Publications by authors named "Yun-Hsiang Chen"

24 Publications

  • Page 1 of 1

Downregulation of α-Synuclein Protein Levels by an Intracellular Single-Chain Antibody.

J Parkinsons Dis 2020 ;10(2):573-590

Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan, Taiwan.

Background: Accumulation of α-synuclein (αSyn) in the dopaminergic neurons is a common pathology seen in patients with Parkinson's disease (PD). Overproduction of αSyn potentiates the formation of oligomeric αSyn aggregates and enhances dopaminergic neuron degeneration. Downregulating intracellular monomeric αSyn prevents the formation of αSyn oligomers and is a potential therapeutic strategy to attenuate the progression of PD.

Objective: The purpose of this study is to investigate the efficacy of gene delivery of αSyn-specific single-chain antibodies in vitro and in vivo.

Methods And Results: The plasmids for αSyn and selective antibodies (NAC32, D10, and VH14) were constructed and were transfected to HEK293 and SH-SY5Y cells. Co-expression of αSyn with NAC32, but not D10 or VH14, profoundly downregulated αSyn protein, but not αSyn mRNA levels in these cells. The interaction of αSyn and NAC32 antibody was next examined in vivo. Adeno-associated virus (AAV)-αSyn combined with AAV-NAC32 or AAV-sc6H4 (a negative control virus) were stereotactically injected into the substantia nigra of adult rats. AAV-NAC32 significantly reduced AAV-encoded αSyn levels in the substantia nigra and striatum and increased tyrosine hydroxylase immunoreactivity in the striatum. Also, in the animals injected with AAV-NAC32 alone, endogenous αSyn protein levels were significantly downregulated in the substantia nigra.

Conclusion: Our data suggest that AAV-mediated gene transfer of NAC32 is a feasible approach for reducing the expression of target αSyn protein in brain.
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http://dx.doi.org/10.3233/JPD-191787DOI Listing
January 2020

Human Milk Oligosaccharide 2'-Fucosyllactose Reduces Neurodegeneration in Stroke Brain.

Transl Stroke Res 2020 10 2;11(5):1001-1011. Epub 2020 Jan 2.

Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan, Miaoli, Taiwan.

2'-Fucosyllactose (2'-FL) is a major oligosaccharide in human milk and is present at trace levels in cow milk. 2'-FL reduces inflammation in the gastrointestinal tract. Its action in the central nervous system has not been well characterized. The purpose of this study is to determine 2'-FL-mediated neural protection and repair in culture and stroke brain. In rat primary cortical neuronal cultures, 2'-FL significantly antagonized N-methyl-D-aspartate (NMDA) or glutamate-mediated changes in ATP production, MAP2 immunoreactivity, and TUNEL. The influx of Ca (Cai) was examined in primary cortical neurons expressing GCaMP5, an endogenous calcium probe. NMDA increased Cai; 2'-FL significantly attenuated this reaction. In a rat middle cerebral artery occlusion model of stroke, we found that intracerebroventricular pretreatment or oral posttreatment with 2'-FL significantly reduced brain infarction, mitigated microglial activation, improved locomotor activity, and upregulated brain-derived neurotrophic factor (BDNF) expression. Post-stroke delivery of 2'-FL increased bromodeoxyuridine (BrdU) labeling in the perilesioned area. These BrdU cells co-expressed NeuN, or nestin, or GFAP. Using subventricular Matrigel cultures, we demonstrated that 2'-FL increased cell migration from subventricular zone explant. This response was reduced by anti-BDNF blocking antibody. In conclusion, our data suggest that 2'-FL has neuroprotective action through inhibition of Cai, inflammation, and apoptosis. Posttreatment with 2'-FL facilitates neural repair in stroke brain.
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http://dx.doi.org/10.1007/s12975-019-00774-zDOI Listing
October 2020

Neuronal Activation Stimulates Cytomegalovirus Promoter-Driven Transgene Expression.

Mol Ther Methods Clin Dev 2019 Sep 3;14:180-188. Epub 2019 Jul 3.

Molecular Mechanisms of Cellular Stress and Inflammation Section, National Institute on Drug Abuse, NIH, Baltimore, MD 21224, USA.

The cytomegalovirus (CMV) immediate early promoter has been extensively developed and exploited for transgene expression and , including human clinical trials. The CMV promoter has long been considered a stable, constitutive, and ubiquitous promoter for transgene expression. Using two different CMV-based promoters, we found an increase in CMV-driven transgene expression in the rodent brain and in primary neuronal cultures in response to methamphetamine, glutamate, kainic acid, and activation of G protein-coupled receptor signaling using designer receptors exclusively activated by designer drugs (DREADDs). In contrast, promoters derived from human synapsin 1 (hSYN1) gene or elongation factor 1α (EF1α) did not exhibit altered transgene expression in response to the same neuronal stimulations. Overall, our results suggest that the long-standing assertion that the CMV promoter confers constitutive expression in neurons should be reevaluated, and future studies should empirically determine the activity of the CMV promoter in a given application.
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http://dx.doi.org/10.1016/j.omtm.2019.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6661544PMC
September 2019

Pifithrin-Alpha Reduces Methamphetamine Neurotoxicity in Cultured Dopaminergic Neurons.

