Peptidomimetic fluoromethylketone rescues mice from lethal endotoxic shock.

BACKGROUND: Septic shock is a leading cause of mortality in intensive care units. No new interventions in the last 20 years have made a substantial impact on the outcome of patients with septic shock. Identification of inhibitable pathways that mediate death in shock is an important goal. MATERIALS AND METHODS: Two novel caspase inhibitors, (2-indolyl)-carbonyl-Ala-Asp-fluoromethylketone (IDN 1529) and (1-methyl-3-methyl-2-indolyl)-carbonyl-Val-Asp-fluoromethylketone (IDN 1965), were studied in a murine model of endotoxic shock. RESULTS: IDN 1529 prolonged survival when given before or up to 3 hr after high-dose LPS (p < 0.01) and increased by 2.2-fold the number of animals surviving longterm after a lower dose of LPS (p < 0.01). Despite its similar chemical structure, IDN 1965 lacked these protective effects. Both compounds inhibited caspases 1, 2, 3, 6, 8, and 9, and both afforded comparable reduction in Fas- and LPS-induced caspase 3-like activity and apoptosis. Paradoxically, administration of IDN 1529 but not IDN 1965 led to an increase in the LPS-induced elevation of serum cytokines related directly (IL-1beta, IL-18) or indirectly (IL-1alpha, IL-1Ra) to the action of caspase 1. CONCLUSIONS: A process that appears to be distinct from both apoptosis and the release of inflammatory cytokines is a late-acting requirement for lethality in endotoxic shock. Inhibition of this process can rescue mice even when therapy is initiated after LPS has made the mice severely ill.     

CpG oligodeoxynucleotides protect newborn mice from a lethal challenge with the neurotropic Tacaribe arenavirus

The innate immune system is key to limiting the early spread of most pathogens and directing the development of Ag-specific immunity. Recently, a number of synthetic molecules that activate the innate immune system by stimulating TLRs have been identified. Among them, synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs (CpG ODNs) were shown to activate TLR9-bearing B cells, macrophages, and dendritic cells to induce a strong proinflammatory milieu and a type 1-biased immune response that protects mice from a variety of parasitic, bacterial, and viral infections. Although the protective effect of CpG ODN in adult mice was well established, its effectiveness in neonates, which have lower numbers of dendritic, B, and T cells and tend to favor Th2 responses, was unclear. This study uses the New World arenavirus Tacaribe, a neurotropic pathogen that is lethal in newborn mice, to explore the effectiveness of TLR-mediated innate immune responses. Neonatal BALB/c mice treated with CpG ODN at the time of infection had reduced viral load (p < 0.01) and increased survival (52%, p < 0.001 i.p.; 36%, p < 0.05 intranasally). Protection was achieved in mice treated no later than 3 days postchallenge and appears to be mediated by an increase in Ag-specific Abs (IgG and IgM) and to require inducible NO synthase expression and NO production. To our knowledge, this is the first study assessing the mechanisms by which CpG ODN can protect mice from a neurotropic viral infection.     

Flavonoids protect mice from two types of lethal shock induced by endotoxin

The protective effect of flavonoids on two types of lethal endotoxic shock was studied. A lethal endotoxic shock was induced by administration of lipopolysaccharide (LPS) into D-galactosamine (D-GalN)-sensitized mice and another one was done by administration of a high dose of LPS into normal mice. Pretreatment with a series of flavonoids protected mice from two types of endotoxin lethality. Flavonoid pretreatment reduced the serum tumor necrosis factor-alpha (TNF-alpha) level in mice injected with D-GalN and LPS, but not in mice injected with a high dose of LPS. TNF-alpha-induced lethal shock in D-GalN-sensitized mice was also protected by pretreatment with flavonoids, suggesting that flavonoids augmented the resistance to TNF-alpha lethality. On the other hand, flavonoids reduced the plasma level of lipid peroxides in mice injected with a high dose of LPS, but not in D-GalN-sensitized mice. Taken together, these results indicated that flavonoids might protect mice from two types of endotoxin lethality. The protective mechanism of flavonoids in each endotoxin lethality is discussed.     

