Mechanical and Thermal ON-OFF Switching of the Vapochromic Behavior of a Luminescent Polymorphic Pt(II) Complex.

Vapochromic materials that exhibit color/luminescence changes induced by vapor exposure have attracted considerable attention. Herein, we report the grinding- and heating-induced ON-OFF switching of the vapochromic behavior of [Pt(ppyCl2)(Clacac)] (1; ppyCl2 = 2-(3-chlorophenyl)-4-chloropyridinato, Clacac = 3-chloroacetylacetonato). 1 formed yellow and orange polymorphs (1-Y and 1-O), and 1-Y could be converted to 1-Og, which showed a very similar crystal structure but with a broadened X-ray diffraction pattern compared with that of 1-O. Moreover, 1-Og can be reversibly transformed into 1-O via heating and grinding. Notably, 1-Og underwent a N,N-dimethylacetamide vapor-induced transformation to 1-Y, whereas 1-O did not undergo such a transformation. These results indicate the ON-OFF switching of vapochromic behavior induced via grinding and heating. This finding will be beneficial for developing intelligent molecular devices.

Effect of an Oral Nutrition Supplement Containing Collagen Peptides on Stratum Corneum Hydration and Skin Elasticity in Hospitalized Older Adults: A Multicenter Open-label Randomized Controlled Study.

OBJECTIVE: The purpose of this randomized open-label study was to investigate the effect of an oral nutrition supplement containing collagen peptides on stratum corneum hydration and skin elasticity. METHODS: The study protocol was registered at the UMIN Clinical Trials Registry (UMIN 000027347). Once-a-day oral administration of a nutrition supplement containing collagen peptides (10.0 g) was instituted in 39 inpatients 65 years or older who were assigned to either the intervention or the control group using a block-randomization design. Stratum corneum hydration and skin elasticity were measured at baseline and at 2, 4, 6, and 8 weeks after the start of the intervention. RESULTS: Mean stratum corneum hydration was significantly increased from 43.7 at baseline to 51.7 at postintervention week 8 in the intervention group (P = .001). Differences in skin elasticity from baseline were significant at postintervention week 6 (P = .026) and week 8 (P = .049). CONCLUSIONS: Oral nutrition supplements containing collagen peptides may reduce skin vulnerability in older adults and thus prevent conditions such as skin tears.

Interleukin-10 expression mediated by an adeno-associated virus vector prevents monocrotaline-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is a fatal disease associated with inflammation and pathological remodeling of the pulmonary artery (PA). Interleukin (IL)-10 is a pleiotropic antiinflammatory cytokine with vasculoprotective properties. Here, we report the preventive effects of IL-10 on monocrotaline-induced PAH. Three-week-old Wistar rats were intramuscularly injected with an adeno-associated virus serotype 1 vector expressing IL-10, followed by monocrotaline injection at 7 weeks old. IL-10 transduction significantly improved survival rates of the PAH rats 8 weeks after monocrotaline administration compared with control gene transduction (75% versus 0%, P<0.01). IL-10 also significantly reduced mean PA pressure (22.8+/-1.5 versus 29.7+/-2.8 mm Hg, P<0.05), a weight ratio of right ventricle to left ventricle plus septum (0.35+/-0.04 versus 0.42+/-0.05, P<0.05), and percent medial thickness of the PA (12.9+/-0.3% versus 21.4+/-0.4%, P<0.01) compared with controls. IL-10 significantly reduced macrophage infiltration and vascular cell proliferation in the remodeled PA in vivo. It also significantly decreased the lung levels of transforming growth factor-beta1 and IL-6, which are indicative of PA remodeling. In addition, IL-10 increased the lung level of heme oxygenase-1, which strongly prevents PA remodeling. In vitro analysis revealed that IL-10 significantly inhibited excessive proliferation of cultured human PA smooth muscle cells treated with transforming growth factor-beta1 or the heme oxygenase inhibitor tin protoporphyrin IX. Thus, IL-10 prevented the development of monocrotaline-induced PAH, and these results provide new insights into the molecular mechanisms of human PAH.

Protection against aminoglycoside-induced ototoxicity by regulated AAV vector-mediated GDNF gene transfer into the cochlea.

