, thus contributing to the morbidity and mortality of patients with influenza virus infections.

Safe-in-Man Broad Spectrum Antiviral Agents.

Emerging and re-emerging viral diseases occur with regularity within the human population. The conventional 'one drug, one virus' paradigm for antivirals does not adequately allow for proper preparedness in the face of unknown future epidemics. In addition, drug developers lack the financial incentives to work on antiviral drug discovery, with most pharmaceutical companies choosing to focus on more profitable disease areas. Safe-in-man broad spectrum antiviral agents (BSAAs) can help meet the need for antiviral development by already having passed phase I clinical trials, requiring less time and money to develop, and having the capacity to work against many viruses, allowing for a speedy response when unforeseen epidemics arise. In this chapter, we discuss the benefits of repurposing existing drugs as BSAAs, describe the major steps in safe-in-man BSAA drug development from discovery through clinical trials, and list several database resources that are useful tools for antiviral drug repositioning.

Leaky severe combined immunodeficiency in mice lacking non-homologous end joining factors XLF and MRI.

Non-homologous end-joining (NHEJ) is a DNA repair pathway required to detect, process, and ligate DNA double-stranded breaks (DSBs) throughout the cell cycle. The NHEJ pathway is necessary for V(D)J recombination in developing B and T lymphocytes. During NHEJ, Ku70 and Ku80 form a heterodimer that recognizes DSBs and promotes recruitment and function of downstream factors PAXX, MRI, DNA-PKcs, Artemis, XLF, XRCC4, and LIG4. Mutations in several known NHEJ genes result in severe combined immunodeficiency (SCID). Inactivation of Mri, Paxx or Xlf in mice results in normal or mild phenotype, while combined inactivation of Xlf/Mri, Xlf/Paxx, or Xlf/Dna-pkcs leads to late embryonic lethality. Here, we describe three new mouse models. We demonstrate that deletion of Trp53 rescues embryonic lethality in mice with combined deficiencies of Xlf and Mri. Furthermore, Xlf-/-Mri-/-Trp53+/- and Xlf-/-Paxx-/-Trp53+/- mice possess reduced body weight, severely reduced mature lymphocyte counts, and accumulation of progenitor B cells. We also report that combined inactivation of Mri/Paxx results in live-born mice with modest phenotype, and combined inactivation of Mri/Dna-pkcs results in embryonic lethality. Therefore, we conclude that XLF is functionally redundant with MRI and PAXX during lymphocyte development in vivo. Moreover, Mri genetically interacts with Dna-pkcs and Paxx.

Identification and Tracking of Antiviral Drug Combinations.

Combination therapies have become a standard for the treatment for HIV and hepatitis C virus (HCV) infections. They are advantageous over monotherapies due to better efficacy, reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to treat viral co-infections. Here, we identify new synergistic combinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), echovirus 1 (EV1), hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1) in vitro. We observed synergistic activity of nelfinavir with convalescent serum and with purified neutralizing antibody 23G7 against SARS-CoV-2 in human lung epithelial Calu-3 cells. We also demonstrated synergistic activity of nelfinavir with EIDD-2801 or remdesivir in Calu-3 cells. In addition, we showed synergistic activity of vemurafenib with emetine, homoharringtonine, anisomycin, or cycloheximide against EV1 infection in human lung epithelial A549 cells. We also found that combinations of sofosbuvir with brequinar or niclosamide are synergistic against HCV infection in hepatocyte-derived Huh-7.5 cells, and that combinations of monensin with lamivudine or tenofovir are synergistic against HIV-1 infection in human cervical TZM-bl cells. These results indicate that synergy is achieved when a virus-directed antiviral is combined with another virus- or host-directed agent. Finally, we present an online resource that summarizes novel and known antiviral drug combinations and their developmental status.

Genetic interaction between the non-homologous end-joining factors during B and T lymphocyte development: In vivo mouse models.

Non-homologous end joining (NHEJ) is the main DNA repair mechanism for the repair of double-strand breaks (DSBs) throughout the course of the cell cycle. DSBs are generated in developing B and T lymphocytes during V(D)J recombination to increase the repertoire of B and T cell receptors. DSBs are also generated during the class switch recombination (CSR) process in mature B lymphocytes, providing distinct effector functions of antibody heavy chain constant regions. Thus, NHEJ is important for both V(D)J recombination and CSR. NHEJ comprises core Ku70 and Ku80 subunits that form the Ku heterodimer, which binds DSBs and promotes the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, PAXX, MRI) and downstream core factors (XLF, Lig4 and XRCC4). In recent decades, new NHEJ proteins have been reported, increasing complexity of this molecular pathway. Numerous in vivo mouse models have been generated and characterized to identify the interplay of NHEJ factors and their role in development of adaptive immune system. This review summarizes the currently available mouse models lacking one or several NHEJ factors, with a particular focus on early B cell development. We also underline genetic interactions and redundancy in the NHEJ pathway in mice.

