Spiritual Interventions Among Pediatric Patients With Cancer: A Systematic Review And Meta-Analysis.

CONTEXT: Although spiritual intervention is crucial in the care of childhood cancer patients (CCPs), its effectiveness has not yet been systematically evaluated. OBJECTIVES: To determine the effectiveness of existing spiritual interventions on psychological, spiritual outcomes, and quality of life (QoL) in CCPs. METHODS: We searched eight databases to identify relevant randomized controlled trials and quasi-experimental studies. Risk of bias was assessed using the Cochrane risk-of-bias tool for randomized trials. Results were either synthesized in a systematic narrative synthesis or a meta-analysis using a random effects model, where appropriate. The pooled treatment effect was estimated using the standardized mean difference (SMD) and 95% confidence interval (CI). RESULTS: Twelve studies with 576 CCPs were included. Eight studies showed a high risk of bias. The overall effect of existing spiritual interventions on QoL (Z = 1.05, SMD = 0.64, 95%CI = -0.15 to 1.83, P = 0.29), anxiety (Z = 1.11, SMD = -0.83, 95%CI = -2.30 to 0.64, P = 0.28) and depressive symptoms (Z = 1.06, SMD = -0.49, 95%CI = -1.40 to 0.42, P = 0.12) were statistically nonsignificant. The nonsignificant findings could be attributed to the high heterogeneity among the included studies (QoL: I2 = 85%; anxiety: I2 = 90%; depressive symptoms: I2 = 58%). CONCLUSION: Evidence to support the positive effects of existing spiritual interventions on psychological and spiritual outcomes and QoL in CCPs is insufficient. Future studies should adopt a more rigorous design and unify the outcome measures to reduce the risk of bias and heterogeneity, respectively.

Noninvasive tracking of gene transcript and neuroprotection after gene therapy.

Gene therapy holds exceptional potential for translational medicine by improving the products of defective genes in diseases and/or providing necessary biologics from endogenous sources during recovery processes. However, validating methods for the delivery, distribution and expression of the exogenous genes from such therapy can generally not be applicable to monitor effects over the long term because they are invasive. We report here that human granulocyte colony-stimulating factor (hG-CSF) complimentary DNA (cDNA) encoded in self-complementary adeno-associated virus-type 2 adeno-associated virus, as delivered through eye drops at multiple time points after cerebral ischemia using bilateral carotid occlusion for 60 min (BCAO-60) led to significant reduction in mortality rates, cerebral atrophy and neurological deficits in C57black6 mice. Most importantly, we validated hG-CSF cDNA expression using translatable magnetic resonance imaging (MRI) in living brains. This noninvasive approach for monitoring exogenous gene expression in the brains has potential for great impact in the area of experimental gene therapy in animal models of heart attack, stroke, Alzheimer's dementia, Parkinson's disorder and amyotrophic lateral sclerosis, and the translation of such techniques to emergency medicine.

Neuronal NOS inhibitor that reduces oxidative DNA lesions and neuronal sensitivity increases the expression of intact c-fos transcripts after brain injury.

In response to oxidative stress, the ischemic brain induces immediate early genes when its nuclear genes contain gene damage. Antioxidant that reduces gene damage also reduces cell death. To study the mechanism of neuronal sensitivity, we investigated the transcription of the c-fos gene after brain injury of the ischemia-reperfusion type using focal cerebral ischemia-reperfusion in Long-Evans hooded rats. We observed a significant (p < 0.01) increase in c-fos mRNA in the ischemic cortex immediately after brain injury. However, the c-fos transcript was sensitive to RNase A protection assay (RPA) upon reperfusion. The transcript became significantly resistant to RPA (42%, p < 0.03) when 3-bromo-7-nitroindazole (25 mg/kg, i.p.), known to abolish nitric oxide, gene damage and neuronal sensitivity, was injected. Our data suggest that neuronal nitric oxide synthase and aberrant mRNA from genes with oxidative damage could be associated with neuronal sensitivity.

