Browsing by Author "Huang, Chung-Hao"

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Cyclooxygenase-2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents
(2017-03-20)
Lin, Chun-Kuang
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Tseng, Chin-Kai
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Wu, Yu-Hsuan
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Liaw, Chih-Chuang
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Lin, Chun-Yu
Cyclooxygenase-2 (COX-2) is one of the important mediators of inflammation in response to viral infection, and it contributes to viral replication, for example, cytomegalovirus or hepatitis C virus replication. The role of COX-2 in dengue virus (DENV) replication remains unclear. In the present study, we observed an increased level of COX-2 in patients with dengue fever compared with healthy donors. Consistent with the clinical data, an elevated level of COX-2 expression was also observed in DENV-infected ICR suckling mice. Using cell-based experiments, we revealed that DENV-2 infection significantly induced COX-2 expression and prostaglandin E-2 (PGE(2)) production in human hepatoma Huh-7 cells. The exogenous expression of COX-2 or PGE2 treatment dose-dependently enhanced DENV-2 replication. In contrast, COX-2 gene silencing and catalytic inhibition sufficiently suppressed DENV-2 replication. In an ICR suckling mouse model, we identified that the COX-2 inhibitor NS398 protected mice from succumbing to life-threatening DENV-2 infection. By using COX-2 promoter-based analysis and specific inhibitors against signaling molecules, we identified that NF-kappa B and MAPK/JNK are critical factors for DENV-2-induced COX-2 expression and viral replication. Altogether, our results reveal that COX-2 is an important factor for DENV replication and can serve as a potential target for developing therapeutic agents against DENV infection.
Differences in Mortality and Clinical Manifestations of Dengue Hemorrhagic Fever in Taiwan in Different Years: A Comparison for Cases in 2014 and 2015 Epidemics
(2017-01-01)
Chang, Ko
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Huang, Chung-Hao
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Lee, Ing-Kit
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Lu, Po-Liang
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Lin, Chun-Yu
People in southern Taiwan experienced two major dengue outbreaks in 2014 and 2015. The mortality and clinical features were very different between these 2 years. Dengue virus serotype 1 (DENV-1) caused epidemic outbreak in 2014 and DENV-2 was predominant in 2015. The characteristics of dengue hemorrhagic fever (DHF) cases in the 2 years was analyzed. We conducted a retrospective chart review to analyze the clinical and laboratory features of 206 adult patients with DHF in southern Taiwan in 2014 and 2015. The mortality rate of DHF cases in 2015 was higher than that of cases in 2014 (38.7% versus 12.4%, P < 0.0001). Compared with cases in 2014, DHF cases in 2015 had more complications, such as gastrointestinal bleeding (78.5% versus 61.9%, P = 0.01), severe hepatitis (30.1% versus 8%, P < 0.0001), and myocarditis (14% versus 0.9%, P < 0.0001). Among the mortality cases, diabetes, chronic renal failure, proton-pump inhibitors using, platelet transfusion, and Charlson comorbidity index score (Charlson score) were also higher in 2015. Multivariate analysis for the mortality cases revealed that the risk factors were Charlson score (3) 5 (P = 0.02, odds ratio [OR] = 4.07, 95% confidence interval [CI] = 1.244-13.307), severe hepatitis (P < 0.0001, OR = 11.97, 95% CI = 3.831-37.396), and acute renal failure (P < 0.0001, OR = 98.76, 95% CI = 10.847-899.22). DHF cases in 2015 had higher mortality and more complications, such as gastrointestinal bleeding, severe hepatitis, and myocarditis, than in 2014 in southern Taiwan. In the 2-year DHF case series, Charlson score >= 5, severe hepatitis, and acute renal failure were independent significant variables for mortality.