Exosomes from Plasmodium yoelii-infected reticulocytes protect mice from lethal infections

Exosomes are 30-100-nm membrane vesicles of endocytic origin that are released after the fusion of multivesicular bodies (MVBs) with the plasma membrane. While initial studies suggested that the role of exosomes was limited to the removal of proteins during the maturation of reticulocytes to erythrocytes, recent studies indicate that they are produced by different types of cells and are involved in promoting inter-cellular communication and antigen presentation. Here, we describe the isolation and characterization of exosomes from peripheral blood of BALB/c mice infected with the reticulocyte-prone non-lethal Plasmodium yoelii 17X strain. Importantly, proteomic analysis revealed the presence of parasite proteins in these vesicles. Moreover, immunization of mice with purified exosomes elicited IgG antibodies capable of recognizing P. yoelii-infected red blood cells. Furthermore, lethal challenge of immunized mice with the normocyte-prone lethal P. yoelii 17XL strain caused a significant attenuation in the course of parasitaemia, increased survival time, and altered the cell tropism to reticulocytes. These results were obtained also when the exosomes were isolated from a P. yoelii-infected reticulocyte culture indicating that reticulocyte-derived exosomes carry antigens and are involved in immune modulation. Moreover, inclusion of CpG ODN 1826 in exosome immunizations elicited IgG2a and IgG2b antibodies and promoted survival, clearance of parasites and subsequent sterile protection of 83% of the animals challenged with P. yoelli 17XL. To our knowledge, this is the first report of immune responses elicited by exosomes derived from reticulocytes opening new avenues for the modulation of anti-malaria responses.     

CpG oligodeoxynucleotides protect mice from lethal challenge with Candida albicans via a pathway involving tumor necrosis factor-alpha-dependent interleukin-12 induction

In this study, we have attempted to determine whether the systemic administration of CpG oligodeoxynucleotide (CpG-ODN) 1826 would protect mice against systemic lethal Candida albicans infection. CpG-ODNs were found completely to protect mice from death and also reduced the growth of C. albicans in the kidneys. The administration of CpG-ODNs resulted in early interleukin (IL)-12 mRNA expression in the kidneys and an increase in serum IL-12 levels. The protective activity of CpG-ODN was abolished in IL-12-deficient (IL-12-/-) mice, thereby indicating the IL-12-dependency inherent to the effects of CpG-ODN. The protective effect of CpG-ODN was not associated with the activity of NF-kappaB. Interestingly, in tumor necrosis factor (TNF)-alpha-deficient (TNF-/-) mice CpG-ODN neither exerted protective effects nor induced IL-12 expression. These data indicate that CpG-ODN protects animals against lethal C. albicans challenge via a pathway that involves the TNF-alpha-dependent induction of IL-12.     

Suppressive oligodeoxynucleotides inhibit silica-induced pulmonary inflammation

Inhalation of silica-containing dust particles induces silicosis, an inflammatory disease of the lungs characterized by the infiltration of macrophages and neutrophils into the lungs and the production of proinflammatory cytokines, chemokines, and reactive oxygen species (ROS). Synthetic oligodeoxynucleotides (ODN) expressing "immunosuppressive motifs" were recently shown to block pathologic inflammatory reactions in murine models of autoimmune disease. Based on those findings, the potential of suppressive ODN to prevent acute murine silicosis was examined. In vitro studies indicate that suppressive ODN blunt silica-induced macrophage toxicity. This effect was associated with a reduction in ROS production and p47phox expression (a subunit of NADPH oxidase key to ROS generation). In vivo studies show that pretreatment with suppressive (but not control) ODN reduces silica-dependent pulmonary inflammation, as manifest by fewer infiltrating cells, less cytokine/chemokine production, and lower levels of ROS (p < 0.01 for all parameters). Treatment with suppressive ODN also reduced disease severity and improved the survival (p < 0.05) of mice exposed to silica.     

Apoptotic cells protect mice against lipopolysaccharide-induced shock

LPS is a main causative agent of septic shock. There is a lack of effective therapies. In vitro studies have shown that uptake of apoptotic cells actively inhibits the secretion by activated macrophages (Mphi) of proinflammatory mediators such as TNF-alpha and that such uptake increases the antiinflammatory and immunosuppressive cytokine TGF-beta. We therefore investigated the protective effect of apoptotic cells against LPS-induced endotoxic shock in mice. The current report is the first study to demonstrate that administration of apoptotic cells can protect mice from LPS-induced death, even when apoptotic cells were administered 24 h after LPS challenge. The beneficial effects of administration of apoptotic cells included 1) reduced circulating proinflammatory cytokines, 2) suppression of polymorphonuclear neutrophil infiltration in target organs, and 3) decreased serum LPS levels. LPS can quickly bind to apoptotic cells and these LPS-coated apoptotic cells can be recognized and cleared by Mphi in a CD14/thrombospondin/vitronectin receptor-dependent manner, accompanied with suppression of TNF-alpha and enhancement of IL-10 expression by LPS-activated Mphi. Apoptotic cells may therefore have therapeutic potential for the treatment of septic shock.     