Since standard aminoglycoside treatment progressively causes hearing disturbance with hair cell degeneration, systemic use of the drugs is limited. Adeno-associated virus (AAV)-based vectors have been of great interest because they mediate stable transgene expression in a variety of postmitotic cells with minimal toxicity. In this study, we investigated the effects of regulated AAV1-mediated glial cell line-derived neurotrophic factor (GDNF) expression in the cochlea on aminoglycoside-induced damage. AAV1-based vectors encoding GDNF or vectors encoding GDNF with an rtTA2s-S2 Tet-on regulation system were directly microinjected into the rat cochleae through the round window at 5 x 10(10) genome copies/body. Seven days after the virus injection, a dose of 333 mg/kg of kanamycin was subcutaneously given twice daily for 12 consecutive days. GDNF expression in the cochlea was confirmed and successfully modulated by the Tet-on system. Monitoring of the auditory brain stem response revealed an improvement of cochlear function after GDNF transduction over the frequencies tested. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression. Our results suggest that AAV1-mediated expression of GDNF using a regulated expression system in the cochlea is a promising strategy to protect the cochlea from aminoglycoside-induced damage.

Protection Against Aminoglycoside-induced Ototoxicity by Regulated AAV Vector-mediated GDNF Gene Transfer Into the Cochlea.

Since standard aminoglycoside treatment progressively causes hearing disturbance with hair cell degeneration, systemic use of the drugs is limited. Adeno-associated virus (AAV)-based vectors have been of great interest because they mediate stable transgene expression in a variety of postmitotic cells with minimal toxicity. In this study, we investigated the effects of regulated AAV1-mediated glial cell line-derived neurotrophic factor (GDNF) expression in the cochlea on aminoglycoside-induced damage. AAV1-based vectors encoding GDNF or vectors encoding GDNF with an rtTA2s-S2 Tet-on regulation system were directly microinjected into the rat cochleae through the round window at 5 × 1010 genome copies/body. Seven days after the virus injection, a dose of 333 mg/kg of kanamycin was subcutaneously given twice daily for 12 consecutive days. GDNF expression in the cochlea was confirmed and successfully modulated by the Tet-on system. Monitoring of the auditory brain stem response revealed an improvement of cochlear function after GDNF transduction over the frequencies tested. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression. Our results suggest that AAV1-mediated expression of GDNF using a regulated expression system in the cochlea is a promising strategy to protect the cochlea from aminoglycoside-induced damage.

Adeno-associated virus vector-mediated systemic interleukin-10 expression ameliorates hypertensive organ damage in Dahl salt-sensitive rats.

BACKGROUND: Inflammation plays an important role in the pathogenesis of hypertension and hypertensive organ damage. Interleukin (IL)-10, a pleiotropic anti-inflammatory cytokine, exerts vasculoprotective effects in many animal models. In the present study, we examined the preventive effects of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against hypertensive heart disease and renal dysfunction in Dahl salt-sensitive rats. METHODS: We injected the rats intramuscularly with an AAV type 1-based vector encoding rat IL-10 or enhanced green fluorescent protein (EGFP) at 5 weeks of age; subsequently, the rats were fed a high-sodium diet from 6 weeks of age. RESULTS: Sustained IL-10 expression significantly improved survival rate of Dahl salt-sensitive rats compared with EGFP expression (62.5% versus 0%, p < 0.001); it also caused 26.0% reduction in systolic blood pressure at 15 weeks (p < 0.0001). Echocardiography exhibited a 22.0% reduction in hypertrophy (p < 0.0001) and a 26.3% improvement in fractional shortening (p < 0.0001) of the rat left ventricle in the IL-10 group compared to the EGFP group. IL-10 expression also caused a 21.7% decrease in the heart weight/body weight index and cardiac atrial natriuretic peptide levels. Histopathological studies revealed that IL-10 decreased inflammatory cell infiltration, fibrosis, and transforming growth factor-beta(1) levels in the failing heart. Furthermore, IL-10 expression significantly reduced urine protein excretion with increased glomerular filtration rates. CONCLUSIONS: This is the first study to demonstrate that the anti-inflammatory cytokine IL-10 has a significant anti-hypertensive effect. AAV vector-mediated IL-10 expression potentially prevents the progression of refractory hypertension and hypertensive organ damage in humans.

Promoter effects of adeno-associated viral vector for transgene expression in the cochlea in vivo.

