Effective virus inactivation and removal by steps of Biotest Pharmaceuticals IGIV production process.

The virus validation of three steps of Biotest Pharmaceuticals IGIV production process is described here. The steps validated are precipitation and removal of fraction III of the cold ethanol fractionation process, solvent/detergent treatment and 35 nm virus filtration. Virus validation was performed considering combined worst case conditions. By these validated steps sufficient virus inactivation/removal is achieved, resulting in a virus safe product.

c-Jun N-terminal kinase (JNK-1) confers protection against brief but not extended ischemia during acute myocardial infarction.

Brief periods of ischemia do not damage the heart and can actually protect against reperfusion injury caused by extended ischemia. It is not known what causes the transition from protection to irreversible damage as ischemia progresses. c-Jun N-terminal kinase-1 (JNK-1) is a stress-regulated kinase that is activated by reactive oxygen and thought to promote injury during severe acute myocardial infarction. Because some reports suggest that JNK-1 can also be protective, we hypothesized that the function of JNK-1 depends on the metabolic state of the heart at the time of reperfusion, a condition that changes progressively with duration of ischemia. Mice treated with JNK-1 inhibitors or transgenic mice wherein the JNK-1 gene was ablated were subjected to 5 or 20 min of ischemia followed by reperfusion. When JNK-1 was inactive, ischemia of only 5 min duration caused massive apoptosis, infarction, and negative remodeling that was equivalent to or greater than extended ischemia. Conversely, when ischemia was extended JNK-1 inactivation was protective. Mechanisms of the JNK-1 switch in function were investigated in vivo and in cultured cardiac myocytes. In vitro there was a comparable switch in the function of JNK-1 from protective when ATP levels were maintained during hypoxia to injurious when reoxygenation followed glucose and ATP depletion. Both apoptotic and necrotic death pathways were affected and responded reciprocally to JNK-1 inhibitors. JNK-1 differentially regulated Akt phosphorylation of the regulatory sites Ser-473 and Thr-450 and the catalytic Thr-308 site in vivo. The studies define a novel role for JNK-1 as a conditional survival kinase that protects the heart against brief but not protracted ischemia.

Selective degeneration of central photoreceptors after hyperbaric oxygen in normal and metallothionein-knockout mice.

PURPOSE: Metallothioneins (MTs) in the brain and retina are believed to bind metals and reduce free radicals, thereby protecting neurons from oxidative damage. This study was undertaken to investigate whether retinal photoreceptor (PR) cells lacking MTs are more susceptible to hyperbaric oxygen (HBO)-induced cell death in vivo. METHODS: Wild-type (WT) and MT-knockout (MT-KO) mice lacking metallothionein (MT)-1 and MT-2 were exposed to three atmospheres of 100% oxygen for 3 hours, 3 times per week for 1, 3, or 5 weeks. The control animals were not exposed. Histologic analysis of PR viability was performed by counting rows of nuclei in the outer nuclear layer (ONL). Ultrastructure studies verified PR damage. RESULTS: HBO exposure produced a major loss of PR cells in the central retinas of WT and MT-KO mice, with no effect on the peripheral retina even at the longest (5 weeks) exposures. The degree of PR damage and cell death increased with duration of HBO exposure. One week of HBO exposure was insufficient to cause PR death, but tissue damage was observed in the inner and outer segments. At 3 weeks, the rows of PR nuclei in the central retina were significantly reduced by 38% in WT and 28% in MT-KO animals. At 5 weeks, PR loss was identical in WT (34%) and MT-KO (34%) animals and was comparable to that in WT at 3 weeks. CONCLUSIONS: The data suggest that MT-1 and -2 alone are not sufficient for protecting PRs against HBO-induced cell death. The selective degeneration of central PRs may provide clues to mechanisms of oxidative damage in retinal disease.

Selective apoptosis of breast cancer cells by siRNA targeting of BORIS.

Brother of the regulator of imprinted sites (BORIS) is an epigenetically acting transcription factor which represses the tumor inhibitor functions of the tumor suppressor protein CTCF. BORIS expression has not been documented in adult females, making it an exciting molecular target for drug development in breast cancer. Previously, we demonstrated that vaccination of mice with zing-finger (ZF)-deleted non-functional BORIS results in regression of breast cancer and generation of potent anti-tumor immune responses. RNAi induction can be used as an alternative approach for selective tumor cell killing. Short interfering RNA (siRNA) molecules targeting BORIS were generated and their efficacy was tested in MDA-MB-231 breast cancer and non-malignant epithelial cell lines. Treatment with BORIS-specific siRNA, but not control siRNA led to a concentration-dependent reduction in BORIS expression and proportional apoptotic death of the cancer but not control cells. To our knowledge this is first report demonstrating a critical role of BORIS in maintaining tumor cell viability.

Robust hypoxia-selective regulation of a retinal pigment epithelium-specific adeno-associated virus vector.