Oxidative damage to the c-fos gene and reduction of its transcription after focal cerebral ischemia.

We investigated oxidative damage to the c-fos gene and to its transcription in the brain of Long-Evans rats using a transient focal cerebral ischemia and reperfusion (FCIR) model. We observed a significant (p < 0.001) increase in the immunoreactivity to 8-hydroxy-2'-guanine (oh8G) and its deoxy form (oh8dG) in the ischemic cortex at 0-30 min of reperfusion in all 27 animals treated with 15-90 min of ischemia. Treatment with a neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole (60 mg/kg, i.p.), abolished the majority but not all of the oh8G/oh8dG immunoreactivity. Treatment with RNase A reduced the oh8G immunoreactivity, suggesting that RNA may be targeted. This observation was further supported by decreased levels of mRNA transcripts of the c-fos and actin genes in the ischemic core within 30 min of reperfusion using in situ hybridization. The reduction in mRNA transcription occurred at a time when nuclear gene damage, detected as sensitive sites to Escherichia coli Fpg protein in the transcribed strand of the c-fos gene, was increased 13-fold (p < 0.01). Our results suggest that inhibiting nNOS partially attenuates FCIR-induced oxidative damage and that nNOS or other mechanisms induce nuclear gene damage that interferes with gene transcription in the brain.

Suppression of postischemic hippocampal nerve growth factor expression by a c-fos antisense oligodeoxynucleotide.

We examined the uptake and distribution of an antisense phosphorothioated oligodeoxynucleotide (s-ODN) to c-fos, rncfosr115, infused into the left cerebral ventricle of male Long-Evans rats and the effect of this s-ODN on subsequent Fos, NGF, neurotrophin-3 (NT-3), and actin expression. To establish the uptake and turnover of s-ODN in the brain, we studied the copurification of the immunoreactivity of biotin with biotinylated s-ODN that was recovered from different regions of the brain. A time-dependent diffusion and the localization of s-ODN were further demonstrated by labeling the 3'-OH terminus of s-ODN in situ with digoxigenin-dUTP using terminal transferase and detection using anti-digoxigenin IgG-FITC. Cellular uptake of the s-ODN was evident in both the hippocampal and cortical regions, consistent with a gradient originating at the ventricular surface. Degradation of the s-ODN was observed beginning 48 hr after delivery. The effectiveness of c-fos antisense s-ODN was demonstrated by its suppression of postischemic Fos expression, which was accompanied by an inhibition of ischemia-induced NGF mRNA expression in the dentate gyrus. Infusion of saline, the sense s-ODN, or a mismatch antisense s-ODN did not suppress Fos expression. That this effect of c-fos antisense s-ODN was specific to NGF was demonstrated by its lack of effect on the postischemic expression of the NT-3 and beta-actin genes. Our results demonstrate that c-fos antisense s-ODN blocks selected downstream events and support the contention that postischemic Fos regulates the subsequent expression of the NGF gene and that Fos expression may have a functional component in neuroregeneration after focal cerebral ischemia-reperfusion.

Gene expression and apoptosis in the spinal cord neurons after sciatic nerve injury.

To demonstrate a dependence of spinal cord motoneurons on the communication with their targets, the expression of immediate early gene c-fos and neurotrophin genes in the lumbar (L3-L6) spinal cord neurons was examined in Sprague-Dawley rats (male > or = 9-weeks-old) with unilateral sciatic nerve transection. Using in situ hybridization, we detected the expression of c-fos mRNA in the motoneurons of the spinal cord segments within 45 min to 3 h of peripheral nerve transection (n = 4 in each time point). The expression of c-fos mRNA was also correlated positively with the expression of Fos antigen using immunohistochemistry, while no change in calbindin and parvalbumin antigens were noted. The expression of BDNF mRNA increased at 90 min after sciatic nerve transection. However, no detectable enhancement in the expression of NGF mRNA was observed. DNA fragmentation in neurons was observed using the incorporation of digoxigenin-dUTP by terminal transferase into 3'-OH terminals of DNA fragments in the ipsilateral section of the spinal cords 48h after nerve injury. Nuclei that exhibited DNA fragmentation were not observed in the spinal cord of the control animals. Lastly, we observed that the majority of astrocytes did not have DNA fragmentation. Because the detection of DNA fragmentation using this assay is one of early detections of apoptosis or programmed cell death, the result suggested we could detect early cell death in spinal cord, and indicated a target dependence of the neurons in the spinal cord after transection of sciatic nerve.

