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1.  HIV: seek, test, treat, and retain 
The “HIV: Seek, Test, Treat, and Retain” session was chaired by Dr. Jacques Normand, the Director of AIDS Research at the U.S. National Institute on Drug Abuse. Dr. Yi-Ming Chen served as the discussant. The three presenters (and their presentation topics) were: Dr. Julio Montaner (Treatment as Prevention—The Key to an AIDS-free Generation), Dr. Chi-Tai Fang (Population-level Effect of Free Access to HAART on Reducing HIV Transmission in Taiwan), and Dr. Zunyou Wu (Challenges in Promoting HIV Test & Treat Strategy in China).
PMCID: PMC4179106  PMID: 25278735
HIV; seek; test; treat; retain
2.  Risk Factors of Tuberculosis Infection Among HIV/AIDS Patients in Burkina Faso 
AIDS Research and Human Retroviruses  2013;29(7):1045-1055.
Tuberculosis (TB) and HIV coinfection is the leading cause of mortality among TB patients and people living with HIV/AIDS (PLWHAs). There is still a need to look for cognitive and behavioral determinants of TB among PLWHAs. This study aims at identifying risk factors of TB infection among PLWHAs in Burkina Faso. A cross-sectional study design and consecutive recruitment method were employed. Adult patients attending TB hospitals or HIV clinics were recruited in two main regions (Hauts-Bassins and Centre) of Burkina Faso from August to October 2010. Stepwise logistic regression models were used for statistical analysis. In total, 734 PLWHAs, including 181 (24.7%) coinfected with TB, participated in this study. Of the latter, 53.4% were from the Hauts-Bassins region and 46.6% from the Centre region. Adjusted TB risk factors among PLWHAs were urban setting, TB history, higher number of persons living in the household, and poor geographic access to care. Moreover adjusted TB risk factors among PLWHAs consisted of CD4 cell counts below 200/μl, a history of sexually transmissible infections, and a past or present history of pulmonary asthma. In addition, lack of education and arterial hypertension were additional risk factors in the Hauts-Bassins region; for PLWHAs in the Centre region, male gender, jobs not in the private and public sector, and past or present history of cardiovascular disease were additional risk factors for TB. Common and different risk factors for TB were identified for PLWHAs in the Hauts-Bassins and Centre regions. This information will be incorporated into the HIV/TB control programs in the future.
PMCID: PMC3685687  PMID: 23517547
3.  Deficiency of Glycine N-Methyltransferase Aggravates Atherosclerosis in Apolipoprotein E–Null Mice 
Molecular Medicine  2012;18(1):744-752.
The mechanism underlying the dysregulation of cholesterol metabolism and inflammation in atherogenesis is not understood fully. Glycine N-methyltransferase (GNMT) has been implicated in hepatic lipid metabolism and the pathogenesis of liver diseases. However, little is known about the significance of GNMT in atherosclerosis. We showed the predominant expression of GNMT in foamy macrophages of mouse atherosclerotic aortas. Genetic deletion of GNMT exacerbated the hyperlipidemia, inflammation and development of atherosclerosis in apolipoprotein E–deficient mice. In addition, ablation of GNMT in macrophages aggravated oxidized low-density lipoprotein-mediated cholesterol accumulation in macrophage foam cells by downregulating the expression of reverse cholesterol transporters including ATP-binding cassette transporters-A1 and G1 and scavenger receptor BI. Furthermore, tumor necrosis factor-α–induced inflammatory response was promoted in GNMT-null macrophages. Collectively, our data suggest that GNMT is a crucial regulator in cholesterol metabolism and in inflammation, and contributes to the pathogenesis of atherosclerosis. This finding may reveal a potential therapeutic target for atherosclerosis.
PMCID: PMC3409278  PMID: 22415010
4.  Glycine N-Methyltransferase Deficiency Affects Niemann-Pick Type C2 Protein Stability and Regulates Hepatic Cholesterol Homeostasis 
Molecular Medicine  2011;18(1):412-422.
Nonalcoholic fatty liver disease (NAFLD) is associated with the development of metabolic syndromes and hepatocellular carcinoma (HCC). Cholesterol accumulation is related to NAFLD, whereas its detailed mechanism is not fully understood. Previously, we reported that glycine N-methyltransferase (GNMT) knockout (Gnmt−/−) mice develop chronic hepatitis and HCC. In this study, we showed that Gnmt−/− mice had hyperlipidemia and steatohepatitis. Single photon emission computed tomography images of mice injected with 131I-labeled 6β-iodocholesterol demonstrated that Gnmt−/− mice had slower hepatic cholesterol uptake and excretion rates than wild-type mice. In addition, genes related to cholesterol uptake (scavenger receptor class B type 1 [SR-B1] and ATP-binding cassette A1 [ABCA1]), intracellular trafficking (Niemann-Pick type C1 protein [NPC1] and Niemann-Pick type C2 protein [NPC2]) and excretion (ATP-binding cassette G1 [ABCG1]) were downregulated in Gnmt−/− mice. Yeast two-hybrid screenings and coimmunoprecipitation assays elucidated that the C conserved region (81–105 amino acids) of NPC2 interacts with the carboxyl-terminal fragment (171–295 amino acids) of GNMT. Confocal microscopy demonstrated that when cells were treated with low-density lipoprotein, NPC2 was released from lysosomes and interacts with GNMT in the cytosol. Overexpression of GNMT doubled the half-lives of both NPC2 isoforms and reduced cholesterol accumulation in cells. Furthermore, GNMT was downregulated in the liver tissues from patients suffering with NAFLD as well as from mice fed a high-fat diet, high-cholesterol diet or methionine/choline-deficient diet. In conclusion, our study demonstrated that GNMT regulates the homeostasis of cholesterol metabolism, and hepatic cholesterol accumulation may result from downregulation of GNMT and instability of its interactive protein NPC2. Novel therapeutics for steatohepatitis and HCC may be developed by using this concept.
