Gene therapy of Parkinson's disease using adeno-associated virus (AAV) vectors

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra and a severe decrease in dopamine in the striatum. A promising approach to the gene therapy of PD is intrastriatal expression of dopamine-synthesizing enzymes [tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC)]. The most appropriate gene-delivery vehicles for neurons are adeno-associated virus (AAV) vectors, which are derived from non-pathogenic virus. Therefore, TH and AADC genes were introduced into the striatum in the lesioned side using separate AAV vectors in parkinsonian rats, and the coexpression of TH and AADC resulted in better behavioral recovery compared with TH alone. Another strategy for gene therapy of PD is the protection of dopaminergic neurons in the substantia nigra using an AAV vector containing a glial cell line-derived neurotrophic factor (GDNF) gene. Combination of dopamine-supplement gene therapy and GDNF gene therapy would be a logical approach to the treatment of PD.     

Nitric oxide synthase activity and inhibition after neonatal hypoxia ischemia in the mouse brain

Despite the emergence of therapies for hypoxic-ischemic injury to the mature nervous system, there have been no proven efficacious therapies for the developing nervous system. Recent studies have shown that pharmacological blockade of neuronal nitric oxide synthase (nNOS) activity can ameliorate damage after ischemia in the mature rodent. We have previously shown that elimination of nNOS neurons, either by targeted disruption of the gene or by pharmacological depletion with intraparenchymal quisqualate, can decrease injury after hypoxia-ischemia. Using a simpler pharmacological approach, we studied the efficacy of a systemically administered NOS inhibitor, 7-nitroindazole, a relatively selective inhibitor of nNOS activity. Using multiple doses and concentrations administered after the insult, we found that there was only a trend for protection with higher doses of the drug. A significant decrease in NOS activity was seen at 18 h and 5 days in the cortex, and at 2 h and 18 h in the hippocampus after the hypoxia-ischemia. nNOS expression decreased and remained depressed for at least 18 h after the insult. When nNOS expression was normalized to MAP2 expression, a decrease was seen at 18 h in the cortex and at 2 and 18 h in the hippocampus. These data suggest that further inhibition of NOS activity at early timepoints may not provide substantial benefit. At 5 days after the insult, however, NOS activity and normalized nNOS expression returned to baseline or higher in the hippocampus, the region showing the most damage. These data suggest that delayed administration of nNOS inhibitor after hypoxic-ischemic injury might be beneficial.     

TH1/TH2 and regulatory cytokines in adults with otitis media with effusion.

OBJECTIVE: Otitis media with effusion is one of the most common and intractable ear diseases. However, the role of Th1, Th2, and immunoregulatory cytokines on the pathogenesis of the disease in adult patients remains to be determined. The aim of this study is to disclose the cytokine expression in middle ear effusions (MEEs) in adults and to compare the profile on the basis of the presence of allergic rhinitis and the type of effusions. STUDY DESIGN: A prospective controlled clinical study. PATIENTS: MEEs were collected from 80 adult subjects. The concentration of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12, and interferon (IFN)-gamma in MEEs were determined by using enzyme-linked immunosorbent assay. RESULTS: IL-2, IL-4, IL-5, IL-10, IL-12, and IFN-gamma in MEEs were detected in 60 (75.0%), 33 (41.3%), 42 (52.5%), 14 (17.5%), 80 (100%), and 66 (82.5%) samples, respectively. Among these cytokines, only the concentration of IL-4 in the allergic rhinitis-positive group was significantly higher than that in the allergic rhinitis-negative group. On the other hand, IL-2, IL-12, and IFN-gamma were detected, regardless of the presence of allergic rhinitis, and the concentration of these cytokines correlated with each other. The correlation between the concentration of IL-4 and IL-5 was also detected. In addition, both the incidence rate and the concentration of IL-10 in MEEs were significantly higher in the mucoid type compared with those in the serous type effusions. CONCLUSION: Regardless of allergic status, IL-12 may play a critical role in the pathogenesis of otitis media with effusion by affecting the production of IL-2 and IFN-gamma. In addition, IL-4 may have some impact on the immunologic condition in adults with allergic rhinitis. IL-10 potentially affects the viscosity of MEEs.     

Effects of Inhalation or Incubation of Oxitropium Bromide on Diaphragm Muscle Contractility in Mice

BackgroundAlthough oxitropium bromide is used clinically as an anticholinergic drug (i.e., parasympathetic antagonist) to relax airway smooth muscle, we examined whether it has or does not have any effects on diaphragm muscle.MethodsThree treatment sets, an oxitropium bromide inhalation only group, an oxitropium bromide inhalation plus endotoxin injection group (in vivo) and an oxitropium bromide incubation group (in vitro) were studied as to diaphragm muscle contractile properties.ResultsOxitropium bromide inhalation shifted force-frequency curves upward at 2 h after inhalation (p < 0.05) and inhibited the decrease of force-frequency curves due to endotoxin injection in vivo. Incubation with oxitropium bromide of untreated diaphragm muscle and diaphragm muscle injected with endotoxin did not increase the force-frequency curves dose-dependently in vitro; however, it caused both types of muscle to be fatigue resistant.ConclusionsWe speculate that the increment of muscle contractility with the inhalation of oxitropium bromide was induced by the antagonization of musucarinic acetylcholine receptors (mAChR). In addition, the changes of fatigue resistance provoked by oxitropium bromide, which also is speculated to antagonize mAChR, may be beneficial in the treatment of patients with COPD.     