Neurotox Res 2019 Aug 8;36(2):347-356. Epub 2019 May 8.

Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan, Taiwan.

Methamphetamine (Meth) is a widely abused stimulant. High-dose Meth induces degeneration of dopaminergic neurons through p53-mediated apoptosis. A recent study indicated that treatment with the p53 inhibitor, pifithrin-alpha (PFT-α), antagonized Meth-mediated behavioral deficits in mice. The mechanisms underpinning the protective action of PFT-α against Meth have not been identified, and hence, their investigation is the focus of this study. Primary dopaminergic neuronal cultures were prepared from rat embryonic ventral mesencephalic tissue. High-dose Meth challenge reduced tyrosine hydroxylase immunoreactivity and increased terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling (TUNEL) labeling. PFT-α significantly antagonized these responses. PFT-α also reduced Meth-activated translocation of p53 to the nucleus, an initial step before transcription. Previous studies have indicated that p53 can also activate cell death through transcription-independent pathways. We found that PFT-α attenuated endoplasmic reticulum (ER) stressor thapsigargin (Tg)-mediated loss of dopaminergic neurons. ER stress was further monitored through the release of Gaussia luciferase (GLuc) from SH-SY5Y cells overexpressing GLuc-based Secreted ER Calcium-Modulated Protein (GLuc-SERCaMP). Meth or Tg significantly increased GLuc release in to the media, with PFT-α significantly reducing GLuc release. Additionally, PFT-α significantly attenuated Meth-induced CHOP expression. In conclusion, our data indicate that PFT-α is neuroprotective against Meth-mediated neurodegeneration via transcription-dependent nuclear and -independent cytosolic ER stress pathways.
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http://dx.doi.org/10.1007/s12640-019-00050-wDOI Listing
August 2019

Recombinant Adeno-Associated Virus-Mediated Expression of Methamphetamine Antibody Attenuates Methamphetamine-Induced Hyperactivity in Mice.

Sci Rep 2017 04 7;7:46301. Epub 2017 Apr 7.

Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan, Taiwan.

Methamphetamine (Meth) is one of the most frequently abused drugs worldwide. Recent studies have indicated that antibodies with high affinity for Meth reduce its pharmacological effects. The purpose of this study was to develop a technique for virus-based passive immunization against Meth effects. We generated a recombinant adeno-associated virus serotype-8 vector (AAV-MethAb) carrying the gene for a Meth-specific monoclonal antibody (MethAb). Infection of 293 cells with AAV-MethAb resulted in the expression and secretion of antibodies which bind to Meth. The viral vector was then examined in adult ICR mice. Systemic administration of AAV-MethAb resulted in long-term expression of MethAb in the serum for up to 29 weeks. Serum collected from the animals receiving AAV-MethAb retained a high specificity for (+)-Meth. Animals were challenged with Meth five weeks after viral injection. Meth levels in the brain and serum were reduced while Meth-induced locomotor activity was significantly attenuated. In conclusion, AAV-MethAb administration effectively depletes Meth from brain and serum while reducing the behavioral response to Meth, and thus is a potential therapeutic approach for Meth abuse.
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http://dx.doi.org/10.1038/srep46301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384190PMC
April 2017

Methadone enhances human influenza A virus replication.

Addict Biol 2017 Jan 8;22(1):257-271. Epub 2015 Sep 8.

Center for Neuropsychiatric Research, National Health Research Institutes, Taiwan.

Growing evidence has indicated that opioids enhance replication of human immunodeficiency virus and hepatitis C virus in target cells. However, it is unknown whether opioids can enhance replication of other clinically important viral pathogens. In this study, the interaction of opioid agonists and human influenza A/WSN/33 (H1N1) virus was examined in human lung epithelial A549 cells. Cells were exposed to morphine, methadone or buprenorphine followed by human H1N1 viral infection. Exposure to methadone differentially enhanced viral propagation, consistent with an increase in virus adsorption, susceptibility to virus infection and viral protein synthesis. In contrast, morphine or buprenorphine did not alter H1N1 replication. Because A549 cells do not express opioid receptors, methadone-enhanced H1N1 replication in human lung cells may not be mediated through these receptors. The interaction of methadone and H1N1 virus was also examined in adult mice. Treatment with methadone significantly increased H1N1 viral replication in lungs. Our data suggest that use of methadone facilitates influenza A viral infection in lungs and might raise concerns regarding the possible consequence of an increased risk of serious influenza A virus infection in people who receive treatment in methadone maintenance programs.
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http://dx.doi.org/10.1111/adb.12305DOI Listing
January 2017

Signaling adaptor protein SH2B1 enhances neurite outgrowth and accelerates the maturation of human induced neurons.