PMCID: PMC3356423  PMID: 22183894
5.  Functional Characterization of Glycine N-Methyltransferase and Its Interactive Protein DEPDC6/DEPTOR in Hepatocellular Carcinoma 
Molecular Medicine  2011;18(1):286-296.
Glycine N-methyltransferase (GNMT) is a tumor suppressor for hepatocellular carcinoma (HCC). High rates of Gnmt knockout mice developed HCC. Epigenetic alteration and dysregulation of several pathways including wingless-type MMTV integration site (Wnt), mitogen-activated protein kinase (MAPK) and Janus kinase and signal transducer and activator of transcription (JAK-STAT) are associated with HCC development in Gnmt knockout mice. We hypothesized that GNMT may regulate signal transduction through interacting with other proteins directly. In this report, we identified a mammalian target of rapamycin (mTOR) inhibitor (DEP domain containing MTOR-interacting protein [DEPDC6/DEPTOR]) as a GNMT-binding protein by using yeast two-hybrid screening. Fluorescence resonance energy transfer assay demonstrated that the C-terminal half of GNMT interact with the PSD-95/Dlg1/ZO-1 (PDZ) domain of DEPDC6/DEPTOR. Immunohistochemical staining showed that 27.5% (14/51) of HCC patients had higher expression levels of DEPDC6/DEPTOR in the tumorous tissues than in tumor-adjacent tissues, especially among HCC patients with hepatitis B viral infection (odds ratio 10.3, 95% confidence interval [CI] 1.05–11.3) or patients with poor prognosis (death hazard ratio 4.51, 95% CI 1.60–12.7). In terms of molecular mechanism, knockdown of DEPDC6/DEPTOR expression in HuH-7 cells caused S6K and 4E-BP activation, but suppressed Akt. Overexpression of DEPDC6/DEPTOR activated Akt and increased survival of HCC cells. Overexpression of GNMT caused activation of mTOR/raptor downstream signaling and delayed G2/M cell cycle progression, which altogether resulted in cellular senescence. Furthermore, GNMT reduced proliferation of HuH-7 cells and sensitized them to rapamycin treatment both in vitro and in vivo. In conclusion, GNMT regulates HCC growth in part through interacting with DEPDC6/DEPTOR and modulating mTOR/raptor signaling pathway. Both GNMT and DEPDC6/DEPTOR are potential targets for developing therapeutics for HCC.
PMCID: PMC3324955  PMID: 22160218
6.  Fucoidan Supplementation Improves Exercise Performance and Exhibits Anti-Fatigue Action in Mice 
Nutrients  2014;7(1):239-252.
Fucoidan (FCD) is a well-known bioactive constituent of seaweed extract that possess a wide spectrum of activities in biological systems, including anti-cancer, anti-inflammation and modulation of immune systems. However, evidence on the effects of FCD on exercise performance and physical fatigue is limited. Therefore, we investigated the potential beneficial effects of FCD on ergogenic and anti-fatigue functions following physiological challenge. Male ICR mice from three groups (n = 8 per group) were orally administered FCD for 21 days at 0, 310 and 620 mg/kg/day, which were, respectively, designated the vehicle, FCD-1X and FCD-2X groups. The results indicated that the FCD supplementations increased the grip strength (p = 0.0002) and endurance swimming time (p = 0.0195) in a dose-depend manner. FCD treatments also produced dose-dependent decreases in serum levels of lactate (p < 0.0001) and ammonia (p = 0.0025), and also an increase in glucose level (p < 0.0001) after the 15-min swimming test. In addition, FCD supplementation had few subchronic toxic effects. Therefore, we suggest that long-term supplementation with FCD can have a wide spectrum of bioactivities on health promotion, performance improvement and anti-fatigue.
PMCID: PMC4303836  PMID: 25558908
brown seaweed extract; exercise performance; forelimb grip strength
7.  Patients Infected with CRF07_BC Have Significantly Lower Viral Loads than Patients with HIV-1 Subtype B: Mechanism and Impact on Disease Progression 
PLoS ONE  2014;9(12):e114441.
The circulating recombinant form (CRF) 07_BC is the most prevalent HIV-1 strain among injection drug users (IDUs) in Taiwan. It contains a 7 amino-acid deletion in its p6gag. We conducted a cohort study to compare viral loads and CD4 cell count changes between patients infected with subtype B and CRF07_BC and to elucidate its mechanism. Twenty-one patients infected with CRF07_BC and 59 patients with subtype B were selected from a cohort of 667 HIV-1/AIDS patients whom have been followed up for 3 years. Generalized estimated equation was used to analyze their clinical data and the results showed that patients infected with CRF07_BC had significantly lower viral loads (about 58,000 copies per ml less) than patients with subtype B infection (p = 0.002). The replicative capacity of nine CRF07_BC and four subtype B isolates were compared and the results showed that the former had significantly lower replicative capacity than the latter although all of them were CCR5- tropic and non-syncytium inducing viruses. An HIV-1-NL4-3 mutant virus which contains a 7 amino-acid deletion in p6gag (designated as 7d virus) was generated and its live cycle was investigated. The results showed that 7d virus had significantly lower replication capacity, poorer protease-mediated processing and viral proteins production. Electron microscopic examination of cells infected with wild-type or 7d virus demonstrated that the 7d virus had poorer and slower viral maturation processes: more viruses attached to the cell membrane and higher proportion of immature virions outside the cells. The interaction between p6gag and Alix protein was less efficient in cells infected with 7d virus. In conclusion, patients infected with CRF07_BC had significantly lower viral loads than patients infected with subtype B and it may due to the deletion of 7 amino acids which overlaps with Alix protein-binding domain of the p6gag.