Enhanced dendritic cell survival attenuates lipopolysaccharide-induced immunosuppression and increases resistance to lethal endotoxic shock

Impaired immune function and associated immunosuppression are hallmarks of septic syndromes. As part of an overall deactivation of the immune system, profound depletion of dendritic cells (DCs) occurs in both septic patients and septic mice. Such depletion of DCs is potentially associated with immunosuppression and with failure to induce a protective Th1 immune response; it may equally be predictive of fatal outcome in septic patients. To evaluate the impact of enhanced DC survival on LPS-induced immunosuppression and on survival after LPS-induced septic shock, we created a transgenic mouse model specifically overexpressing the human form of the antiapoptotic protein Bcl-2 in DCs (DC-hBcl-2 mice). DCs derived from DC-hBcl-2 mice exhibited higher resistance to maturation-induced apoptosis after LPS treatment both in vitro and in vivo. Moreover, prolongation of DC survival diminished sublethal LPS-induced DC loss and immunosuppression, with maintenance of the differentiation potential of Th1 cells and enhanced T cell activation. Such modulation of the immune response appears to constitute a key feature of the attenuated mortality observed after LPS-induced shock in DC-hBcl-2 mice. Our study therefore identifies DC death as a key determinant of endotoxin-induced immunosuppression and mortality in mice.     

Ebola GP-specific monoclonal antibodies protect mice and guinea pigs from lethal Ebola virus infection

Ebola virus (EBOV) causes acute hemorrhagic fever in humans and non-human primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (MAbs) against Ebola glycoprotein in mice and guinea pigs. Immunocompetent mice or guinea pigs were given MAbs i.p. in various doses individually or as pools of 3-4 MAbs to test their protection against a lethal challenge with mouse- or guinea pig-adapted EBOV. Each of the 8 MAbs (100 μg) protected mice from a lethal EBOV challenge when administered 1 day before or after challenge. Seven MAbs were effective 2 days post-infection (dpi), with 1 MAb demonstrating partial protection 3 dpi. In the guinea pigs each MAb showed partial protection at 1 dpi, however the mean time to death was significantly prolonged compared to the control group. Moreover, treatment with pools of 3-4 MAbs completely protected the majority of animals, while administration at 2-3 dpi achieved 50-100% protection. This data suggests that the MAbs generated are capable of protecting both animal species against lethal Ebola virus challenge. These results indicate that MAbs particularly when used as an oligoclonal set are a potential therapeutic for post-exposure treatment of EBOV infection.     

Nitric oxide hemoglobin in mice and rats in endotoxic shock.

Mice given ip bacterial endotoxin (LPS) at 10 mg/kg showed a statistically significant decrease in plasma glucose and an increase in hematocrit at 2 h after injection. Glucose was still decreased at 4 h, but the hematocrit had returned to control values. Nitrosylated hemoglobin (HbNO) was detected at 3, but not at 2 h. By 4 h it had increased 5-fold. When N-monomethylarginine (NMMA) at 100 mg/kg, ip was given 2 h after LPS in mice, the HbNO concentration at 4 h was significantly reduced, but the hypoglycemia was worsened because NMMA itself produced a significant hypoglycemia. Rats given iv LPS, 20 mg/kg, showed a fleeting, transient rise in mean arterial pressure (MAP) lasting only a few min. Thereafter, the MAP tended to drift slowly downward over 4 h, but when the MAP at 30 min intervals was compared to the pre-LPS MAP, there were no significant differences. Plasma glucose in unanesthetized rats was significantly elevated at 1 h, back to control at 2 h, and significantly decreased at 3 h. HbNO was detected as early as 1 h after injection. By 2 h the HbNO concentrations exceeded the highest levels found in mice, and they were still increasing as late as 5 h after injection. Unanesthetized rats showed toxic signs and 3/12 rats died within 4 hours of LPS administration. These results are consistent with a model for endotoxic shock in which LPS stimulates an inducible pathway for NO synthesis.     