Establishment and Comparison of Two Different Diagnostic Platforms for Detection of DENV1 NS1 Protein
(2015-11-01)
Tang, Yin-Liang
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Chiu, Chien-Yu
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Lin, Chun-Yu
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Huang, Chung-Hao
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Chen, Yen-Hsu
Dengue virus (DENV) infection is currently at pandemic levels, with populations in tropical and subtropical regions at greatest risk of infection. Early diagnosis and management remain the cornerstone for good clinical outcomes, thus efficient and accurate diagnostic technology in the early stage of the disease is urgently needed. Serotype-specific monoclonal antibodies (mAbs) against the DENV1 nonstructural protein 1 (NS1), DA12-4, DA13-2, and DA15-3, which were recently generated using the hybridoma technique, are suitable for use in diagnostic platforms. Immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and Western blot analysis further confirmed the serotype specificity of these three monoclonal antibodies. The ELISA-based diagnostic platform was established using the combination of two highly sensitive mAbs (DA15-3 and DB20-6). The same combination was also used for the flow cytometry-based diagnostic platform. We report here the detection limits of flow cytometry-based and ELISA-based diagnostic platforms using these mAbs to be 0.1 and 1 ng/mL, respectively. The collected clinical patient serum samples were also assayed by these two serotyping diagnostic platforms. The sensitivity and specificity for detecting NS1 protein of DENV1 are 90% and 96%, respectively. The accuracy of our platform for testing clinical samples is more advanced than that of the two commercial NS1 diagnostic platforms. In conclusion, our platforms are suitable for the early detection of NS1 protein in DENV1 infected patients.
Generation and Characterization of Antinonstructural Protein 1 Monoclonal Antibodies and Development of Diagnostics for Dengue Virus Serotype 2
(2017-01-01)
Tang, Yin-Liang
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Liu, I-Ju
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Li, Pi-Chun
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Chiu, Chien-Yu
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Lin, Chun-Yu
Dengue virus (DENV) circulates in tropical and subtropical areas around the world, where it causes high morbidity and mortality. There is no effective treatment of infection, with supportive care being the only option. Furthermore, early detection and diagnosis are important to facilitate clinical decisions. In this study, seven monoclonal antibodies (mAbs) recognizing nonstructural protein 1 (NS1) of DENV were generated by hybridoma techniques. These antibodies can be divided into two groups: serotype-specific (DB6-1, DB12-3, and DB38-1) and nonspecific (consisting of antibodies DB16-1, DB20-6, DB29-1, and DB41-2). The B-cell epitopes of DB20-6 and DB29-1 were identified by phage display and site-directed mutagenesis, and its binding motif, WXXWGK, was revealed to correspond to amino acid residues 115-120 of the DENV-2 NS1 protein. A diagnostic platform, consisting of a serotype-specific capture antibody and a complex detection antibody, exhibited a detection limit of about 1 ng/mL, which is sufficient to detect NS1 in clinical serum samples from dengue patients. This diagnostic platform displayed better specificity and sensitivity than two examined commercial NS1 diagnostic platforms. In summary, our results indicate that these newly generated mAbs are suitable for detection of NS1 protein of DENV-2 in clinical samples.
High levels of serum hyaluronan is an early predictor of dengue warning signs and perturbs vascular integrity
(2019-10-01)
Lin, Chun-Yu
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Kolliopoulos, Constantinos
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Huang, Chung-Hao
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Tenhunen, Jyrki
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Heldin, Carl-Henrik
Background: A main pathological feature of severe dengue virus infection is endothelial hyper-permeability. The dengue virus nonstructural protein 1 (NS1) has been implicated in the vascular leakage that characterizes severe dengue virus infection, however, the molecular mechanisms involved are not known. Methods: A cohort of 250 dengue patients has been followed from the onset of symptoms to the recovery phase. Set urn hyaluronan levels and several other clinical parameters were recorded. The effect of NS1 treatment of cultured fibroblasts and endothelial cells on the expressions of hyaluronan synthetic and catabolic enzymes and the hyaluronan receptor CD44, were determined, as have the effects on the formation of hyaluronan-rich matrices and endothelial permeability. Findings: Elevated serum hyaluronan levels (70 ng/ml) during early infection was found to be an independent predictor for occurrence of warning signs, and thus severe dengue fever. High circulating levels of the viral protein NS1, indicative of disease severity, correlated with high concentrations of serum hyaluronan. NS1 exposure decreased the expression of CD44 in differentiating endothelial cells impairing the integrity of vessel-like structures, and promoted the synthesis of hyaluronan in dermal fibroblasts and endothelial cells in synergy with dengue-induced pro-inflammatory mediators. Deposited hyaluronan-rich matrices around cells cultured in vitro recruited CD44-expressing macrophage-like cells, suggesting a mechanism for enhancement of inflammation. In cultured endothelial cells, perturbed hyaluronan-CD44 interactions enhanced endothelial permeability through modulation of VE-cadherin and cytoskeleton re-organization, and exacerbated the NS1-induced disruption of endothelial integrity. Interpretation: Pharmacological targeting of hyaluronan biosynthesis and/or its CD44-mediated signaling may limit the life-threatening vascular leakiness during moderate-to-severe dengue virus infection. (C) 2019 Published by Elsevier B.V.
