Gene expression profiling to monitor therapeutic and adverse effects of antisense therapies for Duchenne muscular dystrophy

OBJECTIVES: The objective of this study was to assess the utility of the gene expression profiling technique for the preclinical evaluation of drug efficacy and safety, taking a new therapeutic approach for Duchenne muscular dystrophy (DMD) as an example. METHODS: Muscles from dystrophin-deficient (mdx) mice, a well-characterized animal model for DMD, were injected with antisense constructs that restore the open reading frame in the Dmd gene. Synthetic antisense oligonucleotides (AONs) complexed with different carriers to enhance cellular uptake and recombinant adeno-associated virus (rAAV)-expressed antisense sequences were evaluated. Muscular gene expression profiles were analyzed on oligonucleotide microarrays. RESULTS: Polyethylenimine (PEI)-complexed AONs restored the reading frame slightly more effectively than uncomplexed, F127- or Optison-complexed AONs. However, PEI induced the expression of many immune genes, reflecting an aggravation of the inflammation present in untreated mdx mice. Expression profiles in Optison and F127-injected muscles were similar to those of saline treated muscles, implying that these carriers did not evoke adverse responses. Due to moderate levels of exon skipping, a significant shift toward wild-type expression levels was not detected. Injection with rAAV vectors resulted in much higher production of dystrophin and greatly improved the histological appearance of the muscle. Depending on the efficacy of the treatment, the expression of genes previously shown to be elevated in muscular dystrophies, partly or completely returned to wild-type expression levels. Reductions in inflammation and fibrosis were among the most prominent changes observed. CONCLUSION: Expression profiling is a powerful tool for the evaluation of both desired and adverse effects of new pharmacological therapies. It is sensitive and detects changes that are not histologically visible. In addition, its ability to simultaneously monitor a large number of different biological processes not only reduces the number of different assays required in preclinical research and clinical trials, but may also assist in the early detection of potential side effects.     

When children of problem drinkers grow old: Does the increased risk of mental disorders persist?

It is well established that children of problem drinkers have an increased risk of developing mental health problems, not only during childhood but also when they grow up into adolescents and adults. However, it has not been examined whether this risk is also present during the old age of these children. In this study, we examine the question whether this increased risk is present in inhabitants of eleven residential homes (mean age 85 years). A total of 355 residents indicated whether one of their parents ever had problems with alcohol. We also used the MINI diagnostic interview to assess the presence of mental disorders. We found that parental problem drinking was significantly associated with having a major depression (current and lifetime), and with the number of drinks in the past week. No significant relationship was found with alcohol-related disorders and anxiety disorders. It was already known that parental problem drinking results in mental health problems in children. We found clear indications that these problems do not disappear when these children grow old.     

Requests from professional care providers for consultation with palliative care consultation teams

Goals of work Professional care providers need a substantial basis of competence and expertise to provide appropriate palliative care. Little is known about the problems professionals experience in their palliative care provision in daily practice or about the nature of the advice and support they request from experts. Our aim was to investigate the extent to which professionals requested assistance from palliative care consultation teams and the reasons behind these requests to trace any gaps they experience in the provision of palliative care.Methods As part of a large national palliative care development programme, we studied requests for consultation made by professional care providers over a 2-year period. The requests for consultation were recorded on a specially developed standard registration form and classified according to 11 domains relevant to palliative care.Main results Professional care providers requested 4351 consultations on account of 8413 specific problems in 11 quality-of-life and quality-of-care domains. The distribution of problems over these domains was unbalanced: 42.2% of the specific problems were physical, while the percentages of psychological, pharmacological and organizational problems were 7.7, 12.5 and 12.8%, respectively. In contrast, issues of a spiritual nature or concerned with daily functioning were raised infrequently (1.1 and 0.9%). Details of the specific problems in all the domains are described in the text and tables.Conclusions The results of our study form a valid basis on which to develop and implement improvements in palliative care. We recommend that future well-founded policies for palliative care should incorporate palliative care consultation as well as educational and organizational interventions.     

Interleukin-1 beta down-regulates the expression of metabotropic glutamate receptor 5 in cultured human astrocytes

Expression of metabotropic glutamate receptor 5 (mGluR5) protein is known to be plastic and to depend critically on the astrocytes' microenvironment. In the present study we investigated whether interleukins, which are involved in the immune response following brain injury, could contribute to the regulation of mGluR5 protein in human astrocytes in culture. Using Western blotting and immunocytochemistry, no detectable changes in the expression of the mGluR5 protein were observed with both interleukin 1β and interleukin 6 in undifferentiated cultures (growing in serum free media). In contrast, in cultures that had been morphologically differentiated by exposure to epidermal growth factor (EGF), addition of interleukin 1β (but not interleukin 6) reduced mGluR5 protein expression. In addition, stimulation of phosphoinositide hydrolysis by the selective group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) was reduced after exposure to interleukin 1β. The suppressive effect on mGluR5 was prevented by the interleukin 1 receptor antagonist. Thus, interleukin 1β may represent an additional pathway through which mGluR5 expression and function can be modulated in astrocytes under different pathological conditions associated with an inflammatory response.     

