Ying-Hsien Huang

High-Dose Aspirin Is Associated with Anemia and Does Not Confer Benefit to Disease Outcomes in Kawasaki Disease

Background Kawasaki disease (KD) is also known as multiple mucocutaneous lymph node syndrome of systemic vasculitis and is a leading cause of coronary artery lesions (CAL) in childhood. Intravenous immunoglobulin (IVIG) has been proven to effectively reduce the incidence of CAL, but the role and effect dose of aspirin in KD is still unclear. Moreover, overt bleeding and anemia are associated with the use of aspirin, and anemia is common in patients with KD. Thus, the aim of this study was conducted to compare the treatment efficacy, degree of anemia and inflammation, and changes in serum hepcidin in children who received a combination of high-dose aspirin and IVIG in the acute stage of KD, and those who received IVIG alone. Materials and Methods KD patients from two medical centers were retrospectively analyzed from 1999–2009. All patients were initially treated with a single dose of IVIG (2 g/kg) as the standard care of treatment. In group 1, high-dose aspirin was prescribed (> 30 mg/kg/day) until the fever subsided, and then low-dose aspirin (3–5 mg/kg/day) was prescribed until all the inflammation signs had resolved. In group 2, low-dose aspirin was prescribed without high-dose aspirin. Laboratory data were collected for analysis in both groups. Results A total of 851 KD patients (group 1, N = 305, group 2, N = 546) were enrolled in this study. There were no significant differences between group 1 and group 2 in terms of gender (p = 0.51), IVIG resistance rate (31/305 vs. 38/546, p = 0.07), CAL formation (52/305 vs. 84/546, p = 0.67), and duration of hospitalization (6.3 ± 0.2 vs. 6.7 ± 0.2 days, p = 0.13). There were also initially no significant differences in total white blood cell count, hemoglobin level, platelet count, and CRP before IVIG treatment between groups (all p>0.1). After IVIG treatment, group 1 had significantly lower levels of hemoglobin (p = 0.006) and higher CRP (p<0.001) as well as a smaller decrease in CRP level (p = 0.012). Furthermore, there was also a higher serum level of hepcidin and a delayed decrease in hepcidin level after receiving IVIG in group 1 (p = 0.04 and 0.02, respectively). Conclusions These results provide evidence demonstrating that high-dose aspirin in the acute phase of KD does not confer any benefit with regards to inflammation and it does not appear to improve treatment outcomes. Therefore, high-dose aspirin is unnecessary in acute phase KD.

Activation of Mir-29a in Activated Hepatic Stellate Cells Modulates Its Profibrogenic Phenotype through Inhibition of Histone Deacetylases 4

Background Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis and that its downregulation influences the activation of hepatic stellate cells (HSCs). In addition, inhibition of the activity of histone deacetylases 4 (HDAC4) has been shown to strongly reduce HSC activation in the context of liver fibrosis. Objectives In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs. Methods We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates to clarify the role of miR-29a in cholestatic liver fibrosis, using the bile duct-ligation (BDL) mouse model. Primary HSCs from both mice were treated with a miR-29a mimic and antisense inhibitor in order to analyze changes in profibrogenic gene expression and HSC activation using real-time quantitative RT-PCR, immunofluorescence staining, western blotting, and cell proliferation and migration assays. Results After BDL, overexpression of miR-29a decreased collagen-1α1, HDAC4 and activated HSC markers of glial fibrillary acidic protein expression in miR-29aTg mice compared to wild-type littermates. Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation. In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation. Conclusions Our results indicate that miR-29a ameliorates cholestatic liver fibrosis after BDL, at least partially, by modulating the profibrogenic phenotype of HSCs through inhibition of HDAC4 function.