IL-28 and IL-29 as protective markers in subject with dengue fever
(2017-06-01)
Hung, Chih-Hsing
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Huang, Chung-Hao
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Wang, Lin
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Huang, Chun-Chi
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Wu, Meng-Chieh
About 400 million people every year are estimated to contract dengue virus infection, which causes prolonged morbidity and sometimes mortality. Interleukin (IL)-28 and IL-29 are relatively newly discovered cytokines and play an important role in our immune defense against pathogens, especially for viral infection. In the present study, we investigated serum IL-28 and IL-29 expression and the relationship to clinical and laboratory parameters in patients with dengue virus infection. Adult patients with dengue (n = 45) and control group (n = 24) were included prospectively. Clinical symptoms and laboratory data were collected from every patient. We investigated IL-28 and IL-29 levels in serum by ELISA. The concentrations of serum IL-28 and IL-29 were significantly higher in subjects with dengue when compared to those of control group. The patients with higher serum IL-28 and IL-29 levels had significantly lower ALAT and peripheral blood neutrophil percentage, but higher peripheral platelet, total white blood cell (WBC), monocyte, and lymphocyte counts. Patients with higher serum IL-28 and IL-29 levels also had more flu-like symptoms, but less vomiting. Increased level of IL-28 and IL-29 was associated with better liver function, platelet and WBC numbers and clinical symptom in subjects with dengue and could potentially serve as a protective marker.
Microbiology of peritoneal dialysis-related infection and factors of refractory peritoneal dialysis related peritonitis: A ten-year single-center study in Taiwan
(2019-10-01)
Wang, Hsin-Hui
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Huang, Chung-Hao
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Kuo, Mei-Chuan
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Lin, Shang-Yi
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Hsu, Chin-Huei
Background: Peritoneal dialysis (PD)-related infection is a serious complication of patients with PD. Refractory peritonitis may lead to failure of PD, shift to hemodialysis (HD) or death. Besides, microbiologic resistance increased worldwide that might impact the treatment choice for such infections. Investigating the causative pathogens and risk factors of PD-related infections in Taiwan was warranted. Methods: This is a retrospective study involving patients with PD from 2007 to 2016 in a southern Taiwan hospital. Patient characteristics, microbiological data, outcomes, and factors associated with refractory peritonitis were analyzed. Results: There were 190 episodes of PD-related peritonitis in 110 patients from this cohort. Gram-positive organisms were the leading cause of PD-related peritonitis, but gram-negative organisms, esp. Pseudomonas aeruginosa, were predominant for exit site infection and tunnel infection. The incidence of peritonitis was 0.25 episode per patient-year (1 episode per 47.69 months). The refractory rate was 14.2% (27/190). Methicillin resistance was noted in 2 (13.3%) of 15 Staphylococcus aureus isolates. Of 114 isolates, 72.8% (83) were susceptible to either cefazolin or gentamicin. Staphylococcus spp. and Escherichia coli infections were significantly associated with refractory peritonitis. Baseline hyponatremia (<130 mmol/L) was independently associated with refractory peritonitis. Conclusion: Gram-positive organisms remained major cause of PD-related peritonitis. About three quarters of causative pathogens were susceptible to the recommended empirical treatment for PD-related peritonitis. Baseline hyponatremia (<130 mmol/L) was independently associated with refractory peritonitis. Staphylococcus spp. and E. coli infections had important roles for refractory peritonitis. Copyright (C) 2019, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC.
