Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase-independent pathway involving intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2-72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large-scale DNA fragmentation ( 50 kbp), a signature event in AIF-mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron-enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large-scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large-scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury. Keywords: apoptosis, brain injury, controlled cortical impact, DNA damage, programmed cell death. Programmed cell death is an essential mechanism for the selective elimination of cells during development, homeostasis of tissues with cell turnover and removal of aging and abnormal cells (Steller 1995). In general, the process of programmed cell death is strictly regulated, with dysregulation occurring in, and contributing to, many acute and chronic pathological conditions (Thompson 1995). In the CNS, programmed cell death contributes to neuronal death after ischemia (Graham and Chen Abbreviations used: AIF, apoptosis inducing factor; CAD, of caspaseactivated deoxyribonuclease; kbp, kilo-basepair; PFGE, pulsed field gel electrophoresis; TBI, traumatic brain injury.

Congenital muscular dystrophy (CMD) is characterized by severe muscle wasting, premature death in early childhood, and lack of effective treatment. Most of the CMD cases are caused by genetic mutations of laminin-␣2, which is essential for the structural integrity of muscle extracellular matrix. Here, we report that somatic gene delivery of a structurally unrelated protein, a miniature version of agrin, functionally compensates for laminin-␣2 deficiency in the murine models of CMD. Adeno-associated virusmediated overexpression of miniagrin restored the structural integrity of myofiber basal lamina, inhibited interstitial fibrosis, and ameliorated dystrophic pathology. Furthermore, systemic gene delivery of miniagrin into multiple vital muscles significantly improved whole body growth and motility and quadrupled the lifespan (50% survival) of the dystrophic mice. Thus, our study demonstrated the efficacy of somatic gene therapy in a mouse model of CMD.adeno-associated virus ͉ integrity ͉ myofiber basal laming ͉ gene therapy

DNA damage is a common sequela of traumatic brain injury (TBI). Available techniques for the in situ identification of DNA damage include DNA polymerase I-mediated biotin-dATP nick-translation (PANT), the Klenow fragment of DNA polymerase I-mediated biotin-dATP nick-end labeling (Klenow), and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). While TUNEL has been widely utilized to detect primarily double-strand DNA breaks, the use of PANT to detect primarily single-strand DNA breaks and Klenow to detect both single- and double-strand DNA breaks has not been reported after TBI. Accordingly, coronal brain sections from naive rats and rats at 0, 0.5, 1, 2, 6, 24, and 72 h (n = 3-5/group) after controlled cortical impact with imposed secondary insult were processed using the PANT, Klenow, and TUNEL methods. Cells with DNA breaks were detected by PANT in the ipsilateral hemisphere as early as 0.5 h after injury and were maximal at 6 h (cortex = 66.3+/-15.8, dentate gyrus 58.6+/-12.8, CA1 = 15.8+/-5.9, CA3 = 12.8+/-4.2 cells/x 400 field, mean +/- SEM, all p < 0.05 versus naive). Cells with DNA breaks were detected by Klenow as early as 30 min and were maximal at 24 h (cortex = 56.3+/-14.3, dentate gyrus 78.0+/-16.7, CA1 = 25.8+/-4.7, CA3 = 29.3+/-15.1 cells/x 400 field, all p < 0.05 versus naive). Cells with DNA breaks were not detected by TUNEL until 2 h and were maximal at 24 h (cortex = 47.7+/-21.4, dentate gyrus 63.0+/-11.9, CA1 = 5.6+/-5.4, CA3 = 6.9+/-3.7 cells/x 400 field, cortex and dentate gyrus p < 0.05 versus naive). Dual-label immunofluorescence revealed that PANT-positive cells were predominately neurons. These data demonstrate that TBI results in extensive DNA damage, which includes both single- and double-strand breaks in injured cortex and hippocampus. The presence of multiple types of DNA breaks implicate several pathways in the evolution of DNA damage after TBI.

Reverse transcription-polymerase chain reaction and immunofluorescence analysis of D2XRII murine bone marrow stromal cells showed that gamma irradiation with doses of 2-50 Gy from (137)Cs stimulated expression of nitric oxide synthase 2 (Nos2, also known as iNos). The activation of Nos2 was accompanied by an increase in the fluorescence of 4,5-diaminofluorescein diacetate, a nitric oxide trap, and accumulation of 3-nitrotyrosine within cellular proteins in a dose-dependent manner. These effects were inhibited by actinomycin D and by N-[3-(aminomethyl)benzyl]acetamidine dihydrochloride, a specific inhibitor of Nos2. The induction of Nos2 expression and Nos2-dependent release of nitric oxide in D2XRII cells was observed within 24 h after irradiation and was similar in magnitude to that observed in cultures incubated with Il1b and Tnf. We conducted (1) confocal fluorescence imaging of 3-nitrotyrosine in bone marrow cells of irradiated C57BL/6J mice and (2) 3-nitrotyrosine fluorescence imaging of FDC-P1JL26 hematopoietic cells that were cocultured with previously irradiated D2XRII bone marrow stromal cells. Exposure to ionizing radiation increased the production of 3-nitrotyrosine in irradiated bone marrow cells in vivo and in nonirradiated FDC-P1JL26 cells cocultured with irradiated D2XRII cells for 1 or 4 h. We suggest that nitrative/oxidative stress to the transplanted multilineage hematopoietic cells due to exposure to nitric oxide released by host bone marrow stromal cells may contribute to the genotoxic events associated with malignant alterations in bone marrow tissue of transplant recipients who are prepared for engraftment by total-body irradiation.

These data demonstrate that tolerance can be induced by vaginal Ag exposure. In these experiments, mice were given vaginal agarose gel suppositories containing either 5 mg OVA or saline for 6 h. Mice were given suppositories either during the estrous (estrogen dominant) or diestrous (progesterone dominant) stage of the estrous cycle. Mice were restrained during the inoculation period to prevent orovaginal transmission of the Ag. After 1 wk, mice were immunized s.c. with OVA in CFA. After 3 wk, mice were tested for delayed-type hypersensitivity responses by measuring footpad swelling and measuring in vitro proliferation of lymphocytes to Ag. Using ELISA, the magnitude of the serum Ab response was also measured. In some mice, FITC conjugated to OVA was used to track the dissemination of the protein into the systemic tissues. The magnitude of footpad swelling was significantly reduced in mice receiving OVA-containing suppositories during estrus compared with mice receiving saline suppositories. Concomitant decreases in the Ag-specific proliferative response were also observed in lymph node lymphocytes and splenocytes. Conversely, mice inoculated during diestrus did not show a decreased response to Ag by either footpad response or in vitro proliferation. Serum Ab titers in the estrus-inoculated mice did not decrease significantly. These data demonstrate that the reproductive tract can be an inductive site for mucosally induced tolerance. However, unlike other mucosal sites such as the lung and gastrointestinal tract, reproductive tract tolerance induction is hormonally regulated.