Expression of nitric oxide synthase isoforms and nitrotyrosine formation after hypoxia-ischemia in the neonatal rat brain

BACKGROUND AND PURPOSE: Production of nitric oxide is thought to play an important role in neuroinflammation. Previously, we have shown that combined inhibition of neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) can reduce hypoxia-ischemia-induced brain injury in 12-day-old rats. The aim of this study was to analyze changes in expression of nNOS, iNOS and endothelial NOS (eNOS), and nitrotyrosine (NT) formation in proteins in neonatal rats up to 48 h after cerebral hypoxia-ischemia. METHODS: Twelve-day-old rats were subjected to unilateral carotid artery occlusion and hypoxia, resulting in unilateral cerebral damage. NOS and nitrotyrosine expression were determined by immunohistochemistry and Western blot analysis at 30 min-48 h after hypoxia-ischemia. RESULTS: nNOS was increased in both hemispheres from 30 min to 3 h after hypoxia-ischemia. In the contralateral hemisphere, eNOS was decreased 1-3 h after hypoxia-ischemia. In the ipsilateral hemisphere, eNOS was decreased at 0.5 h after hypoxia-ischemia, normalized at 1-3 h and was increased 6-12 h after hypoxia-ischemia. At 24 and 48 h after hypoxia-ischemia, eNOS levels normalized. Surprisingly, iNOS expression did not change from 30 min up to 48 h after hypoxia-ischemia in the ipsi- or contralateral hemisphere. In addition, the regional expression of iNOS in the brain as determined by immunohistochemistry did not change after hypoxia-ischemia. Expression of nitrotyrosine was slightly increased in both hemispheres only at 30 min after hypoxia-ischemia. CONCLUSION: In 12-day-old rat pups, cerebral hypoxia-ischemia induced a transient increase in nNOS, eNOS, and nitrotyrosine in proteins, but no change in iNOS expression up to 48 h after the insult.     

Preventing the incidence of new cases of mental disorders: a meta-analytic review

To assess the results of studies examining the effects of preventive interventions on the incidence of mental disorders, we conducted a systematic review. A literature search resulted in 13 high-quality randomized trials, six on depressive disorder (including postpartum depression), one on anxiety, one examining both anxiety and depression, three on posttraumatic stress disorders, one on psychosis, and one on any mental disorder. The overall relative risk (RR) was 0.73 (95% CI, 0.56-0.95), indicating a reduction of the risk to become a new case of a mental disorder. The seven studies on prevention of depressive disorder resulted in a RR of 0.72 (95% CI, 0.54-0.96). The risk of posttraumatic stress disorder was somewhat increased after debriefing, but not significantly (RR=1.33), indicating a possible adverse effect. Prevention of new cases of mental disorders seems to be possible and may be an important way of reducing the enormous burden of these disorders.     

Identification of tumor-specific antibodies in patients with breast cancer vaccinated with gene-modified allogeneic tumor cells

Thirty HLA-A2 women with metastatic breast cancer received up to 14 vaccinations with MDA-MB-231-CD80, an HLA-A2 allogeneic breast cancer cell line, which had been lipofected with the cDNA for the CD80 costimulatory molecule. Tumor cells were administered with BCG or GM-CSF as an adjuvant. Sera obtained before and after vaccination were analyzed for antibodies to tumor cell lysate, MUC1, HER2/neu and p53. Since the cell line was grown in fetal bovine serum (FBS), sera were also analyzed for antibodies to FBS. Eighteen of 24 patients for whom sera were available exhibited anti-FBS activity at baseline. Eleven of these 18 patients and all six patients without baseline anti-FBS activity showed an increased titer after vaccination. The anti-FBS activity required that serum samples be absorbed in excess FBS to detect specific antibodies to tumor cell lysate. A two-fold increase in the titer of IgG specific to tumor cell lysate was observed in 6 patients. Eight of 24 patients made an antibody response to HER-2/neu, four of 24 to MUC1 and one of 24 to p53. Although antibody production to a variety of tumor cell-associated antigens was detected our results suggest that a whole cell vaccine comprising a CD80-transfected allogeneic breast cancer cell line with adjuvant BCG or GM-CSF was not a reliable method to induce significant antibody responses in women with advanced breast cancer.     

Changes in the G-protein-coupled receptor desensitization machinery during relapsing-progressive experimental allergic encephalomyelitis

G-protein-coupled receptors (GPCR) play an important role in inflammation. Their responsiveness is regulated by G-protein-coupled receptor kinases (GRKs) and beta-arrestins. We show here that induction of experimental autoimmune encephalomyelitis (EAE) by myelin oligodendrocyte glycoprotein (MOG) resulted in a profound decrease in GRK2 and GRK6 protein in splenocytes during all phases of disease. GRK2 mRNA was also lower during EAE, although the decrease in mRNA was less pronounced than the decrease in GRK2 protein. Interestingly, beta-arrestin protein expression was significantly increased. Downregulation of GRK2 was restricted to the spleen and mesenteric lymph nodes and was not observed in peritoneal macrophages. Furthermore, EAE did not induce alterations in GRK2 expression in heart, liver and pituitary.