Prognostic Factors in Adult Patients with Dengue: Developing Risk Scoring Models and Emphasizing Factors Associated with Death <= 7 Days after Illness Onset and <= 3 Days after Presentation
(2018-11-01)
Lee, Ing-Kit
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Huang, Chung-Hao
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Huang, Wen-Chi
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Chen, Yi-Chun
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Tsai, Ching-Yen
Dengue is a mosquito-borne viral disease that is a threat to global health. However, information relating to mortality <= 7 days after dengue onset and <= 3 days after presentation is limited. We retrospectively analyzed 1086 adults with dengue during a 12-year period. Three scoring models were established: model-1 (death <= 3 days after presentation), model-2 (death <= 7 days after illness onset), and model-3 (overall fatality). In total, 39 patients with fatal dengue were identified, of which 17 and 14 patients died <= 7 days after illness onset and <= 3 days after presentation, respectively. In model-1 (range: 0-4 points), gastrointestinal bleeding <= 72 h after presentation, thrombocytopenia (<50 x 10(9) cells/L) at presentation, and acute kidney injury after hospitalization, using a cutoff level of 2 points, exhibited good discrimination (area under the receiver curve (AUC): 0.975) between survivors and non-survivors. In model-2, the significant predictors were gastrointestinal bleeding <= 72 h after presentation, and hemoconcentration and leukocytosis after hospitalization. Model-2 (range: 0-4 points) showed an AUC of 0.974, with a cutoff value of 2 points. The independent factors in model-2 were the predictors of overall mortality (model-3), which include thrombocytopenia (<50 x 10(9) cells/L) at presentation. Using a cutoff value of 2 points, model-3 (range: 0-7 points) revealed an excellent discrimination between survivors and non-survivors (AUC: 0.963).
Prostasin Impairs Epithelial Growth Factor Receptor Activation to Suppress Dengue Virus Propagation
(2019-05-01)
Lin, Chun-Kuang
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Tseng, Chin-Kai
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Wu, Yu-Hsuan
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Lin, Chun-Yu
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Huang, Chung-Hao
Background. Dengue virus (DENV), a common and widely spread arbovirus, causes life-threatening diseases, such as dengue hemorrhagic fever or dengue shock syndrome. There is currently no effective therapeutic or preventive treatment for DENV infection. Methods. Next-generation sequencing analysis revealed that prostasin expression was decreased upon DENV infection. Prostasin expression levels were confirmed by real-time quantitative polymerase chain reaction in patients with dengue fever and a DENV-infected mice model. Short hairpin RNA against EGFR and LY294002 were used to investigate the molecular mechanism. Results. Based on clinical studies, we first found relatively low expression of prostasin, a glycosylphosphatidyl inositol-anchored membrane protease, in blood samples from patients with dengue fever compared with healthy individuals and a high correlation of prostasin expression and DENV-2 RNA copy number. DENV infection significantly decreased prostasin RNA levels of in vivo and in vitro models. By contrast, exogenous expression of prostasin could protect ICR suckling mice from life-threatening DENV-2 infection. Mechanistic studies showed that inhibition of DENV propagation by prostasin was due to reducing expression of epithelial growth factor receptor, leading to suppression of the Akt/NF-kappa B-mediated cyclooxygenase-2 signaling pathway. Conclusion. Our results demonstrate that prostasin expression is a noteworthy clinical feature and a potential therapeutic target against DENV infection.