PMCID: PMC4263662  PMID: 25502811
8.  Th17-Related Cytokines in Systemic Lupus Erythematosus Patients with Dilated Cardiomyopathies: A Possible Linkage to Parvovirus B19 Infection 
PLoS ONE  2014;9(12):e113889.
Dilated cardiomyopathies (DCM) are a major cause of mortality in patients with systemic lupus erythematosus (SLE). Immune responses induced by human parvovirus B19 (B19) are considered an important pathogenic mechanism in myocarditis or DCM. However, little is known about Th17-related cytokines in SLE patients with DCM about the linkage with B19 infection. IgM and IgG against B19 viral protein, and serum levels of Th17-related cytokines were determined using ELISA in eight SLE patients with DCM and six patients with valvular heart disease (VHD). Humoral responses of anti-B19-VP1u and anti-B19-NS1 antibody were assessed using Western blot and B19 DNA was detected by nested Polymerase Chain Reaction (PCR). Levels of interleukin (IL)-17, IL-6, IL-1β, and tumor necrosis factor (TNF)-α were significantly higher in SLE patients with DCM (mean ± SEM, 390.99±125.48 pg/ml, 370.24±114.09 pg/ml, 36.01±16.90 pg/ml, and 183.84±82.94 pg/ml, respectively) compared to healthy controls (51.32±3.04 pg/ml, p<0.001; 36.88±6.64 pg/ml, p<0.001; 5.39±0.62 pg/ml, p<0.005; and 82.13±2.42 pg/ml, p<0.005, respectively). Levels of IL-17 and IL-6 were higher in SLE patients with DCM versus those with VHD (both p<0.01). Five (62.5%) of DCM patients had detectable anti-B19-NS1 IgG and four (50.0%) of them had anti-B19-VP1u IgG, whereas only one (16.7%) of VHD patients had detectable anti-B19-NS1 IgG and anti-B19-VP1u IgG. Serum levels of IL-17, IL-6 and IL-1β were markedly higher in SLE patients with anti-B19-VP1u IgG and anti-B19-NS1 IgG compared to those without anti-B19-VP1u IgG or anti-B19-NS1 IgG, respectively. These suggest a potential association of B19 with DCM and Th17-related cytokines implicated in the pathogenesis of DCM in SLE patients.
PMCID: PMC4252071  PMID: 25462010
9.  Epidemiological and Genetic Correlates of Severe Acute Respiratory Syndrome Coronavirus Infection in the Hospital with the Highest Nosocomial Infection Rate in Taiwan in 2003†  
Journal of Clinical Microbiology  2006;44(2):359-365.
Taiwan experienced a series of outbreaks of nosocomial severe acute respiratory syndrome (SARS) infections in 2003. Two months after the final outbreak, we recruited 658 employees from the hospital that suffered the first and most severe SARS infections to help us investigate epidemiological and genetic factors associated with the SARS coronavirus (SARS-CoV). SARS-CoV infections were detected by using enzyme immunoassays and confirmed by a combination of Western blot assays, neutralizing antibody tests, and commercial SARS tests. Risk factors were analyzed via questionnaire responses and sequence-specific oligonucleotide probes of human leukocyte antigen (HLA) alleles. Our results indicate that 3% (20/658) of the study participants were seropositive, with one female nurse identified as a subclinical case. Identified SARS-CoV infection risk factors include working in the same building as the hospital's emergency room and infection ward, providing direct care to SARS patients, and carrying a Cw*0801 HLA allele. The odds ratio for contracting a SARS-CoV infection among persons with either a homozygous or a heterozygous Cw*0801 genotype was 4.4 (95% confidence interval, 1.5 to 12.9; P = 0.007).
PMCID: PMC1392693  PMID: 16455884
10.  HIV-1 CRF07_BC Infections, Injecting Drug Users, Taiwan 
Emerging Infectious Diseases  2006;12(4):703-705.
PMCID: PMC3294689  PMID: 16715583
HIV-1; CRF07_BC; injecting drug user; harm reduction; Taiwan; letter
11.  Effect of the Herbal Drug Guilu Erxian Jiao on Muscle Strength, Articular Pain, and Disability in Elderly Men with Knee Osteoarthritis 
Background. Guilu Erxian Jiao (GEJ) is a widely used Chinese herbal remedy for knee osteoarthritis, but its clinical efficacy is unknown. Methods. We enrolled 42 elderly male patients with knee OA, including 21 patients who received the herbal drug GEJ as the case group and 21 patients who did not receive GEJ as the control group. The effects of 12 weeks of GEJ treatment on muscle strength of lower limbs were measured by a Biodex dynamometer, with disability evaluated on the Lequesne index and articular pain measured on the visual analog scale (VAS) between the two groups on the baseline and after treatment. Results. There were significant increases in the levels of muscle strength of TQ/BW-ext-dominant and TQ/BW-flex-dominant between the two groups after treatment (P < 0.05). There were also significant increases in muscle strength of knee extensor muscles in the GEJ-treated group (n = 21) self-controlled before and after 12 weeks of treatment (all P < 0.01). There were significant decreases in articular pain (P < 0.01) and Lequesne index scores (P < 0.01) in the GEJ-treated group when compared to the non-GEJ-treated group. Conclusions. Our results showed that GEJ is effective and is tolerated well in elderly men with knee OA.