Stem Cells Transl Med 2014 Jun 15;3(6):713-22. Epub 2014 Apr 15.

Division of Regenerative Medicine, Institute of Cellular and System Medicine, and Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China; Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, Institute of Molecular Medicine, and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China; Department of Psychiatry, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China; Department of Pharmacology, Tzu Chi University, Hualien, Taiwan, Republic of China; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China

Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1β (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling.
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http://dx.doi.org/10.5966/sctm.2013-0111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4039450PMC
June 2014

Treatment of methamphetamine abuse: an antibody-based immunotherapy approach.

J Food Drug Anal 2013 Dec;21(4):S82-S86

Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan, Taiwan ; Department of Psychiatry, Chang Gung Memorial Hospital at Linkou and Chang Gung University School of Medicine, Taoyuan, Taiwan.

Methamphetamine is a highly addictive psychostimulant with tens of millions of abusers around the world, and currently there is no effective or approved medication for addiction to it. Monoclonal antibodies with a high affinity for methamphetamine have the potential to sequester the drug in the vascular compartment and reduce entry into the brain, acting as peripheral pharmacokinetic antagonists without inducing adverse effects on neurons. However, in order to maintain the antibodies at an effective level, repeated administration is required, which would be expensive and problematic for patient compliance. In this study, we intended to investigate whether using a recombinant adeno-associated virus-mediated gene transfer technique can be an effective approach to achieve long-term expression of anti-methamphetamine monoclonal antibodies in mouse models. We generated a recombinant adeno-associated virus vector encoding the heavy and light chains of an anti-methamphetamine monoclonal antibody, which were constructed in a single open reading frame and linked with a 2A self-processing sequence. In the context of virus-mediated gene transfer, expression of full-length and functional monoclonal antibodies was successfully demonstrated and . Further investigations on dose optimization, long-term expression, and protection from methamphetamine challenge in mouse models are ongoing.
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http://dx.doi.org/10.1016/j.jfda.2013.09.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072129PMC
December 2013

Increased plasma level of tumor necrosis factor α in patients with narcolepsy in Taiwan.

Sleep Med 2013 Dec 20;14(12):1272-6. Epub 2013 Sep 20.

Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan, Taiwan.

Background: Narcolepsy is a neuropsychiatric disorder characterized by excessive daytime sleepiness, cataplexy, hypnagogic hallucinations, and abnormal rapid eye movement (REM) sleep. Tumor necrosis factor α (TNF α) and its cognate receptors have been reported to be involved in the pathophysiology of narcolepsy in addition to the HLA antigen system. Our study aimed to determine if the TNF-α system was associated with narcolepsy in our patients.

Methods: We first measured the plasma level of TNF α in 56 narcoleptic patients and 53 control subjects using a highly sensitive enzyme-linked immunosorbent assay. We next determined the genotype of three single nucleotide polymorphisms (SNPs) (T-1031C, C-863A, and C-857T) at the promoter region of the TNF-α gene and one missense SNP (T587G, M196R) at the exon 6 of the tumor necrosis factor receptor 2 gene, TNFR2, in a sample of 75 narcoleptic patients and 201 control subjects by direct sequencing analysis.

Results: We found a significant elevation of plasma level of TNF α in patients with narcolepsy compared with the control subjects (4.64pg/mL vs 1.06pg/mL; P=.0013). However, we did not find significant differences between these two groups in the allelic and genotypic distributions of the investigated polymorphisms.

Conclusions: Our study suggests that an increased TNF-α level was associated with narcolepsy in our patients, and that chronic inflammation due to various factors might have led to the increased TNF-α levels found in our patients.
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http://dx.doi.org/10.1016/j.sleep.2013.04.030DOI Listing
December 2013

Association analysis of the major histocompatibility complex, class II, DQ β1 gene, HLA-DQB1, with narcolepsy in Han Chinese patients from Taiwan.

Sleep Med 2013 Dec 29;14(12):1393-7. Epub 2013 Sep 29.

Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan, Taiwan.

Background: Narcolepsy is a rare, chronic, disabling neuropsychiatric disorder characterized by excessive daytime sleepiness, cataplexy, hypnagogic hallucinations, sleep paralysis, and abnormal rapid eye movement sleep. It is strongly associated with the HLA-DQB1(∗)06:02 allele in various ethnic groups. Our study aimed to investigate the allelic spectrum of HLA-DQB1 in a sample of Han Chinese patients with narcolepsy and control subjects from Taiwan.

