Stimulation Of Resident Cells Research Articles

An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

Effects of 1,25(OH)2D3 in experimental mesangial proliferative nephritis in rats

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active form of vitamin D3, is a potent immunomodulatory agent on several cell types such as monocytes and mesangial cells. Recruitment of inflammatory cells, as well as stimulation of resident cells and mesangial matrix accumulation are key features of various experimental and human glomerular diseases. Here we show that 1,25(OH)2D3 attenuates the morphologic and functional alterations in anti-Thy-1.1. nephritis, an experimental model of mesangial proliferative glomerulonephritis. The anti-Thy-1.1 group (group I) comprised 24 rats that at day 0 received 0.5 mL of saline containing 400 microg of monoclonal antibodies (mAb) anti-Thy-1.1 IgG. The anti-Thy-1.1 treated with 1,25(OH)2D3 group (group II) were 24 rats given 1,25(OH)2D3 at the dose of 25 ng/100 g body wt/day, from day -3 to day 14. At day 0, the rats received 400 microg of anti-Thy-1.1 monoclonal IgG. The control group (group III) comprised 12 rats injected with vehicle alone; the control group treated with 1,25(OH)2D3 (group IV)-12 rats were given 1,25(OH)2D3 as in group II without mAb administration. Proteinuria and urinary interleukin-6 excretion were measured daily. Blood urea nitrogen and creatinine, creatinine clearance, calcium, and phosphate were measured at days 0, 4, 7, and 14. In addition to conventional periodic acid-Schiff staining, binding of anti-Thy-1.1 IgG and C3b complement fraction, His48- and ED1-positive cells were studied by immunofluorescence. Mesangial proliferation was studied by the proliferating cell nuclear antigen (PCNA) technique. Apoptosis was evaluated by the TUNEL assay. The anti-Thy-1.1 treated with 1,25(OH)2D3 group versus the anti-Thy-1.1 alone group showed a significant reduction in urinary protein (at day 7, 424 +/- 228 vs. 66 +/- 30 mg/mg urinary creatinine, P = 0.02) and interleukin-6 excretion (at day 3, 537 +/- 360 pg/mL vs. 110 +/- 34 pg/mg urinary creatinine, P = 0.015), reduced glomerular diameters (at day 7, 283 +/- 38 vs. 261 +/- 48 microm, P < 0.01), decreased neutrophil (at day 4, 20 +/- 12 His48-positive cells/glomerulus vs. 3.7 +/- 1.3 His48-positive cells/glomerulus, P < 0.001), and monocyte accumulation (day 7, 4.9 +/- 2.9 ED1-positive cells/glomerulus vs. 2.8 +/- 2.9 ED1-positive cells/glomerulus, P < 0.05), and attenuated glomerular cells proliferation (day 7, 13 +/- 3.2 PCNA-positive cells/glomerulus vs. 9.4 +/- 3 PCNA-positive cells/glomerulus, P < 0.01). Apoptosis showed a biphasic behavior with an early peak at day 4 in the anti-Thy-1.1 group (2.3 +/- 2.2 TUNEL-positive cells/glom) related to cellular lysis and a late peak at day 14 related to the recovery phase. 1,25(OH)2D3 can reduce glomerular hypercellularity, inflammatory infiltration in anti-Thy-1.1 nephritis, preserving the apoptotic response of the reparative phase.