PMCID: PMC4182023  PMID: 25309612
12.  Late-life depression and quality of life in a geriatric evaluation and management unit: an exploratory study 
BMC Geriatrics  2014;14:77.
Late-life depression is common among elderly patients. Ignorance of the health problem, either because of under-diagnosis or under-treatment, causes additional medical cost and comorbidity. For a better health and quality of life (QoL), evaluation, prevention and treatment of late-life depression in elderly patients is essential.
This study examined (1) the differences of clinical characteristics, degree of improvement on QoL and functionality on discharge between non-depressed and depressed elderly inpatients and (2) factors associated with QoL on discharge. Four hundred and seventy-one elderly inpatients admitted to a geriatric evaluation and management unit (GEMU) from 2009 to 2010 were enrolled in this study. Comprehensive geriatric assessment including the activities of daily living (ADL), geriatric depression scale, and mini-mental state examination were conducted. QoL was assessed using the European Quality of Life-5 Dimensions and the European Quality of Life-5 Dimensions Visual Analog Scale on discharge. Information on hospital stay and Charlson comorbidity index were obtained by chart review. Chi-square tests, independent t-tests, Mann–Whitney U tests and multiple linear regressions were used in statistical analysis.
Worse QoL and ADL on discharge were found among the depressed. Depressive symptoms, female gender, duration of hospital stay, and rehabilitation were significant factors affecting QoL on discharge in linear regression models.
The importance of the diagnosis and treatment of depression among elderly inpatients should not be overlooked during hospital stay and after discharge. Greater efforts should be made to improve intervention with depressed elderly inpatients.
PMCID: PMC4085690  PMID: 24941865
Elderly; Late-life depression; Geriatric depression; Quality of life; EQ-5D; Geriatric evaluation and management unit; GEMU; ADL; GDS
13.  Genetic Polymorphisms of the Glycine N-Methyltransferase and Prostate Cancer Risk in the Health Professionals Follow-Up Study 
PLoS ONE  2014;9(5):e94683.
Glycine N-methyltransferase (GNMT) affects genetic stability by regulating the ratio of S-adenosylmethionine to S-adenosylhomocysteine, by binding to folate, and by interacting with environmental carcinogens. In Taiwanese men, GNMT was found to be a tumor susceptibility gene for prostate cancer. However, the association of GNMT with prostate cancer risk in other ethnicities has not been studied. It was recently reported that sarcosine, which is regulated by GNMT, increased markedly in metastatic prostate cancer. We hereby explored the association of GNMT polymorphisms with prostate cancer risk in individuals of European descent from the Health Professionals Follow-up Study (HPFS).
A total of 661 incident prostate cancer cases and 656 controls were identified from HPFS. The GNMT short tandem repeat polymorphism 1 (STRP1), 4-bp insertion/deletion polymorphisms (INS/DEL) and the single nucleotide polymorphism rs10948059 were genotyped to test for their association with prostate cancer risk.
The rs10948059 T/T genotype was associated with a 1.62-fold increase in prostate cancer risk (95% confidence interval (CI): 1.18, 2.22) when compared with the C/C genotype. The STRP1 ≥16GAs/≥16GAs genotype was associated with decreased risk of prostate cancer when compared with the <16GAs/<16GAs genotype (odds ratio (OR) = 0.68; 95% CI: 0.46, 1.01). INS/DEL was not associated with prostate cancer risk. Haplotypes containing the rs10948059 T allele were significantly associated with increased prostate cancer risk.
In men of European descent, the GNMT rs10948059 and STRP1 were associated with prostate cancer risk. Compared to the study conducted in Taiwanese men, the susceptibility GNMT alleles for prostate cancer had a reverse relationship. This study highlights the differences in allelic frequencies and prostate cancer susceptibility in different ethnicities.
PMCID: PMC4011739  PMID: 24800880
14.  Evaluating immunologic response and clinical deterioration in treatment-naïve patients initiating first-line therapies infected with HIV-1 CRF01_AE and subtype B 
HIV-1 group M viruses diverge 25%–35% in envelope, important for viral attachment during infection, and 10–15% in the pol region, under selection pressure from common antiretrovirals. In Asia, subtypes B and CRF01_AE are common genotypes. Our objectives were to determine whether clinical, immunologic or virologic treatment responses differed by genotype in treatment-naïve patients initiating first-line therapy.
Prospectively collected, longitudinal data from patients in Thailand, Hong Kong, Malaysia, Japan, Taiwan and South Korea were provided for analysis. Covariates included demographics, hepatitis B and C coinfections, baseline CD4 T lymphocyte count and plasma HIV-1 RNA levels. Clinical deterioration (a new diagnosis of CDC category B/AIDS-defining illness or death) was assessed by proportional hazards models. Surrogate endpoints were 12-month change in CD4 cell count and virologic suppression post-therapy, evaluated by linear and logistic regression, respectively.