Analysis of genomic instability in Li-Fraumeni fibroblasts with germline p53 mutations.

Germline p53 mutations are frequently observed in the normal DNA of cancer-prone patients with Li-Fraumeni syndrome (LFS). Fibroblasts from LFS patients develop chromosomal aberrations, loss of cell cycle control, and spontaneous immortalization. We transfected four different mutant p53 genes into human skin fibroblasts from normal donors with two copies of wild-type p53 (p53(wt/wt)). Each mutant p53 expression-plasmid induced genomic instability equivalent to that seen in LFS cells. To test the role of wild-type and mutant p53 alleles in DNA replication and fidelity in LFS cells, we analysed the replication of the SV40-based shuttle vector pZ189 in four types of cells. We used p53(wt/mut) and p53(mut/-) LFS fibroblasts, and p53(-/-) non-LFS cells. Replication of pZ189 in vivo was significantly reduced by the presence of a p53(wt) allele. To show that this was not just due to inhibition of the function of T-antigen in SV40-based replication, we constructed a shuttle vector, pZ402, that contains a mutation in SV40 T-antigen which blocks its ability to interact with p53. Replication of pZ402 in LFS cells was also reduced by the presence of p53(wt), indicating that p53 can inhibit replication by interacting with proteins within the cellular replication machinery. Replicative errors in this shuttle vector are detected as mutations in a marker gene, supF. In addition to supF mutations, we observed deletion of a portion of the SV40 T-antigen gene in 100% of replicated plasmid pZ189 mutants (supF-) from the p53(wt/mut) fibroblasts and in 88% of the supF mutants from the p53(mut/-) (amino acid 175 arg to his) LFS cells. In one cell strain of immortal LFS cells, P53(mut/-) , containing a p53 frameshift mutation at amino acid 184, pZ189 replication yielded very few of these deleted shuttle vector plasmids (15%). These large deletions were not detected in plasmids replicated in p53(-/-) non-LFS cells, Saos-2 cells. Replicated plasmids with a normal supF gene were never found to have this large deletion regardless of the cell from which they were derived. Because the supF gene is not in the same region of the shuttle vector as the T-antigen gene it appears that second, independent gene deletions are frequent when replicative errors in supF occur in cells with a mutant p53. We conclude, therefore, that p53(wt/mut) LFS cells contain an activity that promotes mutations. Such an activity, which is likely to be due to the p53(mut), could result in the high rate of chromosomal instability and allelic loss of the wild-type p53 observed as these cells spontaneously immortalize.

Increased expression of c-fos mRNA and AP-1 transcription factors after cortical impact injury in rats.

Levels of c-fos mRNA and AP-1 transcription factors co-expression were measured in a controlled lateral cortical impact model of traumatic brain injury (TBI) in rats. Ipsilateral cerebral cortex and bilateral hippocampal c-fos mRNA increases were revealed by in situ hybridization after lateral cortical impact injury. Based on regional in situ hybridization data, we employed semi-quantitative RT-PCR methods to study the temporal profile of changes in the ipsilateral cortex at the site of injury. We found that TBI produces transient increases of c-fos mRNA expression in the ipsilateral cerebral cortex at 5 min postinjury, which peaks at 1 h postinjury and subsides by 1 day postinjury. Gel shift nuclear protein binding assays showed that AP-1 transcription factor binding was robustly increased in injured cerebral cortex at 1 h, 3 h, 5 h and 1 day after injury. These data indicate that TBI can produce significant increases in c-fos expression and subsequent upregulation of the AP-1 transcription factors. Thus, AP-1 transcription factors modulation of downstream gene expression may be an important component of pathophysiological responses to TBI.

