A cell culture-derived whole-virus H9N2 vaccine induces high titer antibodies against hemagglutinin and neuraminidase and protects mice from severe lung pathology and weight loss after challenge with a highly virulent H9N2 isolate.

BACKGROUND: Influenza viruses of subtype A/H9N2 are enzootic in poultry across Asia and the Middle East and are considered to have pandemic potential. The development of new vaccine manufacturing technologies is a cornerstone of influenza pandemic preparedness. METHODS: A non-adjuvanted whole-virus H9N2 vaccine was developed using Vero cell culture manufacturing technology. The induction of hemagglutination inhibition (HI) and virus-neutralizing antibodies was assessed in CD1 mice and guinea pigs. A highly sensitive enzyme-linked lectin assay was used to investigate the induction of antibodies capable of inhibiting the enzymatic activity of the H9N2 neuraminidase. Protective efficacy against virus replication in the lung after challenge with the homologous virus was evaluated in BALB/c mice by a TCID(50) assay, and prevention of virus replication in the lung and associated pathology were evaluated by histology and immunohistochemistry. To investigate the ability of the vaccine to prevent severe disease, BALB/c mice were challenged with a highly virulent mouse-adapted H9N2 isolate which was generated by multiple lung-to-lung passage of wild-type virus. RESULTS: The vaccine elicited high titers of functional H9N2-specific HA antibodies in both mice and guinea pigs, as determined by HI and virus neutralization assays. High titer H9N2-specific neuraminidase inhibiting (NAi) antibodies were also induced in both species. Vaccinated mice were protected from lung virus replication in a dose-dependent manner after challenge with the homologous H9N2 virus. Immunohistochemical analyses confirmed the lack of virus replication in the lung and an associated substantial reduction in lung pathology. Dose-dependent protection from severe weight loss was also provided after challenge with the highly virulent mouse-adapted H9N2 virus. CONCLUSIONS: The induction of high titers of H9N2-specific HI, virus-neutralizing and NAi antibodies and dose-dependent protection from virus replication and severe disease in animal models suggest that the Vero cell culture-derived whole-virus vaccine will provide an effective intervention in the event of a H9N2 pandemic situation.

A new mouse model mimicking thrombotic thrombocytopenic purpura: correction of symptoms by recombinant human ADAMTS13.

Deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a VWF-cleaving protease, is the key factor in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), a life-threatening thrombotic microangiopathy. It is well established that ADAMTS13 deficiency results in elevated plasma levels of ultra-large VWF multimers (ULVWF), which are prone to induce platelet aggregation; however, the actual trigger of TTP development remains uncertain. Here we describe a new animal model in which some TTP-like symptoms can be triggered in ADAMTS13 knockout mice by challenge with 2000 units/kg body weight of recombinant human VWF containing ULVWF multimers. Animals rapidly showed clinical symptoms and developed severe thrombocytopenia. Schistocytosis, a decrease in hematocrit, and elevated serum lactate dehydrogenase levels were observed. The heart was identified as the most sensitive target organ with rapid onset of extensive platelet aggregation in the ventricles and myocardial necrosis. Prophylactic administration of 200 units/kg recombinant human ADAMTS13 protected ADAMTS13 knockout mice from developing TTP. Therapeutic administration of 320 units/kg rhADAMTS13 reduced the incidence and severity of TTP findings in a treatment interval-dependent manner. We therefore consider this newly established mouse model of thrombotic microangiopathy highly predictive for investigating the efficacy of new treatments for TTP.

Antimycin A and lipopolysaccharide cause the leakage of superoxide radicals from rat liver mitochondria.

Here we show that both Antimycin A, a respiratory chain inhibitor inducing apoptosis, and endotoxic shock, a syndrome accompanied by both necrosis and apoptosis, cause not only an increase but also the leakage of superoxide radicals (O(2)(*-)) from rat heart mitochondria (RHM), while O(2)(*-) generated in intact RHM do not escape from mitochondria. This was shown by a set of O(2)(*-)-sensitive spin probes with varying hydrophobicity. The levels of O(2)(*-) detected in intact RHM gradually increase as the hydrophobicity of spin probes increases and were not sensitive to superoxide dismutase (SOD) added to the incubation medium. Both Antimycin A and endotoxic shock elevated O(2)(*-) levels. Elevated O(2)(*-) levels became sensitive to SOD but in a different manner. The determination of O(2)(*-) with water-soluble PPH was fully sensitive to SOD, while the determination of O(2)(*-) with the more hydrophobic CMH and CPH was only partially sensitive to SOD, suggesting the release of a portion of O(2)(*-) into the surrounding medium.

Rotenone damages striatal organotypic slice culture.

Epidemiological studies suggest the involvement of pesticides in the etiology of Parkinson's disease. Exposure to rotenone results in degeneration of the nigrostriatal pathway through inhibition of complex I. Organotypic striatal slice cultures were prepared from brains of adult mice and treated with rotenone (0.01, 0.05, 0.1, and 1 mM) for 48 h. Lactate dehydrogenase activity was elevated by 167% at 1 mM of rotenone. Using fluorescent indicators, membrane damage was up to 130% as measured by propidium iodide fluorescence, and superoxide (DHE) and nitric oxide (DAF-FM) formation were increased by 195% and 774% at 1 mM of rotenone, respectively, compared to controls. The study concludes that formation of radicals mediated striatal degeneration by rotenone.

A novel endotoxin-induced pathway: upregulation of heme oxygenase 1, accumulation of free iron, and free iron-mediated mitochondrial dysfunction.