Of 1105 patients, 1036 (93.8%) infected with CRF01_AE or subtype B were eligible for inclusion in clinical deterioration analyses and contributed 1546.7 person-years of follow-up (median:413 days, IQR:169–672 days). Patients >40 years demonstrated smaller immunological increases (p=0.002) and higher risk of clinical deterioration (HR=2.17; p=0.008). Patients with baseline CD4 cell counts >200 cells/μL had lower risk of clinical deterioration (HR=0.373; p=0.003). A total of 532 patients (48.1% of eligible) had CD4 counts available at baseline and 12 months post-therapy for inclusion in immunolgic analyses. Patients infected with subtype B had larger increases in CD4 counts at 12 months (p=0.024). A total of 530 patients (48.0% of eligible) were included in virologic analyses with no differences in response found between genotypes.
Results suggest that patients infected with CRF01_AE have reduced immunologic response to therapy at 12 months, compared to subtype-B-infected counterparts. Clinical deterioration was associated with low baseline CD4 counts and older age. The lack of differences in virologic outcomes suggests that all patients have opportunities for virologic suppression.
PMCID: PMC3964197  PMID: 23138836
HIV-1; Asia; genotype; CRF01_AE; subtype B
15.  A Potential Role of Myeloid DAP12-Associating Lectin (MDL)-1 in the Regulation of Inflammation in Rheumatoid Arthritis Patients 
PLoS ONE  2014;9(1):e86105.
The pathogenic roles of myeloid DAP12-associating lectin-1(MDL-1) and DAP12 in human rheumatoid arthritis (RA) remain unknown. Frequencies of MDL-1-expressing monocytes in 22 active RA patients, 16 inactive RA patients, 12 osteoarthritis (OA) patients and 10 healthy controls (HC) were determined by flow-cytometry analysis. The mRNA expression levels of MDL-1 and DAP12 on PBMCs were evaluated by quantitative PCR, and their protein expression levels in the synovium were examined by immunohistochemistry. Significantly higher median percentages of circulating MDL-1-expressing monocytes were observed in active RA patients (53.6%) compared to inactive RA patients (34.1%), OA patients (27.9%), and HC (21.2%). Levels of MDL-1 and DAP12 gene expression in PBMCs and their protein expression in the synovium were significantly higher in active RA patients than in inactive RA or OA patients. MDL-1 levels were positively correlated with parameters of disease activity, articular damage, and levels of proinflammatory cytokines. MDL-1 activator (Dengue virus type 2 antigen) stimulation on PBMCs resulted in significantly enhanced levels of proinflammatory cytokines in RA patients compared to those in OA patients or HC, indicating that MDL-1 activation is functional. Frequencies of MDL-1-expressing monocytes and levels of MDL-1 and DAP12 gene expression significantly decreased after effective therapy. Concordant overexpression of MDL-1 and DAP12 were correlated with increased production of proinflammatory cytokines in RA patients, suggesting their roles in regulating articular inflammation.
PMCID: PMC3897620  PMID: 24465901
16.  Molecular Epidemiology of Enterovirus 71 Infection in the Central Region of Taiwan from 2002 to 2012 
PLoS ONE  2013;8(12):e83711.
Enterovirus 71 (EV71), a causative agent of hand, foot, and mouth disease can be classified into three genotypes and many subtypes. The objectives of this study were to conduct a molecular epidemiological study of EV71 in the central region of Taiwan from 2002–2012 and to test the hypothesis that whether the alternative appearance of different EV71 subtypes in Taiwan is due to transmission from neighboring countries or from re-emergence of pre-existing local strains. We selected 174 EV71 isolates and used reverse transcription-polymerase chain reaction to amplify their VP1 region for DNA sequencing. Phylogenetic analyses were conducted using Neighbor-Joining, Maximum Likelihood and Bayesian methods. We found that the major subtypes of EV71 in Taiwan were B4 for 2002 epidemic, C4 for 2004–2005 epidemic, B5 for 2008–2009 epidemic, C4 for 2010 epidemic and B5 for 2011–2012 epidemic. Phylogenetic analysis demonstrated that the 2002 and 2008 epidemics were associated with EV71 from Malaysia and Singapore; while both 2010 and 2011–2012 epidemics originated from different regions of mainland China including Shanghai, Henan, Xiamen and Gong-Dong. Furthermore, minor strains have been identified in each epidemic and some of them were correlated with the subsequent outbreaks. Therefore, the EV71 infection in Taiwan may originate from pre-existing minor strains or from other regions in Asia including mainland China. In addition, 101 EV71 isolates were selected for the detection of new recombinant strains using the nucleotide sequences spanning the VP1-2A-2B region. No new recombinant strain was found. Analysis of clinical manifestations showed that patients infected with C4 had significantly higher rates of pharyngeal vesicles or ulcers than patients infected with B5. This is the first study demonstrating that different EV 71 genotypes may have different clinical manifestations and the association of EV71 infections between Taiwan and mainland China.
PMCID: PMC3877097  PMID: 24391812
17.  Characterization of Niemann-Pick Type C2 Protein Expression in Multiple Cancers Using a Novel NPC2 Monoclonal Antibody 
PLoS ONE  2013;8(10):e77586.