Potential Antiviral Options against SARS-CoV-2 Infection.

As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.

Mitochondrial transplantation enhances murine lung viability and recovery after ischemia-reperfusion injury.

The most common cause of acute lung injury is ischemia-reperfusion injury (IRI), during which mitochondrial damage occurs. We have previously demonstrated that mitochondrial transplantation is an efficacious therapy to replace or augment mitochondria damaged by IRI, allowing for enhanced muscle viability and function in cardiac tissue. Here, we investigate the efficacy of mitochondrial transplantation in a murine lung IRI model using male C57BL/6J mice. Transient ischemia was induced by applying a microvascular clamp on the left hilum for 2 h. Upon reperfusion mice received either vehicle or vehicle-containing mitochondria either by vascular delivery (Mito V) through the pulmonary artery or by aerosol delivery (Mito Neb) via the trachea (nebulization). Sham control mice underwent thoracotomy without hilar clamping and were ventilated for 2 h before returning to the cage. After 24 h recovery, lung mechanics were assessed and lungs were collected for analysis. Our results demonstrated that at 24 h of reperfusion, dynamic compliance and inspiratory capacity were significantly increased and resistance, tissue damping, elastance, and peak inspiratory pressure (Mito V only) were significantly decreased (P < 0.05) in Mito groups as compared with their respective vehicle groups. Neutrophil infiltration, interstitial edema, and apoptosis were significantly decreased (P < 0.05) in Mito groups as compared with vehicles. No significant differences in cytokines and chemokines between groups were shown. All lung mechanics results in Mito groups except peak inspiratory pressure in Mito Neb showed no significant differences (P > 0.05) as compared with Sham. These results conclude that mitochondrial transplantation by vascular delivery or nebulization improves lung mechanics and decreases lung tissue injury.

Mitochondrial transplantation prolongs cold ischemia time in murine heart transplantation.

BACKGROUND: Cold ischemia time (CIT) causes ischemia‒reperfusion injury to the mitochondria and detrimentally effects myocardial function and tissue viability. Mitochondrial transplantation replaces damaged mitochondria and enhances myocardial function and tissue viability. Herein we investigated the efficacy of mitochondrial transplantation in enhancing graft function and viability after prolonged CIT. METHODS: Heterotopic heart transplantation was performed in C57BL/6J mice. Upon heart harvesting from C57BL/6J donors, 0.5 ml of either mitochondria (1 × 108 in respiration buffer; mitochondria group) or respiration buffer (vehicle group) was delivered antegrade to the coronary arteries via injection to the coronary ostium. The hearts were excised and preserved for 29 ± 0.3 hours in cold saline (4°C). The hearts were then heterotopically transplanted. A second injection of either mitochondria (1 × 108) or respiration buffer (vehicle) was delivered antegrade to the coronary arteries 5 minutes after transplantation. Grafts were analyzed for 24 hours. Beating score, graft function, and tissue injury were measured. RESULTS: Beating score, calculated ejection fraction, and shortening fraction were significantly enhanced (p < 0.05), whereas necrosis and neutrophil infiltration were significantly decreased (p < 0.05) in the mitochondria group as compared with the vehicle group at 24 hours of reperfusion. Transmission electron microscopy showed the presence of contraction bands in vehicle but not in mitochondria grafts. CONCLUSIONS: Mitochondrial transplantation prolongs CIT to 29 hours in the murine heart transplantation model, significantly enhances graft function, and decreases graft tissue injury. Mitochondrial transplantation may provide a means to reduce graft failure and improve transplantation outcomes after prolonged CIT.

Relaxin Family Member Insulin-Like Peptide 6 Ameliorates Cardiac Fibrosis and Prevents Cardiac Remodeling in Murine Heart Failure Models.

BACKGROUND: The insulin/insulin-like growth factor/relaxin family represents a group of structurally related but functionally diverse proteins. The family member relaxin-2 has been evaluated in clinical trials for its efficacy in the treatment of acute heart failure. In this study, we assessed the role of insulin-like peptide 6 (INSL6), another member of this protein family, in murine heart failure models using genetic loss-of-function and protein delivery methods. METHODS AND RESULTS: Insl6-deficient and wild-type (C57BL/6N) mice were administered angiotensin II or isoproterenol via continuous infusion with an osmotic pump or via intraperitoneal injection once a day, respectively, for 2 weeks. In both models, Insl6-knockout mice exhibited greater cardiac systolic dysfunction and left ventricular dilatation. Cardiac dysfunction in the Insl6-knockout mice was associated with more extensive cardiac fibrosis and greater expression of fibrosis-associated genes. The continuous infusion of chemically synthesized INSL6 significantly attenuated left ventricular systolic dysfunction and cardiac fibrosis induced by isoproterenol infusion. Gene expression profiling suggests liver X receptor/retinoid X receptor signaling is activated in the isoproterenol-challenged hearts treated with INSL6 protein. CONCLUSIONS: Endogenous Insl6 protein inhibits cardiac systolic dysfunction and cardiac fibrosis in angiotensin II- and isoproterenol-induced cardiac stress models. The administration of recombinant INSL6 protein could have utility for the treatment of heart failure and cardiac fibrosis.