Recent developments in antibiotic agents for the treatment of complicated intra-abdominal infections
(2016-02-11)
Lin, Shang-Yi
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Huang, Chung-Hao
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Ko, Wen-Chien
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Chen, Yen-Hsu
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Hsueh, Po-Ren
Introduction: Treatment of complicated intra-abdominal infections (cIAIs) is becoming increasingly difficult because of the widespread emergence of multidrug-resistant organisms.Areas covered: In this review, we discuss the effectiveness of several new antibiotics for the treatment of cIAIs, including new -lactamase inhibitor combinations (BLICs) and tetracycline-class drugs, recently developed aminoglycosides and quinolones, and novel lipoglycopeptides and oxazolidinones.Expert opinion: Of the new BLICs, ceftolozane/tazobactam is associated with adequate clinical cure rates in patients with cIAIs. Currently, two new -lactamase inhibitors, namely avibactam and MK-7655, are under development for clinical use in the treatment of cIAIs. Eravacycline, a novel, fully synthetic tetracycline-class drug, has been shown in Phase II and III clinical trials to be more potent than tigecycline against a significant number of multidrug-resistant organisms causing cIAIs. Plazomicin, a next-generation aminoglycoside, is a promising agent for treatment of cIAIs due to multidrug-resistant pathogens. Of the recently developed quinolones, delafloxacin and finafloxacin have been shown to be effective against pathogens that survive and multiply in mildly acidic environments, although further clinical studies examining their clinical utility in the treatment of cIAIs are warranted. Oritavancin, a new semisynthetic lipoglycopeptide agent, has been demonstrated to be a potent antibiotic in the treatment of cIAIs due to drug-resistant Gram-positive organisms. Several other new antibiotics in development also show promise and will hopefully broaden the possibilities for treatment of complicated intra-abdominal infections due to MDR pathogens.
Role of rifampin for the treatment of bacterial infections other than mycobacteriosis
(2017-11-01)
Lee, Chun-Yuan
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Huang, Chung-Hao
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Lu, Po-Liang
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Ko, Wen-Chien
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Chen, Yen-Hsu
Objectives: Rifampin was initially approved for the treatment of tuberculosis. Because of its low toxicity, broad-spectrum activity, and good bioavailability, rifampin is now commonly administered as combination antimicrobial therapy for the treatment of various infections caused by organisms other than mycobacteria. This review summarizes the most recent clinical studies on the use of rifampin combinations for treating four common non-mycobacterial infections: acute bacterial meningitis, infective endocarditis and bacteraemia, pneumonia, and biofilm-related infections. Methods: We performed a literature search of clinical studies published in English from January 2005 to June 2016 using the PubMed database with the search terms "rifampin" with "meningitis" or "infective endocarditis and bacteraemia" or "pneumonia" or "prosthetic joint infections. Results: Current evidence to support a rifampin combination therapy as a treatment for non-mycobacterial infections was largely based on in vitro/in vivo studies and non-comparable retrospective case series. Additionally, controlled clinical trials that directly compared outcomes resulting from rifampin treatment versus treatment without rifampin were limited. Conclusions: Rifampin combination therapy appears promising for the treatment of non-mycobacterial infections. However, further definitive clinical trials are necessary to validate its use because the risk of adverse drug-drug interactions and of the emergence of rifampin resistance during treatment may outweigh the potential benefits. (C) 2017 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Serum Galectin-9 and Galectin-3-Binding Protein in Acute Dengue Virus Infection
(2016-06-01)
Liu, Kuan-Ting
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Liu, Yao-Hua
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Chen, Yen-Hsu
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Lin, Chun-Yu
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Huang, Chung-Hao
Dengue fever is a serious threat for public health and induces various inflammatory cytokines and mediators, including galectins and glycoproteins. Diverse immune responses and immunological pathways are induced in different phases of dengue fever progression. However, the status of serum galectins and glycoproteins is not fully determined. The aim of this study was to investigate the serum concentration and potential interaction of soluble galectin-1, galectin-3, galectin-9, galectin-3 binding protein (galectin-3BP), glycoprotein 130 (gp130), and E-, L-, and P-selectin in patients with dengue fever in acute febrile phase. In this study, 317 febrile patients (187 dengue patients, 150 non-dengue patients that included 48 patients with bacterial infection and 102 patients with other febrile illness) who presented to the emergency department and 20 healthy controls were enrolled. Our results showed the levels of galectin-9 and galectin-3BP were significantly higher in dengue patients than those in healthy controls. Lower serum levels of galectin-1, galectin-3, and E-, L-, and P-selectin in dengue patients were detected compared to bacteria-infected patients, but not to healthy controls. In addition, strong correlation between galectin-9 and galectin-3BP was observed in dengue patients. In summary, our study suggested galectin-9 and galectin-3BP might be critical inflammatory mediators in acute dengue virus infection.