PURPOSE: To develop an hypoxia-regulated retinal pigment epithelium (RPE)-specific adeno-associated virus (AAV) gene transfer platform that exploits hypoxia as a physiologic trigger for an early antiangiogenic treatment strategy directed at arresting neovascularization in the eye. METHODS: Tissue-specific and hypoxia-regulated expression vectors were constructed with tandem combinations of hypoxia responsive elements and aerobically silenced elements (HRSE) that together induce gene expression in hypoxia and suppress it in normoxia. For RPE-specific expression, the HRSE and a (6x) HRE (hypoxia responsive element) oligomer were ligated upstream of the Rpe65 promoter in a pGL3 firefly luciferase plasmid (pGL3-HRSE-6xHRE-Rpe65). The cell specificity of this novel hybrid promoter was tested in human RPE (ARPE-19), human glioblastoma, rat C6 glioma, mouse hippocampal neurons, and human embryonic kidney cell lines. Expression of all cell types in normoxia, and following 40 h hypoxia, was analyzed by dual luciferase assay. After confirmation of its tissue-specificity and hypoxia-inducibility, the hybrid promoter construct was integrated into a replication-deficient AAV delivery system and tested for cell-selectivity and hypoxia-inducible green fluorescent protein (GFP) reporter expression. RESULTS: The HRSE-6xHRE-Rpe65 promoter was highly selective for RPE cells, strongly induced in hypoxia, and similar in activation strength to the cytomegalovirus (CMV) promoter. The AAV.HRSE.6xHRE.Rpe65 vector induced robust GFP expression in hypoxic ARPE-19 cells, but elicited no GFP expression in hypoxia in other cell types or in normoxic ARPE-19 cells. CONCLUSIONS: The hypoxia-regulated, aerobically-silenced RPE-targeting vector forms a platform for focal autoregulated delivery of antiangiogenic agents in hypoxic regions of the RPE. Such autoinitiated therapy provides a means for early intervention in choroidal neovascularization, when it is most sensitive to inhibition.

PKC-alpha and TAK-1 are intermediates in the activation of c-Jun NH2-terminal kinase by hypoxia-reoxygenation.

c-Jun NH(2)-terminal kinase (JNK), a member of the MAPK family of protein kinases, is a stress-response kinase that is activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through nonreceptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We recently demonstrated that the activation of JNK by this condition is initiated by ROI generated by mitochondrial electron transport and involves sequential activation of the proline-rich kinase 2 and the small GTP-binding factors Rac-1 and Cdc42. Here we present evidence that protein kinase C (PKC) and transforming growth factor-beta-activated kinase-1 (TAK-1) are also components of this pathway. Inhibition of PKC with the broad-range inhibitor calphostin C, the PKC-alpha/beta-selective inhibitor Go9367, or adenovirus-expressing dominant-negative PKC-alpha blocked the phosphorylation of proline-rich kinase 2 and JNK. Reoxygenation activated the mitogen-activated protein kinase kinase kinase, TAK-1, and promoted the formation of a complex containing Rac-1, TAK-1, and JNK but not apoptosis-stimulating kinase-1 or p21-activated kinase-1, which was detected within the first 10 min of reoxygenation. These results identify two new components, PKC and TAK-1, that have not been previously described in this signaling pathway.

Mitochondrial signals initiate the activation of c-Jun N-terminal kinase (JNK) by hypoxia-reoxygenation.

C-Jun N-terminal kinase (JNK) is part of the mitogen-activated protein kinase (MAPK) family of signaling pathways that are induced in response to extracellular stimuli. JNK is primarily a stress-response pathway and can be activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through non-receptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). The molecular initiators of MAPKs by non-receptor stimuli have not been described. Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We show here that the activation of JNK by this condition is initiated in the mitochondria and requires coupled electron transport, ROI generation, and calcium flux. These signals cause the selective, sequential activation of the calcium-dependent, proline-rich kinase Pyk2 and the small GTP binding factors Rac-1 and Cdc42. Interruption of these interactions with inactivated dominant negative mutant proteins, blocking calcium flux, or inhibiting electron transport through mitochondrial complexes II, III, or IV prevents JNK activation and results in a proapoptotic phenotype that is characteristic of JNK inhibition in this model of ischemia-reperfusion. The signaling pathway is unique for the reoxygenation stimulus and provides a framework for other non-receptor-mediated pathways of MAPK activation.

Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress.

Reperfusion injury occurs when ischaemic tissue is reperfused. It involves the generation and release of reactive oxygen that activates numerous signalling pathways and initiates cell death. Exposure of isolated cardiac myocytes to chronic hypoxia followed by reoxygenation results in the early activation of c-Jun N-terminal kinase (JNK) and death by apoptosis of approx. 30% of the myocytes. Although JNK activation has been described in a number of models of ischaemia/reperfusion, the contribution of JNK activation to cell fate has not been established. Here we report that the activation of JNK by reoxygenation correlates with myocyte survival. Transfection of myocytes with JNK pathway interfering plasmid vectors or infection with adenoviral vectors support the hypothesis that JNK is protective. Transfection or infection with JNK inhibitory mutants increased the rates of apoptosis by almost 2-fold compared with control cultures grown aerobically or subjected to hypoxia and reoxygenation. Caspase 9 activity, measured by LEHD cleavage, increased >3-fold during reoxygenation and this activity was enhanced significantly at all times in cultures infected with dominant negative JNK adenovirus. Hypoxia-reoxygenation mediated a biphasic (2.6- and 2.9-fold) activation of p38 mitogen-activated protein kinase, as well as a small increase of tumour necrosis factor alpha (TNFalpha) secretion, but treatments with the p38 MAPK-specific inhibitor SB203580 or saturating levels of a TNFalpha-1 blocking antibody provided only partial protection against apoptosis. The results suggest that JNK activation is protective and that the pathway is largely independent of p38 MAPK or secreted TNFalpha.