Methods: We determined the genotype of the major histocompatibility complex, class II, DQ β1 gene, HLA-DQB1, in 72 narcolepsy subjects (44 men, 28 women), including 52 narcolepsy subjects with cataplexy (narcolepsy+cataplexy), 20 narcolepsy subjects without cataplexy (narcolepsy-cataplexy), and 194 control subjects (94 men, 100 women) using a sequence-specific oligonucleotide-probe hybridization technique.

Results: We found a strong HLA-DQB1(∗)06:02 association in narcolepsy+cataplexy subjects (odds ratio [OR], 321.4 [95% confidence interval {CI}, 70.7-1461.4]). The association was less prominent in narcolepsy-cataplexy subjects (OR, 6.9 [95% CI, 2.4-20.1]). In addition to the DQB1(∗)06:02, we found that (∗)03:01 also was a predisposing allele (OR, 2.0 [95% CI, 1.1-3.7]) in narcolepsy+cataplexy subjects, though the (∗)06:01 was a predisposing allele (OR, 2.8 [95% CI, 1.2-6.7]) in narcolepsy-cataplexy subjects. Furthermore, we found a significant overrepresentation of DQB1(∗)06:02 homozygotes in narcolepsy+cataplexy subjects.

Conclusions: Our data add further support to the strong association of the HLA-DQB1(∗)06:02 allele with narcolepsy, especially in narcolepsy+cataplexy patients. Our study also indicates additional HLA-DQB1 alleles may modify the presentation of narcolepsy+cataplexy patients, such as DQB1(∗)03:01 and DQB1(∗)06:01 in our study. Our results are limited by the small sample size and can only be considered as preliminary findings.
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http://dx.doi.org/10.1016/j.sleep.2013.06.017DOI Listing
December 2013

The development of antibody-based immunotherapy for methamphetamine abuse: immunization, and virus-mediated gene transfer approaches.

Curr Gene Ther 2013 Feb;13(1):39-50

Division of Mental Health and Addiction Medicine, Institute of Population Health Sciences, Zhunan, Miaoli County, Taiwan.

Methamphetamine is a highly addictive psychostimulant that has been seriously abused worldwide, and currently there are no approved medications for the treatment of its abuse. Conventional treatments for drug addiction mainly seek to use small molecule agonists or antagonists to target the drug receptors in the brain, but unfortunately it is difficult to find a similar small molecule for the treatment of methamphetamine dependence. Alternatively, anti-methamphetamine antibodies can sequester the drug in the bloodstream and reduce the amount of drug available to the central nervous system, acting as peripheral pharmacokinetic antagonists. This review describes the development of antibody-based immunotherapies, classified into active and passive immunizations, for the treatment of methamphetamine addiction. Furthermore, an alternative therapeutic approach, using a recombinant adeno-associated virus-mediated gene transfer technique to achieve in vivo expression of characterized anti-methamphetamine monoclonal antibodies, is proposed in this article.
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http://dx.doi.org/10.2174/156652313804806552DOI Listing
February 2013

Methamphetamine reduces human influenza A virus replication.

PLoS One 2012 6;7(11):e48335. Epub 2012 Nov 6.

Division of Mental Health and Addiction Medicine, Institute of Population Health Sciences, National Health Research Institutes, Zhunan, Taiwan.

Methamphetamine (meth) is a highly addictive psychostimulant that is among the most widely abused illicit drugs, with an estimated over 35 million users in the world. Several lines of evidence suggest that chronic meth abuse is a major factor for increased risk of infections with human immunodeficiency virus and possibly other pathogens, due to its immunosuppressive property. Influenza A virus infections frequently cause epidemics and pandemics of respiratory diseases among human populations. However, little is known about whether meth has the ability to enhance influenza A virus replication, thus increasing severity of influenza illness in meth abusers. Herein, we investigated the effects of meth on influenza A virus replication in human lung epithelial A549 cells. The cells were exposed to meth and infected with human influenza A/WSN/33 (H1N1) virus. The viral progenies were titrated by plaque assays, and the expression of viral proteins and cellular proteins involved in interferon responses was examined by Western blotting and immunofluorescence staining. We report the first evidence that meth significantly reduces, rather than increases, virus propagation and the susceptibility to influenza infection in the human lung epithelial cell line, consistent with a decrease in viral protein synthesis. These effects were apparently not caused by meth's effects on enhancing virus-induced interferon responses in the host cells, reducing viral biological activities, or reducing cell viability. Our results suggest that meth might not be a great risk factor for influenza A virus infection among meth abusers. Although the underlying mechanism responsible for the action of meth on attenuating virus replication requires further investigation, these findings prompt the study to examine whether other structurally similar compounds could be used as anti-influenza agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048335PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3491060PMC
April 2013

Genetic analysis of AUTS2 as a susceptibility gene of heroin dependence.

