In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters.

The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruses to neonatal rodents as an alternative to the existing technology of generating germline transgenic light producing rodents. At this age, neonates acquire immune tolerance to the conditionally responsive luciferase reporter. This simple and transferrable procedure permits surrogate quantitation of transcription factor activity over the lifetime of the animal. We show principal efficacy by temporally quantifying NFκB activity in the brain, liver and lungs of somatotransgenic reporter mice subjected to lipopolysaccharide (LPS)-induced inflammation. This response is ablated in Tlr4(-/-) mice or when co-administered with the anti-inflammatory glucocorticoid analogue dexamethasone. Furthermore, we show the malleability of this technology by quantifying NFκB-mediated luciferase expression in outbred rats. Finally, we use somatotransgenic bioimaging to longitudinally quantify LPS- and ActivinA-induced upregulation of liver specific glucocorticoid receptor and Smad2/3 reporter constructs in somatotransgenic mice, respectively

Lipoxygenases and Poly(ADP-Ribose) Polymerase in Amyloid Beta Cytotoxicity

The 12/15-lipoxygenase(s) (LOX), poly(ADP-ribose) polymerase (PARP-1) activity and mitochondrial apoptosis inducing factor (AIF) protein in the amyloid β (Aβ) toxicity were investigated in PC12 cells that express either wild-type (APPwt) or double Swedish mutation (APPsw) forms of human Aβ precursor protein. Different levels of Aβ secretion and free radicals formation characterize these cells. The results demonstrated a relationship between the Aβ levels and LOX protein expression and activity. High Aβ concentration in APPsw cells correlated with a significant increase in free radicals and LOX activation, which leads to translocation of p65/NF-κB into the nucleus. An increase in AIF expression in mitochondria was observed concurrently with inhibition of PARP-1 activity in the nuclear fraction of APPsw cells. We suggested that AIF accumulation in mitochondria may be involved in adaptive/protective processes. However, inhibition of PARP-1 may be responsible for the disturbances in transcription and DNA repair as well as the degeneration of APP cells. Under conditions of increased nitrosative stress, evoked by the nitric oxide donor, sodium nitroprusside (SNP, 0.5 mM), 70–80% of all cells types died after 24 h, significantly more in APPsw cells. There was no further significant change in mitochondrial AIF level and PARP-1 activity compared to corresponding non-treated cells. Only one exception was observed in PC12 control, where SNP significantly inhibits PARP-1 activity. Moreover, SNP significantly activated gene expression for 12/15-LOX in all types of investigated cells. Inhibitors of all LOX isoforms and specific inhibitor of 12-LOX enhanced the survival of cells that were subjected to SNP. We conclude that the LOX pathways may play a role in Aβ toxicity and in nitrosative-stress-induced cell death and that inhibition of these pathways offers novel protective strategies

Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice

Inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. Prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. To this end high dose attenuated VSV-G pseudotyped equine infectious anaemia virus (EIAV) encoding ß-galactosidase under the CMV promoter was injected into the fetal circulation of immuno-competent MF1 mice. We saw prolonged, extensive gene expression in the liver, heart, brain, and muscle and to a lesser extent in the kidney and lung of postnatal mice. Progressive clustered hepatocyte staining suggests clonal expansion of cells stably transduced. We thus provide proof of principle for efficient gene delivery and persistent transgene expression after prenatal application of the EIAV vector and its potential for permanent correction of genetic diseases

Protective effect of ethanolic and water extracts of sea buckthorn (<i style="">Hippophae rhamnoides</i> L.) against the toxic effects of mustard gas

821-831Ethanolic extract of H. rhamnoides L. leaf (HL-EOH), water and ethanolic extract of H. rhamnoides fruit (HF-W and HF-EOH), and H. rhamnoides flavone from fruit (HR-flavone) were evaluated against percutaneously administered sulphur mustard (SM), a chemical warfare agent. The animals administered with SM (9.7, 19.3 and 38.7 mg/kg) died at various days depending upon the dose and there was a significant reduction in the body weight. The H. rhamnoides extracts (1g/kg; 3 doses; po) significantly protected the lethality, with a protective index of 2.4, 1.7, 1.7 and 2.2 for HL-EOH, HF-W, HF-EOH and HR-flavone respectively. Reduced glutathione (GSH) and oxidized glutalthione (GSSG) levels were reduced, and malondialdehyde (MDA) was elevated after percutaneous administration of SM. Oral administration of HL-EOH and HR-flavone significantly protected the body weight loss. Recovery in the levels of GSH, GSSG and MDA were also observed following oral administration of HL-EOH and HR-flavone. All the extracts were non-toxic and the LD50 was more than 5 g/kg. The present study shows that percutaneous administration of SM induces oxidative stress and ethanolic extract of leaf of H. rhamnoides and H. rhamnoides flavone from fruit can significantly protect it