The aims of this study were to evaluate the expression of enhanced green fluorescent protein (EGFP) driven by 6 different promoters, including cytomegalovirus IE enhancer and chicken beta-actin promoter (CAG), cytomegalovirus promoter (CMV), neuron-specific enolase promoter (NSE), myosin 7A promoter (Myo), elongation factor 1alpha promoter (EF-1alpha), and Rous sarcoma virus promoter (RSV), and assess the dose response of CAG promoter to transgene expression in the cochlea. Serotype 1 adeno-associated virus (AAV1) vectors with various constructs were transduced into the cochleae, and the level of EGFP expression was examined. We found the highest EGFP expression in the inner hair cells and other cochlear cells when CAG promoter was used. The CMV and NSE promoter drove the higher EGFP expression, but only a marginal activity was observed in EF-1alpha promoter driven constructs. RSV promoter failed to driven the EGFP expression. Myo promoter driven EGFP was exclusively expressed in the inner hair cells of the cochlea. When driven by CAG promoter, reporter gene expression was detected in inner hair cells at a dose as low as 3x10(7) genome copies, and continued to increase in a dose-dependent manner. Our data showed that individual promoter has different ability to drive reporter gene expression in the cochlear cells. Our results might provide important information with regard to the role of promoters in regulating transgene expression and for the proper design of vectors for gene expression and gene therapy.

A case of reversible encephalopathy accompanied by demyelination occurring after ingestion of Sugihiratake mushrooms.

Recently, an outbreak of acute encephalopathy associated with Sugihiratake mushroom ingestion has been reported in northern Japan. Patients with chronic kidney diseases are thought to be at risk for severe encephalopathy following Sugihiratake mushroom ingestion. We report a case of encephalopathy associated with Sugihiratake mushroom ingestion in a patient with diabetic nephropathy. Brain magnetic resonance imaging showed discriminative intensity in the medial temporal lobe, claustrum, and insula cortex bilaterally. Cerebrospinal fluid examination revealed mildly elevated protein and marked elevation of myelin basic protein without pleocytosis. Twenty-five days after admission, these signal-intensity changes had markedly improved, and the patient was discharged without sequelae. Although the exact mechanism of this acute encephalopathy remains undetermined, demyelination is believed to be a possible associated pathological change. In cases of encephalopathy of undetermined cause with distinct magnetic resonance findings, Sugihiratake mushroom intoxication should be considered in areas where ingestion of this mushroom is common.

Cerebral arteriopathy with extracranial artery involvement in a patient with ulcerative colitis.

Arteriopathy of the central nervous system (CNS) complicated with ulcerative colitis is a rare condition, moreover the involvement of extracranial arteries has not been documented. An 18-year-old female complained of a severe pulsatile headache and nausea. She had been diagnosed and treated for ulcerative colitis for four years. Magnetic resonance imaging of the brain showed normal results; however, magnetic resonance angiography (MRA) revealed severe irregularity of the intracerebral arteries. After treatment with prednisolone, the patient fully recovered and the irregularity of the intracerebral arteries was dramatically improved. Vasculitis was strongly suggested as the cause of arteriopathy of the CNS in the present case. Involvement of extracranial arteries such as the carotid artery was also incidentally discovered by duplex ultrasonography and the HLA typing suggested genetic susceptibility to Takayasu's arteritis. Findings from our patient suggest that extracranial arterial involvement should be considered in the case of arteriopathy of the CNS associated with ulcerative colitis.

Large-scale production of recombinant viruses by use of a large culture vessel with active gassing.

Adenovirus and adeno-associated virus (AAV) vectors are increasingly used for gene transduction experiments. However, to produce a sufficient amount of these vectors for in vivo experiments requires large-capacity tissue culture facilities, which may not be practical in limited laboratory space. We describe here a large-scale method to produce adenovirus and AAV vectors with an active gassing system that uses large culture vessels to process labor- and cost-effective infection or transfection in a closed system. Development of this system was based on the infection or transfection of 293 cells on a large scale, using a large culture vessel with a surface area of 6320 cm2. A minipump was connected to the gas inlet of the large vessel, which was placed inside the incubator, so that the incubator atmosphere was circulated through the vessel. When active gassing was employed, the productivity of the adenovirus and AAV vectors significantly increased. This vector production system was achieved by improved CO2 and air exchange and maintenance of pH in the culture medium. Viral production with active gassing is particularly promising, as it can be used with existing incubators and the large culture vessel can readily be converted for use with the active gassing system.

Distinct patterns of gene transfer to gerbil hippocampus with recombinant adeno-associated virus type 2 and 5.