Drug Alcohol Depend 2013 Mar 17;128(3):238-42. Epub 2012 Sep 17.

Division of Mental Health and Addiction Medicine, Institute of Population Health Sciences, National Health Research Institutes, Zhunan, Taiwan.

Background: Both alcoholism and heroin dependence are common substance use disorders with a high genetic basis. A recent genetic study reported that the autism susceptibility candidate 2 gene (AUTS2) was involved in regulating the alcohol drinking behavior. In our previous total gene expression profiling study, we found that the AUTS2 transcript was significantly down-regulated in lymphoblastoid cell lines (LCL) in heroin dependent individuals compared with control subjects, which prompted us to investigate whether AUTS2 is associated with heroin dependence.

Methods: We compared the AUTS2 transcript level of LCL between 124 heroin dependent males and 116 control males using real-time quantitative PCR, and conducted a genetic association study of the rs6943555 of AUTS2 with heroin dependence using a sample of 546 heroin dependent males and 373 control males.

Results: We first verified that the average transcript level of AUTS2 in the heroin dependent group was significantly lower than that in the control group (p=0.017). In the genetic association analysis, we found that AA homozygotes of rs6943555 were significantly over-represented in the heroin dependent subjects compared with the control subjects (odds ratio=1.7, 95% confidence interval: 1.08-2.74, p=0.017). Analyzing the sample from the AUTS2 transcript experiment, we found that AA carriers (n=19) had significantly lower AUTS2 mRNA levels in their LCL compared to TT carriers (n=97, p=0.002) and AT carriers (n=91, p=0.005).

Conclusions: Our data indicate that the AUTS2 gene might be associated with heroin dependence, and reduced AUTS2 gene expression might confer increased susceptibility to heroin dependence.
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http://dx.doi.org/10.1016/j.drugalcdep.2012.08.029DOI Listing
March 2013

Shikonin induces immunogenic cell death in tumor cells and enhances dendritic cell-based cancer vaccine.

Cancer Immunol Immunother 2012 Nov 19;61(11):1989-2002. Epub 2012 Apr 19.

Department and Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan, ROC.

Immunogenic cell death is characterized by damage-associated molecular patterns, which can enhance the maturation and antigen uptake of dendritic cells. Shikonin, an anti-inflammatory and antitumor phytochemical, was exploited here as an adjuvant for dendritic cell-based cancer vaccines via induction of immunogenic cell death. Shikonin can effectively activate both receptor- and mitochondria-mediated apoptosis and increase the expression of all five tested damage-associated molecular patterns in the resultant tumor cell lysates. The combination treatment with damage-associated molecular patterns and LPS activates dendritic cells to a high maturation status and enhances the priming of Th1/Th17 effector cells. Shikonin-tumor cell lysate-loaded mature dendritic cells exhibit a high level of CD86 and MHC class II and activate Th1 cells. The shikonin-tumor cell lysate-loaded dendritic cell vaccines result in a strong induction of cytotoxic activity of splenocytes against target tumor cells, a retardation in tumor growth, and an increase in the survival of test mice. The much enhanced immunogenicity and efficacy of the current cancer vaccine formulation, that is, the use of shikonin-treated tumor cells as cell lysates for the pulse of dendritic cells in culture, may suggest a new ex vivo approach for developing individualized, dendritic cells-based anticancer vaccines.
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http://dx.doi.org/10.1007/s00262-012-1258-9DOI Listing
November 2012

Shikonin enhances efficacy of a gene-based cancer vaccine via induction of RANTES.

J Biomed Sci 2012 Apr 12;19:42. Epub 2012 Apr 12.

Department and Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan.

Background: Shikonin, a phytochemical purified from Lithospermum erythrorhizon, has been shown to confer diverse pharmacological activities, including accelerating granuloma formation, wound healing, anti-inflammation and others, and is explored for immune-modifier activities for vaccination in this study. Transdermal gene-based vaccine is an attractive approach for delivery of DNA transgenes encoding specific tumor antigens to host skin tissues. Skin dendritic cells (DCs), a potent antigen-presenting cell type, is known to play a critical role in transmitting and orchestrating tumor antigen-specific immunities against cancers. The present study hence employs these various components for experimentation.

Method: The mRNA and protein expression of RANTES were detected by RT-PCR and ELISA, respectively. The regional expression of RANTES and tissue damage in test skin were evaluated via immunohistochemistry assay. Fluorescein isothiocyanate sensitization assay was performed to trace the trafficking of DCs from the skin vaccination site to draining lymph nodes. Adjuvantic effect of shikonin on gene gun-delivered human gp100 (hgp100) DNA cancer vaccine was studied in a human gp100-transfected B16 (B16/hgp100) tumor model.