Niemann-Pick Type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in cancer. In this study, we have pinpointed the impact of various different cancers on NPC2 expression. A series of anti-NPC2 monoclonal antibodies (mAbs) with the IgG2a isotype were generated and peptide screening demonstrated that the reactive epitope were amino acid residues 31-40 of the human NPC2 protein. The specificity of these mAbs was confirmed by Western blotting using shRNA mediated knock-down of NPC2 in human SK-Hep1 cells. By immunohistochemical staining, NPC2 is expressed in normal kidney, liver, breast, colon, lung, esophageal, uterine cervical, pancreatic and stomach tissue. Strong expression of NPC2 was found in the distal and proximal convoluted tubule of kidney and the hepatocytes of liver. Normal esophageal, uterine cervical, pancreatic, stomach, breast, colon and lung tissue stained moderately to weakly. When compared to their normal tissue equivalents, NPC2 overexpression was observed in cancers of the breast, colon and lung. Regarding to breast cancer, NPC2 up-regulation is associated with estrogen receptor (-), progesterone receptor (-) and human epidermal growth factor receptor (+). On the other hand, NPC2 was found to be down-regulated in renal cell carcinoma, liver cirrhosis and hepatoma tissues. By antigen-capture enzyme immunoassay ELISA, the serum NPC2 is increased in patients with cirrhosis and liver cancer. According to western blot data, the change of glycosylated pattern of NPC2 in serum is associated with cirrhosis and liver cancer. To the best of our knowledge, this is the first comprehensive immunohistochemical and serological study investigating the expression of NPC2 in a variety of different human cancers. These novel monoclonal antibodies should help with elucidating the roles of NPC2 in tumor development, especially in liver and breast cancers.
PMCID: PMC3798307  PMID: 24147030
18.  Androgen response element of the glycine N-methyltransferase gene is located in the coding region of its first exon 
Bioscience Reports  2013;33(5):e00070.
Androgen plays an important role in the pathogenesis of PCa (prostate cancer). Previously, we identified GNMT (glycine N-methyltransferase) as a tumour susceptibility gene and characterized its promoter region. Besides, its enzymatic product-sarcosine has been recognized as a marker for prognosis of PCa. The goals of this study were to determine whether GNMT is regulated by androgen and to map its AREs (androgen response elements). Real-time PCR analyses showed that R1881, a synthetic AR (androgen receptor) agonist induced GNMT expression in AR-positive LNCaP cells, but not in AR-negative DU145 cells. In silico prediction showed that there are four putative AREs in GNMT-ARE1, ARE2 and ARE3 are located in the intron 1 and ARE4 is in the intron 2. Consensus ARE motif deduced from published AREs was used to identify the fifth ARE-ARE5 in the coding region of exon 1. Luciferase reporter assay found that only ARE5 mediated the transcriptional activation of R1881. ARE3 overlaps with a YY1 [Yin and Yang 1 (motif (CaCCATGTT, +1118/+1126)] that was further confirmed by antibody supershift and ChIP (chromatin immunoprecipitation) assays. EMSA (electrophoretic mobility shift assay) and ChIP assay confirmed that AR interacts with ARE5 in vitro and in vivo. In summary, GNMT is an AR-targeted gene with its functional ARE located at +19/+33 of the first exon. These results are valuable for the study of the influence of androgen on the gene expression of GNMT especially in the pathogenesis of cancer.
PMCID: PMC3775523  PMID: 23883094
androgen receptor; androgen response element; chromatin immunoprecipitation; electrophoretic mobility shift assay; GNMT; prostate cancer; Yin and Yang 1; AR, androgen receptor; ARE, androgen response element; ChIP, chromatin immunoprecipitation; CS, charcoal-stripped; CT, cycle threshold; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GNMT, glycine N-methyltransferase; mTOR, mammalian target of rapamycin; NF-Y, nuclear factor-Y; PCa, prostate cancer; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B (also called Akt); RGA, reporter gene assay; RT–PCR, reverse transcription–PCR; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SV40, simian virus 40; TBP, TATA-box-binding protein; wt, wild-type; YY1, Yin and Yang 1
19.  Dextromethorphan Inhibits Activations and Functions in Dendritic Cells 
Dendritic cells (DCs) play an important role in connecting innate and adaptive immunity. Thus, DCs have been regarded as a major target for the development of immunomodulators. In this study, we examined the effect of dextromethorphan (DXM), a common cough suppressant with a high safety profile, on the activation and function of DCs. In the presence of DXM, the LPS-induced expression of the costimulatory molecules in murine bone marrow-derived dendritic cells (BMDCs) was significantly suppressed. In addition, DXM treatment reduced the production of reactive oxygen species (ROS), proinflammatory cytokines, and chemokines in maturing BMDCs that were activated by LPS. Therefore, DXM abrogated the ability of LPS-stimulated DCs to induce Ag-specific T-cell activation, as determined by their decreased proliferation and IFN-γ secretion in mixed leukocyte cultures. Moreover, the inhibition of LPS-induced MAPK activation and NF-κB translocation may contribute to the suppressive effect of DXM on BMDCs. Remarkably, DXM decreased the LPS-induced surface expression of CD80, CD83, and HLA-DR and the secretion of IL-6 and IL-12 in human monocyte-derived dendritic cells (MDDCs). These findings provide a new insight into the impact of DXM treatment on DCs and suggest that DXM has the potential to be used in treating DC-related acute and chronic diseases.
PMCID: PMC3679715  PMID: 23781253
20.  Involvement of TLR7 MyD88-dependent signaling pathway in the pathogenesis of adult-onset Still's disease 
The objective of this study was to investigate the potential role of the Toll-like receptor 7 (TLR7) signaling pathway in the pathogenesis of adult-onset Still's disease (AOSD).