Transit and integration of extracellular mitochondria in human heart cells.

Tissue ischemia adversely affects the function of mitochondria, which results in impairment of oxidative phosphorylation and compromised recovery of the affected organ. The impact of ischemia on mitochondrial function has been extensively studied in the heart because of the morbidity and mortality associated with injury to this organ. As conventional methods to preserve cardiac cell viability and contractile function following ischemia are limited in their efficacy, we developed a unique approach to protect the heart by transplanting respiration-competent mitochondria to the injured region. Our previous animal experiments showed that transplantation of isolated mitochondria to ischemic heart tissue leads to decreases in cell death, increases in energy production, and improvements in contractile function. We also discovered that exogenously-derived mitochondria injected or perfused into ischemic hearts were rapidly internalised by cardiac cells. Here, we used three-dimensional super-resolution microscopy and transmission electron microscopy to determine the intracellular fate of endocytosed exogenous mitochondria in human iPS-derived cardiomyocytes and primary cardiac fibroblasts. We found isolated mitochondria are incorporated into cardiac cells within minutes and then transported to endosomes and lysosomes. The majority of exogenous mitochondria escape from these compartments and fuse with the endogenous mitochondrial network, while some of these organelles are degraded through hydrolysis.

Myocardial rescue with autologous mitochondrial transplantation in a porcine model of ischemia/reperfusion.

OBJECTIVE: To demonstrate the clinical efficacy of autologous mitochondrial transplantation in preparation for translation to human application using an in vivo swine model. METHODS: A left mini-thoracotomy was performed on Yorkshire pigs. The pectoralis major was dissected, and skeletal muscle tissue was removed and used for the isolation of autologous mitochondria. The heart was subjected to regional ischemia (RI) by temporarily snaring the circumflex artery. After 24 minutes of RI, hearts received 8 × 0.1 mL injections of vehicle (vehicle-only group; n = 6) or vehicle containing mitochondria (mitochondria group; n = 6) into the area at risk (AAR), and the snare was released. The thoracotomy was closed, and the pigs were allowed to recover for 4 weeks. RESULTS: Levels of creatine kinase-MB isoenzyme and cardiac troponin I were significantly increased (P = .006) in the vehicle-only group compared with the mitochondria group. Immune, inflammatory, and cytokine activation markers showed no significant difference between groups. There was no significant between-group difference in the AAR (P = .48), but infarct size was significantly greater in the vehicle group (P = .004). Echocardiography showed no significant differences in global function. Histochemistry and transmission electron microscopy revealed damaged heart tissue in the vehicle group that was not apparent in the mitochondria group. T2-weighted magnetic resonance imaging and histology demonstrated that the injected mitochondria were present for 4 weeks. CONCLUSIONS: Autologous mitochondrial transplantation provides a novel technique to significantly enhance myocardial cell viability following ischemia and reperfusion in the clinically relevant swine model.

Intracoronary Delivery of Mitochondria to the Ischemic Heart for Cardioprotection.

We have previously shown that transplantation of autologously derived, respiration-competent mitochondria by direct injection into the heart following transient ischemia and reperfusion enhances cell viability and contractile function. To increase the therapeutic potential of this approach, we investigated whether exogenous mitochondria can be effectively delivered through the coronary vasculature to protect the ischemic myocardium and studied the fate of these transplanted organelles in the heart. Langendorff-perfused rabbit hearts were subjected to 30 minutes of ischemia and then reperfused for 10 minutes. Mitochondria were labeled with 18F-rhodamine 6G and iron oxide nanoparticles. The labeled mitochondria were either directly injected into the ischemic region or delivered by vascular perfusion through the coronary arteries at the onset of reperfusion. These hearts were used for positron emission tomography, microcomputed tomography, and magnetic resonance imaging with subsequent microscopic analyses of tissue sections to confirm the uptake and distribution of exogenous mitochondria. Injected mitochondria were localized near the site of delivery; while, vascular perfusion of mitochondria resulted in rapid and extensive dispersal throughout the heart. Both injected and perfused mitochondria were observed in interstitial spaces and were associated with blood vessels and cardiomyocytes. To determine the efficacy of vascular perfusion of mitochondria, an additional group of rabbit hearts were subjected to 30 minutes of regional ischemia and reperfused for 120 minutes. Immediately following regional ischemia, the hearts received unlabeled, autologous mitochondria delivered through the coronary arteries. Autologous mitochondria perfused through the coronary vasculature significantly decreased infarct size and significantly enhanced post-ischemic myocardial function. In conclusion, the delivery of mitochondria through the coronary arteries resulted in their rapid integration and widespread distribution throughout the heart and provided cardioprotection from ischemia-reperfusion injury.