Results: Among various phytochemicals tested, shikonin induced the highest level of expression of RANTES in normal skin tissues. In comparison, mouse RANTES cDNA gene transfection induced a higher level of mRANTES expression for a longer period, but caused more extensive skin damage. Topical application of shikonin onto the immunization site before gene gun-mediated vaccination augmented the population of skin DCs migrating into the draining lymph nodes. A hgp100 cDNA gene vaccination regimen with shikonin pretreatment as an adjuvant in a B16/hgp100 tumor model increased cytotoxic T lymphocyte activities in splenocytes and lymph node cells on target tumor cells.

Conclusion: Together, our findings suggest that shikonin can effectively enhance anti-tumor potency of a gene-based cancer vaccine via the induction of RANTES expression at the skin immunization site.
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http://dx.doi.org/10.1186/1423-0127-19-42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3353861PMC
April 2012

Neuroimmune pharmacology of neurodegenerative and mental diseases.

J Neuroimmune Pharmacol 2011 Mar 7;6(1):28-40. Epub 2010 Sep 7.

Division of Mental Health and Addiction Medicine, Institute of Population Health Sciences, Zhunan, Miaoli County, Taiwan, Republic of China.

Neuroimmune pharmacology is a newly emerging field that intersects with neuroscience, immunology, and pharmacology and that is seeking avenues for translational research and better understanding of disease mechanisms. It focuses on the immunity of the central nervous system (CNS) which is greatly influenced by endogenous effectors, such as cytokines and neurotransmitters, and by exogenous substances, including therapeutic compounds, infectious pathogens, and drugs of abuse. In this article, we attempt to raise awareness of the pivotal discovery of how those mediators affect the immunity of the CNS in both physiological conditions and processes of certain mental illnesses, including psychiatric disorders, neurodegenerative diseases, and cerebral dysfunctions due to drugs of abuse. The abnormality in cytokine networks, neurotransmitter homeostasis, and other immune responses may be involved in the neuropathology associated with those mental illnesses, and the therapeutic effects of the potential treatments can be attributed, at least partially, to their immunomodulatory activities. However, the resulting inflammatory cytokines from certain treatments frequently cause psychiatric complications. In addition, the poor neuropathological outcomes frequently found among drug abusers with HIV-1 infection appear to be related to the neurotoxic and immunomodulatory effects of the drugs used. Importantly, glial cells, especially microglia and astrocytes, are key players in the immunomodulatory activities in the CNS, and the functioning CNS is largely dependent upon the reciprocal interactions between neurons and glial cells. Therefore, glia-neuron interactions have become a critical issue for further understanding the disease mechanism. From this review, readers will gain insights into the new field of neuroimmune pharmacology, with a focus on the impacts of CNS immunity on the mental illnesses.
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http://dx.doi.org/10.1007/s11481-010-9241-8DOI Listing
March 2011

Enhancement of vascular formation but not improvement of ventricular function of infarcted rat hearts by a high dose of adenovirus-carried VEGF transgene.

Chin J Physiol 2009 Nov;52(5 Suppl):384-94

Department of Internal Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.

The purpose of this study was to examine the influence of adenovirus-carried VEGF165 transgene at 5 x 10(10) pfu (Ad-VEGF) on vascular formation, cardiac geometry and ventricular function in infarcted hearts of the rat and to explore the mechanism of Ad-VEGF-mediated actions on ventricular function by quantitative proteomic analysis. Seven days after coronary occlusion, intramyocardial injection with normal saline (vehicle control), adenovirus-carried beta-galactosidase gene (Ad-LacZ, vector control) or Ad-VEGF to infarcted hearts was conducted. Seven days after intramyocardial injection, ventricular function, cardiac morphology and vascular density were assessed after echocardiographic analysis and immunohistological staining. One dimensional gel electrophoresis coupled with stable isotope dimethyl labeling and LC/MS/MS was used to quantify the abundance ratio of each protein pair in Ad-VEGF- and Ad-LacZ-treated hearts. Our data indicated that both Ad-VEGF and Ad-LacZ increased arteriolar densities. However, the former increased arterial densities but the latter did not. Compared with the vehicle control, Ad-LacZ reversed occlusion-induced wall thinning and functional impairment but Ad-VEGF did not. Quantitative proteomic analysis showed increased ratios of plasma proteins (such as albumin) and oxygen carriers (such as myoglobin) by Ad-VEGF and decreased ratios of proteins involved in glycolysis, calcium homeostasis and lipolysis by Ad-VEGF. Taken together, our functional, morphological and proteomic data suggest that intramuscular delivery of Ad-LacZ at higher doses may improve ventricular function and wall thinning with arteriolar formation. Excessive amounts of VEGF by Ad-VEGF may offset Ad-LacZ-induced improvement in ventricular functions by interfering with calcium homeostasis and lipolysis in infarcted hearts.
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http://dx.doi.org/10.4077/cjp.2009.amk033DOI Listing
November 2009

Improved growth of enteric adenovirus type 40 in a modified cell line that can no longer respond to interferon stimulation.