Cholestasis downregulate hepcidin expression through inhibiting IL-6-induced phosphorylation of signal transducer and activator of transcription 3 signaling

Hepcidin is downregulated during progressive cholestasis in biliary atresia, but the mechanism is unknown. To verify whether downregulation of hepcidin is specific to cholestasis irrespective of the patients age, we first analyzed liver hepcidin mRNA and protein expression in adults with primary biliary cirrhosis (PBC) (n=4), non-cholestatic cirrhosis (n=19) and in controls (n=9). We evaluated the tyrosine phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) expressions in the liver sections. A rat model of cholestasis by ligation of the extrahepatic bile duct (BDL) was created, and lipopolysaccharide (LPS)-induced cholangitis in cholestatic rats 2 weeks after BDL was also established to study the modulation of hepcidin by interleukin-6 (IL-6) and STAT3 signaling pathway in these models, using real-time quantitative reverse transcription–PCR, immunohistochemistry, western blotting and enzyme-linked immunosorbent assay (ELISA). An in vitro study of the effect of bile acids on hepcidin expression was carried out to re-confirm the in vivo findings. There was significantly lower hepcidin mRNA and pSTAT3 protein expression in cholestatic cirrhosis compared with non-cholestatic cirrhosis in adults. BDL caused significant decrease in hepcidin and gp130 mRNA expression compared with sham-operated group and normal control. Furthermore, there was significantly lower pSTAT3 protein expression and nuclear translocation in the cholestatic liver from the patients and the BDL rats, which was comparable to lower liver hepcidin mRNA and plasma hepcidin expression. Furthermore, BDL for 2 weeks attenuated the upregulation of hepcidin expression induced by LPS. Hydrophobic bile acid glycochenodeoxycholate inhibited IL-6-induced pSTAT3 expression in primary hepatocytes and resulted in the downregulation of hepcidin mRNA expression. In conclusion, the study shows that cholestasis or its important component—hydrophobic bile acids—can downregulate hepcidin expression through inhibiting IL-6-induced STAT3 phosphorylation and pSTAT3 protein nuclear translocation.

Hepcidin protects against lipopolysaccharide-induced liver injury in a mouse model of obstructive jaundice

Obstructive jaundice (OJ) increases the risk of liver injury and sepsis, leading to increased mortality. Cholestatic liver injury is associated with a downregulation of hepcidin expression levels. In fact, hepcidin has an important antimicrobial effect, especially against Escherichia coli. It is unknown whether supplementing recombinant hepcidin is effective in alleviating cholestasis-induced liver injury and mortality in mice with superimposed sepsis. A mouse model of cholestasis was developed using extrahepatic bile duct ligation for 3 days. In addition, sepsis due to E. coli 0111:B4 lipopolysaccharide (LPS) was induced in the model. The serum levels of total bilirubin, AST, ALT, and LDH and the mRNA levels of IL-1β, TNF-α, and MCP-1 in the liver were significantly higher in the OJ mice receiving LPS than in the sham-operated mice receiving LPS. Compared to the OJ mice receiving LPS, the hepcidin-pretreated OJ mice receiving LPS showed a significant decrease in the above mentioned parameters, as well as a reversal in the downregulation of LC3B-II and upregulation of cleaved caspase-3; this, in turn, led to significantly decreased lethality in 24h. In conclusion, these results indicate that hepcidin pretreatment significantly reduced hepatic proinflammatory cytokine expression and liver injury, leading to reduced early lethality in OJ mice receiving LPS. Enhanced autophagy and reduced apoptosis may account for the protective effects of hepcidin.

Melatonin in the Regulation of Liver Steatosis following Prenatal Glucocorticoid Exposure

Nonalcoholic fatty liver disease patients are characterized by hepatic steatosis. Prenatal glucocorticoid overexposure can result in steatosis. In this study, we aimed to determine the mechanism and cellular apoptosis of prenatal glucocorticoid overexposure in rats and whether melatonin can rescue the prenatal glucocorticoid-induced steatosis and apoptosis in neonatal rats. Pregnant Sprague-Dawley rats at gestational days 14 to 21 were administered dexamethasone. Acute effects of prenatal programming liver were assessed at postnatal day 7. The expression of proteins involved in the apoptotic and methylation pathways was analyzed by RT-PCR and Western blotting. Apoptosis and steatosis were examined by histology staining. The liver steatosis and apoptosis were increased in prenatal glucocorticoid group more than in control group and decreased in melatonin group. The expression of leptin decreased in prenatal glucocorticoid and increased in melatonin group by liver RT-PCR and Western blot study. Caspase 3, TNF-α proteins expression, and TUNEL stains increased in prenatal glucocorticoid compared with control and decreased in melatonin group. The liver histone deacetylase, DNA methyltransferase activity, and DNA methylation were increased in prenatal glucocorticoid and decreased in melatonin group. The present study showed that the prenatal glucocorticoid induced programming liver steatosis at day 7 after delivery, possibly via altered leptin expression. Melatonin can reverse the methylation of leptin and decreased liver steatosis.