Suppression of ischemia-induced fos expression and AP-1 activity by an antisense oligodeoxynucleotide to c-fos mRNA.

Activation of c-fos, an immediate early gene, and the subsequent expression of the Fos protein have been noted following focal cerebral ischemia. Fos and Jun form a heterodimer as activator protein 1 (AP-1), which transregulates the expression of several genes. To study the postischemic events related to c-fos expression, we suppressed the expression of c-fos by intraventricular infusion of an antisense oligodeoxynucleotide (anti-rncfosr115) of c-fos mRNA. The effectiveness of anti-rncfosr115 was confirmed first by its capability to block in vitro c-fos mRNA translation. In vivo, after intraventricular infusion of 32P-labeled anti-rncfosr115, the oligodeoxynucleotide was internalized within 6 hours and detectable also in the nucleic acids fraction up to 41 hours. Treatment of the recovered nucleic acids with RNase H separated the labeled oligodeoxynucleotide from the nucleic acid fraction, indicating an association of the antisense oligodeoxynucleotide and cellular RNA after uptake. When focal cerebral ischemia was induced 16 hours after the infusion of anti-rncfosr115, the postischemic increase in Fos expression and AP-1 binding activity were suppressed. Specificity of the effect of anti-rncfosr115 was suggested by its failure to suppress the DNA binding activity of nuclear cyclic AMP response elements. These results support the hypothesis that increased AP-1 binding activity following focal cerebral ischemia is dependent on Fos expression and can be inhibited in vivo by antisense c-fos oligodeoxynucleotides.

Infidelity of DNA synthesis as a cause of mutagenesis.

The concept underlying these studies is that a major determinant of mutagenesis involves perturbations in the fidelity of DNA replication. i.e., the accuracy by which DNA polymerases copy DNA templates. To investigate this relationship, we have designed in vitro assays to measure the accuracy of DNA replication and used these systems to screen for and to quantitate factors that promote errors in DNA synthesis. Using DNA polymerase from bacteria, the frequency of mistakes with phi X174 DNA as a template approaches 10(-7) and is similar to the spontaneous mutation rates in bacterial cells. In contrast, DNA polymerases from animal cells are more error-prone. The differences in fidelity among mammalian DNA polymerases which lack error-correcting mechanisms suggest that these enzymes enhance accuracy by improving base-selection. Thus, mutants in DNA polymerase-alpha might be altered in base-selection. Chinese hamster V79 cell mutants selected by resistance to aphidicolin, a specific inhibitor of DNA polymerase-alpha, have been reported (Somatic Cell Genet., 7: 235-253, 1981). DNA polymerase-alpha was purified from mitochondria-free crude extracts of these mutants by sequential column chromatography using DEAE-cellulose and phosphocellulose. DNA polymerase-alpha purified from one of the mutants is 10-fold more resistant to aphidicolin than the same enzyme purified from the parental cells. Moreover, the apparent Km for dCTP is 1.0 +/- 0.4 microM for the mutant polymerase and 10 +/- 4 microM for the parental enzyme. These observed differences are in accord with the known competition between aphidicolin and dCTP, and provide a mechanism for the aphidicolin resistance of the mutant, i.e., the decrease in Km for dCTP. The elevated spontaneous and induced mutation rate exhibited by this mutant could be mediated by the alteration in DNA polymerase-alpha. With DNA replicating enzymes from a variety of sources, enhancement of mutagenesis has been demonstrated by alteration in precursor pools, damage to DNA templates, loss of nucleotide bases on DNA, metal ions that interact with nucleotide bases, and organic compounds that intercalate into DNA. The alterations of deoxynucleoside triphosphate pools also occur after treatment of animal cells with known mutagens. This observation may provide a new mechanism for mutagenesis by these agents independent of alterations in DNA.