J Gen Virol 2007 Jan;88(Pt 1):71-76

Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonnington Campus, Loughborough LE12 5RD, UK.

Human enteric adenoviruses propagate poorly in conventional human cell lines used to grow other adenovirus serotypes. As human enteric adenoviruses have a defect in counteracting the cellular interferon (IFN) response in cell culture, to aid in growth of the virus, a 293-based cell line defective in its ability to respond to IFN was constructed. This cell line (293-SV5/V) constitutively expresses V-protein of the paramyxovirus Simian virus 5, which degrades the signal transducer and activator of transcription 1 (STAT1) and thereby prevents the STAT1-mediated IFN response. Analysis of human enteric adenovirus type 40 (HAdV-40)-infected 293-SV5/V cells compared with parental 293 cells shows that the recombinant line allows more rapid production of virus and results in higher titres. These results suggest that the defect in HAdV-40 in counteracting the IFN response can be overcome at least partially through the use of 293-SV5/V cell lines.
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http://dx.doi.org/10.1099/vir.0.82445-0DOI Listing
January 2007

The NPro product of bovine viral diarrhea virus inhibits DNA binding by interferon regulatory factor 3 and targets it for proteasomal degradation.

J Virol 2006 Dec 13;80(23):11723-32. Epub 2006 Sep 13.

Division of Basic Medical Sciences, St. George's, University of London, London SW17 0RE, United Kingdom.

Bovine viral diarrhea virus (BVDV) is a pestivirus that can establish a persistent infection in the developing fetus and has the ability to disable the production of type I interferon. In this report, we extend our previous observations that BVDV encodes a protein able to specifically block the activity of interferon regulatory factor 3 (IRF-3), a transcription factor essential for interferon promoter activation, by demonstrating that this is a property of the N-terminal protease fragment (NPro) of the BVDV polyprotein. Although BVDV infections cause relocalization of cellular IRF-3 from the cytoplasm to the nucleus early in infection, NPro blocks IRF-3 from binding to DNA. NPro has the additional property of targeting IRF-3 for polyubiquitination and subsequent destruction by cellular multicatalytic proteasomes. The autoprotease activity of NPro is not required for the inhibition of type I interferon induction or the targeting of IRF-3 for degradation.
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http://dx.doi.org/10.1128/JVI.01145-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1642611PMC
December 2006

Influenza A virus NS1 protein binds p85beta and activates phosphatidylinositol-3-kinase signaling.

Proc Natl Acad Sci U S A 2006 Sep 8;103(38):14194-9. Epub 2006 Sep 8.

Centre for Biomolecular Sciences, University of St. Andrews, St. Andrews, Fife KY16 9ST, United Kingdom.

Influenza A virus NS1 is a multifunctional protein, and in virus-infected cells NS1 modulates a number of host-cell processes by interacting with cellular factors. Here, we report that NS1 binds directly to p85beta, a regulatory subunit of phosphatidylinositol-3-kinase (PI3K), but not to the related p85alpha subunit. Activation of PI3K in influenza virus-infected cells depended on genome replication, and showed kinetics that correlated with NS1 expression. Additionally, it was found that expression of NS1 alone was sufficient to constitutively activate PI3K, causing the phosphorylation of a downstream mediator of PI3K signal transduction, Akt. Mutational analysis of a potential SH2-binding motif within NS1 indicated that the highly conserved tyrosine at residue 89 is important for both the interaction with p85beta, and the activation of PI3K. A mutant influenza virus (A/Udorn/72) expressing NS1 with the Y89F amino acid substitution exhibited a small-plaque phenotype, and grew more slowly in tissue culture than WT virus. These data suggest that activation of PI3K signaling in influenza A virus-infected cells is important for efficient virus replication.
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http://dx.doi.org/10.1073/pnas.0606109103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1599933PMC
September 2006

Genetic fusion of proteins to the SIV Tat protein enhances their immunogenicity.

Vaccine 2006 Feb 19;24(6):708-15. Epub 2005 Sep 19.

School of Biomedical Sciences, University of St. Andrews, Biomolecular Sciences Bldg., North Haugh, St. Andrews, Fife, Scotland KY16 9ST, United Kingdom.