Increased Risk of Atopic Dermatitis in Preschool Children with Kawasaki Disease: A Population-Based Study in Taiwan

Kawasaki disease (KD) is an acute febrile systemic vasculitis and has been reported to be associated with allergic disease. The risk of atopic dermatitis (AD) in preschool children with KD has not been investigated. The study was to determine the longitudinal risk of the development of AD in preschool children with KD. A nationwide 5-year population-based study was performed using data from the National Health Insurance Database in Taiwan between 1999 and 2003. The risk factors for AD were compared between the 2 study groups during the follow-up period using the Cox proportional hazards model. In addition, plasma interleukin (IL)-5 levels were analyzed in normal subjects and KD patients. Among the 1440 subjects included, 21.6% developed AD during the 5-year follow-up period, of which 30.3% and 18.7% belonged to the study cohort and the comparison group, respectively. Children with KD were 1.25 times more likely to have AD than those in controls (P = 0.04). Levels of IL-5 and IgE were significantly higher in KD patients. Children with KD had a higher risk of developing AD during the 5-year follow-up period than the control group. Increased IL-5 and IgE levels may be key factors contributing to the risk of AD.

Associations of mitochondrial haplogroups b4 and e with biliary atresia and differential susceptibility to hydrophobic bile Acid.

Mitochondrial dysfunction has been implicated in the pathogenesis of biliary atresia (BA). This study aimed to determine whether a specific mitochondrial DNA haplogroup is implicated in the pathogenesis and prognosis of BA. We determined 40 mitochondrial single nucleotide polymorphisms in 15 major mitochondrial haplogroups by the use of 24-plex PCR and fluorescent beads combined with sequence-specific oligonucleotide probes in 71 patients with BA and in 200 controls in the Taiwanese population of ethnic Chinese background. The haplogroup B4 and E prevalence were significantly lower and higher respectively, in the patients with BA than in the controls (odds ratios, 0.82 [p = 0.007] and 7.36 [p = 0.032] respectively) in multivariate logistic-regression analysis. The 3-year survival rate with native liver was significantly lower in haplogroup E than the other haplogroups (P = 0.037). A cytoplasmic hybrid (cybrid) was obtained from human 143B osteosarcoma cells devoid of mtDNA (ρ0 cell) and was fused with specific mtDNA bearing E and B4 haplogroups donated by healthy Taiwanese subjects. Chenodeoxycholic acid treatment resulted in significantly lower free radical production, higher mitochondrial membrane potential, more viable cells, and fewer apoptotic cybrid B4 cells than parental 143B and cybrid E cells. Bile acid treatment resulted in a significantly greater protective mitochondrial reaction with significantly higher mitochondrial DNA copy number and mitofusin 1 and 2 concentrations in cybrid B4 and parental cells than in cybrid E cells. The results of the study suggested that the specific mitochondrial DNA haplogroups B4 and E were not only associated with lower and higher prevalence of BA respectively, in the study population, but also with differential susceptibility to hydrophobic bile acid in the cybrid harboring different haplogroups.