CpG oligodeoxynucleotides protect normal and SIV-infected macaques from Leishmania infection

Oligodeoxynucleotides containing CpG motifs (CpG ODNs) mimic microbial DNA and activate effectors of the innate immune response, which limits the spread of pathogens and promotes an adaptive immune response. CpG ODNs have been shown to protect mice from infection with intracellular pathogens. Unfortunately, CpG motifs that optimally stimulate humans are only weakly active in mice, mandating the use of nonhuman primates to monitor the activity and safety of "human" CpG ODNs in vivo. This study demonstrates that CpG ODN treatment of rhesus macaques significantly reduces the severity of the lesions caused by a challenge with Leishmania: Leishmania superinfection is common in immunocompromised hosts, particularly those infected with HIV. This study shows that PBMCs from HIV-infected subjects respond to stimulation with CpG ODNs. To determine whether CpG ODNs can protect retrovirus-infected primates, SIV-infected macaques were treated with CpG ODNs and then challenged with Leishmania: Both lesion size and parasite load were significantly reduced in the CpG-treated animals. These findings support the clinical development of CpG ODNs as immunoprotective agents in normal and HIV-infected patients.     

Effects of centimeter waves on the immune system of mice in endotoxic shock

The effects of centimeter waves (8.15-18 GHz, 1 microW/cm2, 1 h daily for 10 days; MW) on the production of the tumor necrosis factor alpha, interleukin-lalpha, interleukin-1beta, interleukin-2, and the expression of interleukin-6, interleukin-10, interferon-gamma, nitric oxide and HSP27, HSP72 and HSP90alpha in mice irradiated before or after LPS injection were studied. An acute endotoxic model was produced by a single LPS injection. The effects of microwaves on nitric oxide, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma were dependent on the functional status of exposed animals. Thus, an exposure of healthy mice to microwaves for 10 days was followed by a decrease in nitric oxide and interferon-gamma production, and an increase in the production of the tumor necrosis factor-alpha and interleukin-6. On the contrary, an exposure to MW before intoxication resulted in an increase in the synthesis of nitric oxide and interferon-gamma as well as a decrease in the concentration of the tumor necrosis factor-alpha and interleukin-6 in blood of mice in endotoxic shock. When microwave exposure was used after LPS injection, it did not provide any protective effect, and preliminary irradiation enhanced the resistance of the organism to endotoxic shock.     

BCL-2 and BAX protect adult mice from lethal Sindbis virus infection but do not protect spinal cord motor neurons or prevent paralysis

Cellular proteins that regulate apoptotic cell death can modulate the outcome of Sindbis virus (SV) encephalitis in mice. Both endogenous and overexpressed BCL-2 and BAX proteins protect newborn mice from fatal SV infection by blocking apoptosis in infected neurons. To determine the effects of these cellular factors on the course of infection in older animals, a more neurovirulent SV vector (dsNSV) was constructed from a viral strain that causes both prominent spinal cord infection with hind-limb paralysis and death in weanling mice. This vector has allowed assessment of the effects of BCL-2 and BAX on both mortality and paralysis in these hosts. Similar to newborn hosts, weanling mice infected with dsNSV encoding BCL-2 or BAX survived better than animals infected with control viruses. This finding indicates that BCL-2 and BAX both protect neurons that mediate host survival. Neither cellular factor, however, could suppress the development of hind-limb paralysis or prevent the degeneration of motor neurons in the lumbar spinal cord. Infection of BAX knockout mice with dsNSV demonstrated that endogenous BAX also enhances the survival of animals but has no effect on paralysis. These findings for the spinal cord are consistent with earlier data showing that dying lumbar motor neurons do not exhibit an apoptotic morphology. Thus, divergent cell death pathways are activated in different target populations of neurons during neurovirulent SV infection of weanling mice.     

Anti-IL-6 monoclonal antibodies protect against lethal Escherichia coli infection and lethal tumor necrosis factor-alpha challenge in mice