Frequencies of TLR7-expressing precursor of myeloid dendritic cells (pre-mDCs) and mDCs in 28 AOSD patients, 28 patients with systemic lupus erythematosus (SLE) and 12 healthy controls (HC) were determined by flow cytometry analysis. Transcript and protein levels of TLR7 signaling molecules in peripheral blood mononuclear cells (PBMCs) were evaluated by quantitative PCR and western blotting respectively. Serum cytokines levels were measured by ELISA.
Significantly higher median frequencies of TLR7-expressing pre-mDCs and mDCs were observed in AOSD patients (65.5% and 14.9%, respectively) and in SLE patients (60.3% and 14.4%, respectively) than in HC (42.8% and 8.8%, respectively; both P <0.001). Transcript and protein levels of TLR7-signaling molecules, including MyD88, TRAF6, IRAK4 and IFN-α, were upregulated in AOSD patients and SLE patients compared with those in HC. Disease activity scores were positively correlated with the frequencies of TLR7-expressing mDCs and expression levels of TLR7 signaling molecules in both AOSD and SLE patients. TLR7 ligand (imiquimod) stimulation of PBMCs resulted in significantly enhanced levels of interleukin (IL)-1β, IL-6, IL-18 and IFN-α in AOSD and SLE patients. Frequencies of TLR7-expressing mDCs and expression levels of TLR7 signaling molecules significantly decreased after effective therapy.
Elevated levels of TLR7 signaling molecules and their positive correlation with disease activity in AOSD patients suggest involvement of the TLR7 signaling pathway in the pathogenesis of this disease. The overexpression of TLR7 MyD88-dependent signaling molecules may be a common pathogenic mechanism for both AOSD and SLE.
PMCID: PMC3672755  PMID: 23497717
21.  Germinal center kinase-like kinase (GLK/MAP4K3) expression is increased in adult-onset Still's disease and may act as an activity marker 
BMC Medicine  2012;10:84.
Germinal center kinase-like kinase (GLK, also termed MAP4K3), a member of the MAP4K family, may regulate gene transcription, apoptosis and immune inflammation in response to extracellular signals. The enhanced expression of GLK has been shown to correspond with disease severity in patients with systemic lupus erythematosus. We investigated the role of GLK in the pathogenesis of adult-onset Still's disease, which shares some similar clinical characteristics with systemic lupus erythematosus.
The frequencies of circulating GLK-expressing T-cells in 24 patients with active adult-onset Still's disease and 12 healthy controls were determined by flow cytometry analysis. The expression levels of GLK proteins and transcripts were evaluated in peripheral blood mononuclear cells by immunoblotting and quantitative PCR. Serum levels of T helper (Th)17-related cytokines, including IL-1β, IL-6, IL-17 and TNF-α, were measured by ELISA.
Significantly higher median frequencies of circulating GLK-expressing T-cells were observed in patients with adult-onset Still's disease (31.85%) than in healthy volunteers (8.93%, P <0.001). The relative expression levels of GLK proteins and transcripts were also significantly higher in patients with adult-onset Still's disease (median, 1.74 and 2.35, respectively) compared with those in healthy controls (0.66 and 0.92, respectively, both P <0.001). The disease activity scores were positively correlated with the frequencies of circulating GLK-expressing T-cells (r = 0.599, P <0.005) and the levels of GLK proteins (r = 0.435, P <0.05) or GLK transcripts (r = 0.452, P <0.05) in patients with adult-onset Still's disease. Among the examined Th17-related cytokines, elevated levels of serum IL-6 and IL-17 were positively correlated with the frequencies of circulating GLK-expressing T-cells and the levels of GLK proteins as well as transcripts in patients with adult-onset Still's disease. GLK expression levels decreased significantly after effective therapy in these patients.
Elevated expression levels of GLK and their positive correlation with disease activity in patients with adult-onset Still's disease indicate that GLK may be involved in the pathogenesis and act as a novel activity biomarker of this disease.
PMCID: PMC3424974  PMID: 22867055
Adult-onset Still's disease; GCK-like kinase (GLK, MAP4K3); mitogen-activated protein kinases (MAPKs); pathogenesis; Th17-related cytokines
22.  Prevalence of and risk factors for lipodystrophy among HIV-infected patients receiving combined antiretroviral treatment in the Asia-Pacific region: results from the TREAT Asia HIV Observational Database (TAHOD) 
Endocrine Journal  2011;58(6):475-484.
The prevalence of and risk factors for lipodystrophy (LD) among patients receiving combined antiretroviral treatment (cART) in the Asia-Pacific region are largely unknown. LD diagnosis was based on the adverse event definition from the US NIH Division of AIDS (2004 version), and only cases with a severity grade of ≥ 3 were included. TAHOD patients who had recently commenced cART with ≥ 3 drugs after 1996 from sites which had ever reported LD were included in the analysis. Covariates for the forward multivariate logistic regression model included demographic variables, CDC disease classification, baseline CD4 and viral load, hepatitis B/C virus co-infection, and regimen and duration of cART. LD was diagnosed in 217 (10.5%) of 2072 patients. The median duration of cART was 3.8 (interquartile range, 2.2–5.3) years (stavudine, 2.0 (1.0–3.5) years; zidovudine, 1.8 (0.6–3.9) years; and protease inhibitors (PI), 2.6 (1.3–4.5) years). In the multivariate model, factors independently associated with LD included use of stavudine (≤ 2 years vs. no experience: OR 25.46, p<0.001, > 2 years vs. no experience: OR 14.92, p<0.001), use of PI (> 2.6 years vs. no experience: OR 0.26, p<0.001), and total duration of cART (> vs. ≤ 3.8 years: OR 4.84, p<0.001). The use of stavudine was strongly associated with LD in our cohort. Stavudine-sparing cART strategies are warranted to prevent the occurrence of LD in the Asia-Pacific region.