Alterations in NADPH oxidase expression and blood–brain barrier in bile duct ligation-treated young rats: Effects of melatonin

Bile duct ligation (BDL)-treated rats exhibit cholestasis and increased systemic and brain oxidative stress. Activation of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and disruption of the blood-brain barrier (BBB) are implicated as the pathogenetic mechanisms of brain dysfunction in BDL-treated adult rats. Young rats underwent sham ligation or BDL at day 17 for 2 or 4weeks. Treatment group rats were administered melatonin between day 17 and 45. We found a progressive increase in prefrontal cortex NADPH-dependent superoxide anion (O(2)(-)) production and increased expression of NADPH oxidase subunits in either the prefrontal cortex or the hippocampus in BDL-treated young rats. In addition, expression of intercellular adhesion molecule-1 (ICAM) and tissue plasminogen activator (t-PA) genes were increased in either the prefrontal cortex or the hippocampus. Prefrontal cortex capillary junctional complex proteins expressions including occludin, claudin-5, platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin were not altered. Melatonin lowered the prefrontal cortex NADPH-dependent O(2)(-) production and t-PA gene expression. We conclude that alterations in NADPH oxidase expression and BBB are involved in brain dysfunction in BDL-treated young rats. In addition, melatonin regulates NADPH oxidase activity and t-PA gene expression.

Gamma-Glutamyl Transferase in the Diagnosis of Biliary Atresia

BACKGROUND Early diagnosis of biliary atresia (BA) is important because the prognosis is closely related to timing of Kasai operation. The aim of this study was to test the clinical application of serum gamma-glutamyl transferase (GGT) concentration and the ratio of serum GGT to aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in differentiating BA from neonatal hepatitis (NH). METHODS Ninety-three (46 male and 47 female) cases of BA and 65 (45 male and 20 female) NH were included in this study. Serum concentrations of GGT, AST, and ALT were measured in all cholestatic infants. The results of peak GGT level, GGT/AST ratio, GGT/ALT ratio were compared between groups. RESULTS The serum GGT levels were significantly higher in BA patients than those in NH patients (353.3 +/- 334.4 IU/L vs. 114.8 +/- 86 IU/L, P < 0.001). GGT/AST values were over 2 in 55/68 BA and 15/ 54 NH (OR = 11.0, 95% CI 4.7-25.7, P < 0.001). GGT/ALT values were over 2 in 54/65 BA and 19/50, NH respectively (OR = 8.0, 95% CI 3.4-19.0, P < 0.001). A GGT level greater than 300 IU/L had a sensitivity of 39.7% in the diagnosis of BA, GGT/AST over 2 was 80.9% and GGT/ALT over 2 was 83.1%; the specificities were 98.1%, 72.2% and 62.0%, respectively. The respective accuracies of the diagnosis of BA were 65.6%, 77.1% and 73.9%. CONCLUSIONS GGT/AST ratio over 2 indicates high possibility of biliary atresia and should prompt further investigations to confirm the diagnosis.

Genome-Wide Association Study Identifies Novel Susceptibility Genes Associated with Coronary Artery Aneurysm Formation in Kawasaki Disease

Kawasaki disease (KD) or Kawasaki syndrome is known as a vasculitis of small to medium-sized vessels, and coronary arteries are predominantly involved in childhood. Generally, 20–25% of untreated with IVIG and 3–5% of treated KD patients have been developed coronary artery lesions (CALs), such as dilatation and aneurysm. Understanding how coronary artery aneurysms (CAAs) are established and maintained in KD patients is therefore of great importance. Upon our previous genotyping data of 157 valid KD subjects, a genome-wide association study (GWAS) has been conducted among 11 (7%) CAA-developed KD patients to reveal five significant genetic variants passed pre-defined thresholds and resulted in two novel susceptibility protein-coding genes, which are NEBL (rs16921209 (P = 7.44 × 10−9; OR = 32.22) and rs7922552 (P = 8.43 × 10−9; OR = 32.0)) and TUBA3C (rs17076896 (P = 8.04 × 10−9; OR = 21.03)). Their known functions have been reported to associate with cardiac muscle and tubulin, respectively. As a result, this might imply their putative roles of establishing CAAs during KD progression. Additionally, various model analyses have been utilized to determine dominant and recessive inheritance patterns of identified susceptibility mutations. Finally, all susceptibility genes hit by significant genetic variants were further investigated and the top three representative gene-ontology (GO) clusters were regulation of cell projection organization, neuron recognition, and peptidyl-threonine phosphorylation. Our results help to depict the potential routes of the pathogenesis of CAAs in KD patients and will facilitate researchers to improve the diagnosis and prognosis of KD in personalized medicine.