Mitochondria are involved in the development of organ failure in critical care diseases. However, the mechanisms underlying mitochondrial dysfunction are not clear yet. Inducible hemoxygenase (HO-1), a member of the heat shock protein family, is upregulated in critical care diseases and considered to confer cytoprotection against oxidative stress. However, one of the products of HO-1 is Fe2+ which multiplies the damaging potential of reactive oxygen species catalyzing Fenton reaction. The aim of this study was to clarify the relevance of free iron metabolism to the oxidative damage of the liver in endotoxic shock and its impact on mitochondrial function. Endotoxic shock in rats was induced by injection of lipopolysaccharide (LPS) at a dose of 8 mg/kg (i.v.). We observed that the pro-inflammatory cytokine TNF-alpha and the liver necrosis marker aspartate aminotransferase were increased in blood, confirming inflammatory response to LPS and damage to liver tissue, respectively. The levels of free iron in the liver were significantly increased at 4 and 8 h after onset of endotoxic shock, which did not coincide with the decrease of transferrin iron levels in the blood, but rather with expression of the inducible form of heme oxygenase (HO-1). The proteins important for sequestering free iron (ferritin) and the export of iron out of the cells (ferroportin) were downregulated facilitating the accumulation of free iron in cells. The temporarily increased concentration of free iron in the liver correlated with the temporary impairment of both mitochondrial function and tissue ATP levels. Addition of exogenous iron ions to mitochondria isolated from control animals resulted in an impairment of mitochondrial respiration similar to that observed in endotoxic shock in vivo. Our data suggest that free iron released by HO-1 causes mitochondrial dysfunction in pathological situations accompanied by endotoxic shock.

Blue laser light increases perfusion of a skin flap via release of nitric oxide from hemoglobin.

It has recently been shown that nitrosyl complexes of hemoglobin (NO-Hb) are sensitive to low-level blue laser irradiation, suggesting that laser irradiation can facilitate the release of biologically active nitric oxide (NO), which can affect tissue perfusion. The aim of this study was to evaluate the therapeutic value of blue laser irradiation for local tissue perfusion after surgical intervention. Blood was withdrawn from a rat, exposed to NO and infused back to the same rat or used for in vitro experiments. In vitro, an increase of NO-Hb levels (electron paramagnetic resonance spectroscopy) up to 15 microM in rat blood did not result in the release of detectable amounts of NO (NO selective electrode). Blue laser irradiation of NO-Hb in blood caused decomposition of NO-Hb complexes and release of free NO. Systemic infusion of NO-Hb in rats affected neither systemic circulation (mean arterial pressure) nor local tissue perfusion (Doppler blood flow imaging system). In contrast, a clear enhancement of local tissue perfusion was observed in epigastric flap when elevated NO-Hb levels in blood were combined with local He-Cd laser irradiation focused on the left epigastric artery. The enhancement of regional tissue perfusion was not accompanied by any detectable changes in systemic circulation. This study demonstrates that blue laser irradiation improves local tissue perfusion in a controlled manner stimulating NO release from NO-Hb complexes.

Opposite effects of endotoxin on mitochondrial and endoplasmic reticulum functions.

In this study, we determined functional integrity and reactive oxygen species generation in mitochondria and endoplasmic reticulum in liver of rats subjected to endotoxic shock to clarify whether intracellular reactive oxygen species (ROS) destabilize cellular integrity causing necrosis in rats challenged with lipopolysaccharide (LPS). LPS caused drastically increased plasma levels of alanine aminotransferase, suggesting damage to plasma membranes of liver cells. Liver necrosis was confirmed by histological examination. LPS induced a significant increase in ROS production in rat liver mitochondria (RLM), but did not impair mitochondrial function. In contrast to mitochondria, enzymatic activity and ROS production of cytochrome P450 were lower in microsomal fraction obtained from LPS-treated animals, suggesting the dysfunction of endoplasmic reticulum. Protein patterns obtained from RLM by two-dimensional electrophoresis showed significant upregulation of mitochondrial superoxide dismutase by LPS. We hypothesize that upregulation of this enzyme protects mitochondria against mitochondrial ROS, but does not protect other cellular compartments such as endoplasmic reticulum and plasma membrane causing necrosis.

Different effects of endotoxic shock on the respiratory function of liver and heart mitochondria in rats.

This study was designed to clarify whether mitochondrial function/dysfunction and reactive oxygen species (ROS) production have a temporal relationship with organ failure during endotoxic shock. Adult male Sprague-Dawley rats were divided into three groups receiving 1) isotonic saline (control group, n = 16); 2) 8 mg/kg lipopolysaccharide (LPS; n = 8); or 3) 20 mg/kg LPS (n = 8) intraperitoneally under short anesthesia with 3.5% of isoflurane. After 16 h, animals were killed to analyze plasma, rat liver mitochondria (RLM), and rat heart mitochondria (RHM). In accordance with plasma analysis, LPS-treated rats were divided into "responders" and "nonresponders" with high and low levels of alanine aminotransferase and creatine, respectively. RHM from responders had significantly lower respiratory activity in state 3, suggesting a decreased rate of ATP synthesis. In contrast, RLM from responders had significantly higher respiratory activity in state 3 than both nonresponders and the control group. This increase was accompanied by a decrease in phosphate-to-oxygen ratio values, which was not observed in RHM. ROS generation determined with a spin probe, 1-hydroxy-3-carboxypyrrolidine, neither revealed a difference in RHM between LPS and control groups nor between responders and nonresponders. In contrast, RLM isolated from responders showed a marked increase in ROS production compared with both the control group and nonresponders. Our data demonstrate that 1) RHM and RLM respond to endotoxic shock in a different manner, decreasing and increasing respiratory activity, respectively, and 2) there is a temporal relationship between ROS production in RLM (but not in RHM) and tissue damage in rats subjected to LPS shock.

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock.

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.