PMCID: PMC3329967  PMID: 21521929
Lipodystrophy; HIV; Adverse effects; Combined antiretroviral therapy; Asia-Pacific
23.  Loss to Followup in HIV-Infected Patients from Asia-Pacific Region: Results from TAHOD 
AIDS Research and Treatment  2012;2012:375217.
This study examined characteristics of HIV-infected patients in the TREAT Asia HIV Observational Database who were lost to follow-up (LTFU) from treatment and care. Time from last clinic visit to 31 March 2009 was analysed to determine the interval that best classified LTFU. Patients defined as LTFU were then categorised into permanently LTFU (never returned) and temporary LTFU (re-entered later), and these groups compared. A total of 3626 patients were included (71% male). No clinic visits for 180 days was the best-performing LTFU definition (sensitivity 90.6%, specificity 92.3%). During 7697 person-years of follow-up, 1648 episodes of LFTU were recorded (21.4 per 100-person-years). Patients LFTU were younger (P = 0.002), had HIV viral load ≥500 copies/mL or missing (P = 0.021), had shorter history of HIV infection (P = 0.048), and received no, single- or double-antiretroviral therapy, or a triple-drug regimen containing a protease inhibitor (P < 0.001). 48% of patients LTFU never returned. These patients were more likely to have low or missing haemoglobin (P < 0.001), missing recent HIV viral load (P < 0.001), negative hepatitis C test (P = 0.025), and previous temporary LTFU episodes (P < 0.001). Our analyses suggest that patients not seen at a clinic for 180 days are at high risk of permanent LTFU, and should be aggressively traced.
PMCID: PMC3296146  PMID: 22461979
24.  Failure to prescribe pneumocystis prophylaxis is associated with increased mortality, even in the cART era: results from the Treat Asia HIV observational database 
Pneumocystis jiroveci pneumonia (PCP) prophylaxis is recommended for patients with CD4 counts of less than 200 cells/mm3. This study examines the proportion of patients in the TREAT Asia HIV Observational Database (TAHOD) receiving PCP prophylaxis, and its effect on PCP and mortality.
TAHOD patients with prospective follow up had data extracted for prophylaxis using co-trimoxazole, dapsone or pentamidine. The proportion of patients on prophylaxis was calculated for each calendar year since 2003 among patients with CD4 counts of less than 200 cells/mm3. The effect of prophylaxis on PCP and survival were assessed using random-effect Poisson regression models.
There were a total of 4050 patients on prospective follow up, and 90% of them were receiving combination antiretroviral therapy. Of those with CD4 counts of less than 200 cells/mm3, 58% to 72% in any given year received PCP prophylaxis, predominantly co-trimoxazole. During follow up, 62 patients developed PCP (0.5 per 100 person-years) and 169 died from all causes (1.36/100 person-years). After stratifying by site and adjusting for age, CD4 count, CDC stage and antiretroviral treatment, those without prophylaxis had no higher risk of PCP, but had a significantly higher risk of death (incident rate ratio 10.8, p < 0.001). PCP prophylaxis had greatest absolute benefit in patients with CD4 counts of less than 50 cells/mm3, lowering mortality rates from 33.5 to 6.3 per 100 person-years.
Approximately two-thirds of TAHOD patients with CD4 counts of less than 200 cells/mm3 received PCP prophylaxis. Patients without prophylaxis had significantly higher mortality, even in the era of combination ART. Although PCP may be under-diagnosed, these data suggest that prophylaxis is associated with important survival benefits.
PMCID: PMC3354658  PMID: 22281054
25.  Risk Factors for HIV-1 seroconversion among Taiwanese men visiting gay saunas who have sex with men 
BMC Infectious Diseases  2011;11:334.
Men having sex with men (MSM) accounts for 33.6% of all reported cases of HIV-1 infection in Taiwan. The aim of this study was to investigate the epidemiology of HIV-1 infection among MSM in gay saunas in Taiwan.
Patrons of 5 gay saunas were recruited for a weekly volunteer counseling and testing program from 2001 to 2005. Questionnaires were collected for a risk factor analysis. HIV-1 subtypes were determined using DNA sequencing and phylogenetic analyses.
HIV-1 prevalence rates among MSM in gay saunas in 2001 through 2005 were 3.4%, 5.1%, 8.9%, 8.5%, and 8.3%, respectively. In total, 81 of 1, 093 (7.4%) MSM had HIV-1 infection. Fifty-two HIV-1 strains were genotyped, and all of them were subtype B. HIV-seropositive men were significantly younger than the seronegatives. Only 37.1% used condoms every time during sexual intercourse. A multivariate logistic regression analysis showed that the risk factors for HIV-1 were being uncircumcised (odds ratio (OR) = 2.19; 95% confidence interval (CI), 1.08~4.45); having sexual intercourse with at least 2 partners during each sauna visit (≥ 2 vs. ≤ 1, OR = 1.71; 95% CI, 1.02~2.89); and the role played during anal intercourse (versatile vs. an exclusively insertive role, OR = 2.76; 95% CI, 1.42~5.36).
Overall, 7.4% Taiwanese MSM participating in this study had HIV-1 subtype B infection. Uncircumcised, being versatile role during anal intercourse, and having sex with more than one person during each sauna visit were main risk factors for HIV-1 infection.
PMCID: PMC3295735  PMID: 22136602

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