Potentially fatal physiologic and metabolic derangements can occur in response to bacterial infection in animals and man. Recently it has been shown that alterations in the levels of circulating cytokines such as IL-6 and TNF-alpha occur shortly after bacterial challenge. To understand better the role of IL-6 in inflammation, we investigated the effects of in vivo anti-mouse IL-6 antibody treatment in a mouse model of septic shock. Rat anti-mouse IL-6 neutralizing mAb was produced from splenocytes of an animal immunized with mouse rIL-6. This mAb, MP5-20F3, was a very potent and specific antagonist of mouse IL-6 in vitro bioactivity, demonstrated using the NFS60 myelomonocytic and KD83 plasmacytoma target cell lines, and also immunoprecipitated radiolabeled IL-6. Anti-IL-6 mAb pretreatment of mice subsequently challenged with lethal doses of i.p. Escherichia coli or i.v. TNF-alpha protected mice from death caused by these treatments. Pretreatment of E. coli-challenged mice with anti-IL-6 led to an increase in serum TNF bioactivity, in comparison to isotype control antibody, implicating IL-6 as a negative modulator of TNF in vivo. Anti-TNF-alpha treatment of mice challenged i.p. with live E. coli resulted in a 70% decrease in serum IL-6 levels, determined by immunoenzymetric assay, compared to control antibody, thereby supporting a role for TNF-alpha as a positive regulator of IL-6 levels. We conclude that IL-6 is a mediator in lethal E. coli infection, and suggest that antagonists of IL-6 may be beneficial therapeutically in life-threatening bacterial infection.     

Radiation rescue: mesenchymal stromal cells protect from lethal irradiation

Background

Successful treatment of acute radiation syndromes relies on immediate supportive care. In patients with limited hematopoietic recovery potential, hematopoietic stem cell (HSC) transplantation is the only curative treatment option. Because of time consuming donor search and uncertain outcome we propose MSC treatment as an alternative treatment for severely radiation-affected individuals.

Methods and Findings

Mouse mesenchymal stromal cells (mMSCs) were expanded from bone marrow, retrovirally labeled with eGFP (bulk cultures) and cloned. Bulk and five selected clonal mMSCs populations were characterized in vitro for their multilineage differentiation potential and phenotype showing no contamination with hematopoietic cells. Lethally irradiated recipients were i.v. transplanted with bulk or clonal mMSCs. We found a long-term survival of recipients with fast hematopoietic recovery after the transplantation of MSCs exclusively without support by HSCs. Quantitative PCR based chimerism analysis detected eGFP-positive donor cells in peripheral blood immediately after injection and in lungs within 24 hours. However, no donor cells in any investigated tissue remained long-term. Despite the rapidly disappearing donor cells, microarray and quantitative RT-PCR gene expression analysis in the bone marrow of MSC-transplanted animals displayed enhanced regenerative features characterized by (i) decreased proinflammatory, ECM formation and adhesion properties and (ii) boosted anti-inflammation, detoxification, cell cycle and anti-oxidative stress control as compared to HSC-transplanted animals.

Conclusions

Our data revealed that systemically administered MSCs provoke a protective mechanism counteracting the inflammatory events and also supporting detoxification and stress management after radiation exposure. Further our results suggest that MSCs, their release of trophic factors and their HSC-niche modulating activity rescue endogenous hematopoiesis thereby serving as fast and effective first-line treatment to combat radiation-induced hematopoietic failure.     

Successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus

Newly emerging viruses often circulate as a heterogeneous swarm in wild animal reservoirs prior to their emergence in humans, and their antigenic identities are often unknown until an outbreak situation. The newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV) and reemerging influenza virus cause disproportionate disease in the aged, who are also notoriously difficult to successfully vaccinate, likely due to immunosenescence. To protect against future emerging strains, vaccine platforms should induce broad cross-reactive immunity that is sufficient to protect from homologous and heterologous challenge in all ages. From initial studies, we hypothesized that attenuated Venezuelan equine encephalitis virus (VEE) replicon particle (VRP) vaccine glycoproteins mediated vaccine failure in the aged. We then compared the efficacies of vaccines bearing attenuated (VRP(3014)) or wild-type VEE glycoproteins (VRP(3000)) in young and aged mice within novel models of severe SARS-CoV pathogenesis. Aged animals receiving VRP(3000)-based vaccines were protected from SARS-CoV disease, while animals receiving the VRP(3014)-based vaccines were not. The superior protection for the aged observed with VRP(3000)-based vaccines was confirmed in a lethal influenza virus challenge model. While the VRP(3000) vaccine's immune responses in the aged were sufficient to protect against lethal homologous and heterologous challenge, our data suggest that innate defects within the VRP(3014) platform mediate vaccine failure. Exploration into the mechanism(s) of successful vaccination in the immunosenescent should aid in the development of successful vaccine strategies for other viral diseases disproportionately affecting the elderly, like West Nile virus, influenza virus, norovirus, or other emerging viruses of the future.