Genetic modification of the gerbil hippocampal neuronal cells in vivo helps us understand the mechanisms of neuronal function under various circumstances such as ischemic insult. In this study, we examined the distinct distribution of the recombinant adeno-associated virus type 2 (rAAV2) and rAAV5 vectors for gene delivery to primary cultured cells and the gerbil hippocampus. Mixed cortical cultures containing both neurons and astrocytes from E17 rat embryos were infected with rAAVs containing the Cytomegalovirus virus (CMV) promoter. rAAV2 was preferably transduced to neurons, whereas rAAV5 was inclined to be transduced to astrocytes in vitro. rAAV2 and rAAV5 vectors, each with the CMV or Rous sarcoma virus (RSV) promoter, were injected into the gerbil hippocampus using a stereotaxic apparatus. Five days after injection, transgene expression was analyzed with X-gal staining. In the gerbil hippocampus, rAAV5 with the CMV promoter achieved a higher overall transgene expression than rAAV2 with the CMV promoter. The transgene expression of rAAV2 with the RSV promoter was found in the pyramidal and granular cells, while the transgene expression of rAAV5 with the RSV promoter was preferentially found in the granular cells. These findings would be valuable in optimizing rAAV-mediated gene transfer to the gerbil hippocampus.

[An outbreak of encephalopathy after eating autumn mushroom (Sugihiratake; Pleurocybella porrigens) in patients with renal failure: a clinical analysis of ten cases in Yamagata, Japan].

In September and October, 2004, an outbreak of encephalopathy of unknown etiology occurred in certain areas of Japan including Yamagata, Akita, and Niigata prefectures. These patients had a history of chronic renal failure, most of them had undergone hemodialysis, and also had a history of eating Sugihiratake (Pleurocybella porrigens), an autumn mushroom without known toxicity. Since clinical details of this type of encephalopathy remain unknown, we analyzed the clinical, radiological and electroencephalographic (EEG) features of ten cases of this encephalopathy in Yamagata prefecture. The summary of the present study is as follows: 1. Ten patients had chronic renal failure, and seven underwent hemodialysis. 2. Each patient had a history of eating Sugihiratake within 2-3 weeks of the onset of neurological symptoms. 3. The onset was subacute; the initial symptoms were tremor, dysarthria, and/or weakness of the extremities, which lasted an average of 4.5 days (ranging from 2 to 11 days), followed by severe consciousness disturbance and intractable seizures, resulting in status epilepticus in 5 patients. Myoclonus was also seen in 4 patients and Babinski reflex in 3. 4. Brain CT and MRI examinations were unremarkable in the early stages of the disease. Three to eight days after onset, however, conspicuous lesions appeared in the areas of the insula and basal ganglia in 6 patients. On MRI, these brain lesions were hyperintense on T2-weighted and FLAIR images, and hypointense on T1-weighted images. 5. EEG examination was performed in 6 patients, all of whom showed abnormal EEG findings. Periodic synchronous discharge (PSD) was seen in 2 patients, spike and wave complex in one patient, and non-specific slow waves in 3. 6. Prognosis was different from case to case. Three patients died at 13, 14, and 29 days after onset. Two patients still showed persistent disturbance of consciousness one month after onset. One patient showed parkinsonism after recovering from consciousness disturbance. Four patients recovered nearly completely around one month after onset In 3 of the 4 recovered patients, renal failure was not severe and they did not need to undergo hemodialysis. This suggests that the degree of renal failure is a key for the prognosis of this type of encephalopathy. The present study suggests that this endemic disease is a newly recognized clinical entity of encephalopathy.

A histone deacetylase inhibitor enhances recombinant adeno-associated virus-mediated gene expression in tumor cells.

The transduction of cancer cells using recombinant adeno-associated virus (rAAV) occurs with low efficiency, which limits its utility in cancer gene therapy. We have previously sought to enhance rAAV-mediated transduction of cancer cells by applying DNA-damaging stresses. In this study, we examined the effects of the histone deacetylase inhibitor FR901228 on tumor transduction mediated by rAAV types 2 and 5. FR901228 treatment significantly improved the expression of the transgene in four cancer cell lines. The cell surface levels of alpha v integrin, FGF-R1, and PDGF-R were modestly enhanced by the presence of FR901228. These results suggest that the superior transduction induced by the HDAC inhibitor was due to an enhancement of transgene expression rather than increased viral entry. Furthermore, we characterized the association of the acetylated histone H3 in the episomal AAV vector genome by using the chromatin immunoprecipitation assay. The results suggest that the superior transduction may be related to the proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. In the analysis with subcutaneous tumor models, strong enhancement of the transgene expression as well as therapeutic effect was confirmed in vivo. The use of this HDAC inhibitor may enhance the utility of rAAV-mediated transduction strategies for cancer gene therapy.

Specific and efficient transduction of Cochlear inner hair cells with recombinant adeno-associated virus type 3 vector.