The potential of genetically fusing recombinant proteins to the simian immunodeficiency virus (SIV) Tat protein has been investigated. The recombinant SIV Tat protein was initially expressed in very low amounts in E. coli, but optimization of the coding sequence for translation in the bacterial host significantly improved protein expression. Whilst fusion of SIV Tat to an experimental antigen (GST) facilitated the binding of the antigen to cell surfaces it did not appear to facilitate the transport of the protein into the cytosol. The immunogenicity of GST was significantly enhanced, in the absence of adjuvants, when fused to SIV Tat, with the induction of IgG1 and IgG2a antibodies indicative of a Th1 response being induced. However, no evidence was obtained that such an immunization scheme efficiently induced a CTL response.
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http://dx.doi.org/10.1016/j.vaccine.2005.09.012DOI Listing
February 2006

Susceptibility of B lymphocytes to adenovirus type 5 infection is dependent upon both coxsackie-adenovirus receptor and alphavbeta5 integrin expression.

J Gen Virol 2005 Jun;86(Pt 6):1669-1679

Infection and Immunity, Henry Wellcome Research Building, Wales College of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK.

Human lymphocytes are resistant to genetic modification, particularly from recombinant adenoviruses, thus hampering the analysis of gene function using adenoviral vectors. This study engineered an Epstein-Barr virus-transformed B-lymphoblastoid cell line permissive to adenovirus infection and elucidated key roles for both the coxsackie-adenovirus receptor and alphavbeta5 integrin in mediating entry of adenoviruses into these cells. The work identified a strategy for engineering B cells to become susceptible to adenovirus infection and showed that such a strategy could be useful for the introduction of genes to alter lymphoblastoid-cell gene expression.
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http://dx.doi.org/10.1099/vir.0.80806-0DOI Listing
June 2005

A human immunodeficiency virus 1 (HIV-1) clade A vaccine in clinical trials: stimulation of HIV-specific T-cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans.

J Gen Virol 2004 Apr;85(Pt 4):911-919

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, The John Radcliffe, Oxford OX3 9DS, UK.

The immunogenicities of candidate DNA- and modified vaccinia virus Ankara (MVA)-vectored human immunodeficiency virus (HIV) vaccines were evaluated on their own and in a prime-boost regimen in phase I clinical trials in healthy uninfected individuals in the United Kingdom. Given the current lack of approaches capable of inducing broad HIV-neutralizing antibodies, the pTHr.HIVA DNA and MVA.HIVA vaccines focus solely on the induction of cell-mediated immunity. The vaccines expressed a common immunogen, HIVA, which consists of consensus HIV-1 clade A Gag p24/p17 proteins fused to a string of clade A-derived epitopes recognized by cytotoxic T lymphocytes (CTLs). Volunteers' fresh peripheral blood mononuclear cells were tested for HIV-specific responses in a validated gamma interferon enzyme-linked immunospot (ELISPOT) assay using four overlapping peptide pools across the Gag domain and three pools of known CTL epitopes present in all of the HIVA protein. Both the DNA and the MVA vaccines alone and in a DNA prime-MVA boost combination were safe and induced HIV-specific responses in 14 out of 18, seven out of eight and eight out of nine volunteers, respectively. These results are very encouraging and justify further vaccine development.
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http://dx.doi.org/10.1099/vir.0.19701-0DOI Listing
April 2004

Mutation analysis of synapsin III gene in schizophrenia.

Am J Med Genet 2002 Jan;114(1):79-83

Department of Psychiatry, Tzu Chi General Hospital and Tzu Chi University Medical School, Hualien City, Taiwan.

Synapsin III is a new synapsin family gene with the putative function of synaptogenesis regulation and neurotransmitter release in the brain. The gene was mapped to 22q12-q13, a schizophrenia susceptible region gene as suggested by several linkage studies. Hence, the synapsin III gene is considered a candidate gene of schizophrenia. We systematically sequenced the protein coding and 5'-promoter regions of the synapsin III gene to look for mutations in 62 Han Chinese schizophrenic patients from Taiwan with positive family history. Further case-control association study was performed among 163 patients and 151 controls using the genetic polymorphic markers identified from these 62 patients. Three single nucleotide polymorphisms (SNPs) were identified: g.-631C > G and g.-196G>A at 5'-promoter region, and g.69G>A at exon 1. Besides, no other mutations were identified in these patients. The g.69G>A polymorphism does not alter the amino acid threonine at codon 23 (ACG>ACA). Further case-control association studies also did not find significant differences of genotype or allele frequency distributions of these three polymorphisms between 163 patients and 151 non-psychotic comparison individuals. Hence, our data are not in favor of a large effect of synapsin III gene in the pathogenesis of schizophrenia.
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http://dx.doi.org/10.1002/ajmg.10116DOI Listing
January 2002