The improvement of calvarial bone healing by durable nanogel-crosslinked materials

Abstract

Different approaches have been developed to improve the scaffold properties that provide structural support and biological interaction to achieve the desired environment for tissue regeneration. We previously reported that addition of human fibroblast growth factor 18 (hFGF18) to acryloyl group-modified cholesterol-bearing pullulan (CHPOA) nanogel-crosslinked (NanoClik) hydrogels that contain human bone morphogenetic protein 2 (hBMP2) stabilized bone healing in mouse calvarial defect model. In this study, we evaluated the use of disc-shaped dried nanogel-crosslinked gel as carriers of growth factors in order to seek possible clinical application in future. Both conventionally-dried NanoClik disc and nanogel-crosslinked porous (NanoCliP) disc made by freeze-drying that contained the growth factors induced bone healing but not as much as with NanoClik hydrogel application but addition of RGD peptides (RGD-NanoCliP disc) improved the healing. All type of discs showed the same biphasic ovalbumin-Alexa Fluor 488 protein release profile in vitro, an initial burst followed by a gradual sustained release more than one week, which was confirmed in vivo. Histological analysis showed remarkable new bone formation with more calcification in RGD-NanoCliP disc with the growth factors and the osteogenesis appeared to begin in the dura mater in contact with the disc. These observations suggest: (1) the fitness of the durable discs to the bone defect is a critical factor for bone healing, which is supplemented by addition of RGD peptides, (2) the porosity is suitable for osteoblast recruitment, (3) growth factor release pattern of the CHPOA nanogel based gels is ideal for bone healing.     

Changes of NK Cells in Preeclampsia

The regulation of uterine and circulating peripheral blood natural killer (NK) cells has been associated with reproductive immunology such as recurrent pregnancy losses, implantation failures, or preeclampsia. Preeclampsia is a hypertensive disorder of pregnancy characterized by increased blood pressure accompanied by proteinuria and is a major cause of maternal and fetal mortality. Natural cytotoxicity receptors (NCRs) are unique markers, which regulate NK cell cytotoxicity and cytokine production. The relation of NCRs to reproduction is not fully characterized yet. The different profile of NCRs expression may suggest presence of abnormal regulation of NK cell in women with reproductive failures. Pregnant women with preeclampsia carry immunological abnormalities of NCRs on peripheral blood NK cells during pregnancy. The lower expression of NKp46⁺ NK cells in women with preeclampsia may account for the higher production of NK1 cytokine that is known as NK1 shift in pregnant women with preeclampsia. Evaluation of NKp46 on peripheral blood NK cells may be applicable to find the onset of preeclampsia. In this review, various expressions of NK cell surface markers including NCRs on NK cells, NK cell cytotoxicity, and production of cytokines and angiogenic factors by NK cells were reviewed in relation to preeclampsia.     

Negative regulation of cell motile and invasive activities by lysophosphatidic acid receptor-3 in colon cancer HCT116 cells

Lysophosphatidic acid (LPA) mediates a wide range of biological responses with G protein-coupled transmembrane receptors (LPA receptors). So far, at least six types of LPA receptors (LPA receptor-1 (LPA₁) to LPA₆) have been identified. Recently, it has been reported that LPA₃ indicates opposite effects on cellular functions of cancer cells. In the present study, to assess a biological role of LPA₃ on cell migration ability of colon cancer cells, we generated LPA receptor-3 (LPAR3) knockdown (HCT-sh3-3) cells from HCT116 and measured cell motile and invasion activities. In motility assay with a cell culture insert, HCT-sh3-3 cells showed significantly high cell motile activity, compared with control cells. For invasion assay, the filter was coated with Matrigel. The invasive activity of HCT-sh3-3 cells was significantly higher than that of control cells. Furthermore, we also examined the effects of LPAR3 knockdown on the interaction between colon cancer cells and endothelial F-2 cells. When F-2 cells were cultured with serum-free DMEM containing a supernatant from HCT-sh3-3 cells, the cell growth rate and migration activity of F-2 cells were significantly stimulated, associating with the elevated expressions of vascular endothelial growth factor (VEGF)-A and VEGF-C genes in HCT-sh3-3 cells. These results suggest that LPA₃ may act as a negative regulator on cell motile and invasive abilities of colon cancer HCT116 cells.