Recombinant adeno-associated virus (AAV) vectors are of interest for cochlear gene therapy because of their ability to mediate the efficient transfer and long-term stable expression of therapeutic genes in a wide variety of postmitotic tissues with minimal vector-related cytotoxicity. In the present study, seven AAV serotypes (AAV1-5, 7, 8) were used to construct vectors. The expression of EGFP by the chicken beta-actin promoter associated with the cytomegalovirus immediate-early enhancer in cochlear cells showed that each of these serotypes successfully targets distinct cochlear cell types. In contrast to the other serotypes, the AAV3 vector specifically transduced cochlear inner hair cells with high efficiency in vivo, while the AAV1, 2, 5, 7, and 8 vectors also transduced these and other cell types, including spiral ganglion and spiral ligament cells. There was no loss of cochlear function with respect to evoked auditory brain-stem responses over the range of frequencies tested after the injection of AAV vectors. These findings are of value for further molecular studies of cochlear inner hair cells and for gene replacement strategies to correct recessive genetic hearing loss due to monogenic mutations in these cells.

In situ generation of pseudotyped retroviral progeny by adenovirus-mediated transduction of tumor cells enhances the killing effect of HSV-tk suicide gene therapy in vitro and in vivo.

BACKGROUND: Hybrid adeno-retroviral vector systems utilize the high efficiency of adenovirus transduction to direct the in situ production of retroviral progeny. In this study, we show that a single-step transduction of glioma cells with trans-complementing hybrid adeno-retroviral vectors effectively turns these cells into retrovirus vector-producing cells, which in turn facilitates the transduction of adjacent cells. METHODS: We have adapted the adeno-retroviral hybrid viral vector system to enhance the ganciclovir (GCV) killing of glioma cells following transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene. To assess the effect of the in situ production of retroviral vectors on the transduction efficiency of glioma cells, 9L cells were transduced with adeno-retroviral hybrid vectors that separately express a retroviral genome (AVC2.GCEGFP or AVC2.GCTK) and retroviral packaging proteins (AxTetGP and AxTetVSVG). The generation of an integrated HSV-tk provirus by trans-complementation of the adeno-retroviral vectors was verified by analysis of the flanking retroviral LTR sequences. Tumors established on nu/nu mice were injected with the viruses followed by intraperitoneal injections of either PBS or GCV. We also estimated the copy numbers of the HSV-tk transgene present in the tumors of the treated mice. To determine the expression pattern of the HSV-tk transcripts within a tumor, in situ hybridization analysis was performed using an RNA probe specific for HSV-tk. RESULTS: The co-transduction of rat 9L glioma cells with AVC2.GCEGFP together with vectors expressing packaging proteins of retroviruses increased the transduction efficiency. Transduction with AVC2.GCTK together with packaging vectors increased the in vitro sensitivity of cells to the pro-drug GCV by one log compared with control cells that were incapable of generating retrovirus. In vivo, the injection of established subcutaneous 9L tumors on athymic mice with a combination of AVC2.GCTK and packaging vectors followed by GCV treatment resulted in complete tumor regression in 50% of tumors at day 22, while no tumor regression was observed in control animals. Retroviral sequences diagnostic of 3' LTR reduplication in vivo were detected in genomic DNA extracted from the transduced tumors, indicating pro-viral integration of the retroviral genome derived from the adeno-retroviral hybrid vector. Furthermore, the relative copy number of the HSV-tk gene in tumors treated with the adeno-retroviral vectors was up to approximately 250-fold higher than in control tumors. In situ hybridization suggested dispersion of the HSV-tk product across a wider area of the tumor than in control tumors, which indicates the spread of the in situ generated retroviruses. CONCLUSIONS: Although the efficacy of this system has to be evaluated in orthotopic models, our observations suggest that this hybrid adeno-retroviral vector system could improve the suicide gene therapy of tumors.

Adeno-associated virus vectors for gene transfer to the brain.

Gene therapy is a novel method under investigation for the treatment of neurological disorders. Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system. Adeno-associated virus (AAV) is a potentially useful gene transfer vehicle for neurologic gene therapies. The advantages of AAV vector include the lack of any associated disease with a wild-type virus, the ability to transduce nondividing cells, the possible integration of the gene into the host genome, and the long-term expression of transgenes. The development of novel therapeutic strategies for neurological disorder by using AAV vector has an increasing impact on gene therapy research. This article describes methods that can be used to generate rodent and nonhuman primate models for testing treatment strategies linked to pathophysiological events in the ischemic brain and neurodegenerative disorders such as Parkinson's disease.