Shared IgG Infection Signatures vs. Hemorrhage-Restricted IgA Clusters in Human Dengue: A Phenotype of Differential Class-Switch via TGF beta 1
(2017-12-04)
Huang, Chung-Hao
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Chang, Ya-Hui
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Lin, Chun-Yu
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Wang, Wen-Hung
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Kuan, Hui-Chung
Phenotypic manifestations of infectious diseases are closely related to individual immune responses. Methods to extract information from patients' own immune reactions would be of great use for both diagnosis and treatment. Dengue fever is one of the diseases that clinical aggravations could occur paradoxically after humoral immunity appears. This property makes dengue fever an excellent disease model to explore. A principal component analyses (PCAs)-based framework derived from a prior vaccination study was developed. The framework was verified by successful demonstrations of known IgG signatures from a Mexico Dengue data set. Afterward the pipeline was tested upon de novo IgG and IgA libraries of Dengue patients from southern Taiwan. We discovered four infection signatures within IgG repertoires, two of which were identical to previous reports. However, it was IgA but not IgG that could differentiate hemorrhagic from non-hemorrhagic patients. IgA repertoires were found more diversified among bleeders, from whom seven signature clusters were characterized. The expressions of transforming growth factor beta 1 (TGF beta 1) and accordingly mediated class-switch activity of IgA were distinct only among the PCA-segregated bleeding group. In sum, intercontinental sharing of IgG signatures in dengue fever was demonstrated via a unified working flow. Differential regulation of IgA class-switch with associated diversity expansion plus existences of hemorrhage-restricted clusters were shown. The ability of the framework to find common IgG signatures would implicate applications to infections even from unknown pathogens. The clusters within IgA repertoires could offer perspectives to other IgA-related bleeding disorders such as Henoch-Schonlein purpura or IgA nephropathy. Substantiated grounds for IgA-specific effector function via TGF beta 1-mediated class-switch would be a new factor to consider for infectious diseases.
Suppressed humoral immunity is associated with dengue nonstructural protein NS1-elicited anti-death receptor antibody fractions in mice
(2020-04-14)
Tsai, Chung-Lin
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Sun, Der-Shan
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Su, Mei-Tzu
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Lien, Te-Sheng
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Chen, Yen-Hsu
Dengue virus (DENV) infections may cause life-threatening dengue hemorrhagic fever (DHF). Suppressed protective immunity was shown in these patients. Although several hypotheses have been formulated, the mechanism of DENV-induced immunosuppression remains unclear. Previously, we found that cross-reactive antibodies against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 1 (death receptor 4 [DR4]) were elicited in DHF patients, and that anti-DR4 autoantibody fractions were elicited by nonstructural protein 1 (NS1) immunizations in experimental mice. In this study, we found that anti-DR4 antibodies could suppress B lymphocyte function in vitro and in vivo. Treatment with the anti-DR4 immunoglobulin (Ig) induced caspase-dependent cell death in immortalized B lymphocyte Raji cells in vitro. Anti-DR4 Igs elicited by NS1 and DR4 immunizations markedly suppressed mouse spleen transitional T2 B (IgM(+)IgD(+)), bone marrow pre-pro-B (B220(+)CD43(+)), pre-B (B220(+)CD43(-)), and mature B cell (B220(+)IgD(+)) subsets in mice. Furthermore, functional analysis revealed that the pre-elicitation of anti-NS1 and anti-DR4 Ig titers suppressed subsequently neutralizing antibody production by immunization with DENV envelop protein. Our data suggest that the elicitation of anti-DR4 titers through DENV NS1 immunization plays a suppressive role in humoral immunity in mice.