Hypomethylation of Thyroid Peroxidase as a Biomarker for Hepatocellular Carcinoma with Tumor Thrombosis

Objective Thyroid hormones(THs)regulate multiple physiological activities in the liver,including cellular metabolism,differentiation,and cell growth,and play important roles in the pathogenesis of hepatocellular carcinoma(HCC).Thyroid peroxidase(TPO)is a key molecule involved in the THs synthesis and signaling pathway.As an epigenetic modification,DNA methylation has a critical role in tumorigenesis with diagnostic potential.However,the connection between THs and DNA methylation has been rarely investigated.Methods The methylation of key TH-related genes was analyzed by in-house epigenome-wide scanning,and we further analyzed the methylation levels of the TPO promotor in 164 sample pairs of HCC and adjacent non-cancerous tissues by Sequenom EpiTYPER assays,and evaluated their clinical implications.Results We identified that the methylation of the TPO promoter was downregulated in the HCC tissues(P<0.0001)with a mean difference ranging from 18.5%to 22.3%.This methylation pattern correlated with several clinical factors,including a multi-satellite tumor,fibrous capsule,and the presence of tumor thrombus.The receiver operator characteristic(ROC)curve analysis further confirmed that the percent methylated reference(PMR)values for TPO were predictive of the tumor[the area under the curve(AUC)ranged from 0.755 to 0.818]and the thrombosis in the HCC patients(the AUC ranged from 0.706 to 0.777).Conclusion These findings demonstrated that epigenetic alterations of TPO,as indicated by the PMR values,were a potential biomarker for HCC patients with tumor thrombosis.

Glycosylation of recombinant human thyroid peroxidase ectodomain of insect cell origin has little effect on recognition by serum thyroid peroxidase antibody

Background Thyroid peroxidase （TPO） is an important autoantigen in Hashimoto＇s thyroiditis （HT）, and almost all epitopes are located in TPO ectodomain. The glycosylation of TPO might contribute to breaking self-tolerance, therefore, pudfied glycosylated recombinant TPO ectodomain is prerequisite of elucidating its role in the pathogenesis of HT. The aim of our study was to investigate whether the glycosylation has influence on the antigenic determinants of recombinant TPO. Methods Bac-to-Bac baculovirus expression system was used to generate recombinant human TPO ectodomain. The antigenicity was analyzed by antigen specific enzyme-linked immunosorbant assays （ELISAs）. The glycosylation of recombinant human TPO ectodomain of High Five insect cell origin was detected by lectin-ELISAs. Results TPO ectodomain was recovered from the culture media as a soluble protein, and it was fused with a hexahistidine tag which allowed purification by nickel-affinity chromatography. The recombinant TPO ectodomain could be recognized by all the 54 HT patients and three TPO monoclonal antibodies. Fucose, sialic acid and galactose were all detected on the recombinant TPO ectodomain. Sera TPOAb binding decreased slightly after non-specific deglycosylation of TPO by periodic acid. Conclusions High Five insect cells derived recombinant human TPO ectodomain had N-glycosylation sites, which might have little effect on recognition by serum TPOAb.

Thyroid peroxidase antibody positivity and triiodothyronine levels are associated with pediatric Graves'ophthalmopathy

Background:Graves'ophthalmopathy(GO)occurs commonly in children with Graves'disease(GD).However,there are limited studies on the clinical manifestations and thyroid autoantibodies in pediatric GO.The aim of this study was to investigate the prevalence and risk factors of GO in childhood GD.Methods:Clinical and biochemical data from children and adolescents with GD were retrospectively reviewed.Eighty patients under 19 years of age were included in the present study.We compared the clinical and biochemical differences between patients with and without GO.Results:Thirty-nine percent of the patients had GO,and 81%of the GO patients were females.Of these,two patients showed unilateral GO.Triiodothyronine(T3)levels were higher in GO patients than in those without GO.Anti-thyroglobulin antibody and thyroid stimulating hormone receptor antibody titers were not significantly different between the two groups.Anti-thyroid peroxidase antibody(TPO Ab)positivity was 68%in the patients with GO and only 47%in the patients without GO.In multivariate regression analysis,high T3 levels and TPO Ab positivity were related to the presence of GO.Conclusion:In children and adolescents with GD,TPO Ab positivity and high T3 levels could act as predictive factors for the presence of GO.

Molecular characterization of thyroid peroxidase gene in porcine (sus scrofa)

Thyroid peroxidase （TPO） is the key enzyme involved in thyroid hormone synthesis. Several mutations in the TPO gene may affect the normal growth and development of mammals. In this study, the TPO gene was mapped, its expression level was determined in thyroid grand at the age of 1, 20, 45, 60, 90, 120 and 150 days for Jinhua pig, the alternative splicing form was searched and the single nucleotide polymorphism （SNP, A/G642） of TPO gene was analyzed. The results showed that the porcine TPO was mapped to 3q22-27, the expres- sion level of TPO was relatively stable among the various ages and two novel transcript variants in porcine TPO gene were found： the splicing variant TPO-2 lacked exon 8, while TPO-3 lacked exon 8 and exon 14, 15, 16. Moreover, we found that the SNP of A/G642in the fourteenth exon of TPO gene was significantly associated with ham weight （P 〈 0.05）. Our results provided important basis on the regulation and metabolism of the thyroid gland in animals.

Expression and Antigen Activity Determination of Human Thyroid Peroxidase in Silkworm and Yeast

In this study,we report the expression of human thyroid peroxidase（TPO） in silkworm larvae and Pichia pastoris GS115. Recombinant TPO is sequentially purified from the hemolymph of infected silkworm larvae and yeast using a Ni-NTA resin kit. The concentration of yield of recombinant TPO is 4.87 mg per thousand larvae and 40.83 mg per liter yeast culture. However,the recombinant TPO produced in silkworm show similar binding ability with the specific anti-TPO serum to standard human TPO purified from insect cells. The lower antigen activity indicates the TPO expressed in yeast is not suitable to be used as the coating antigen in enzyme linked immunosorbent assay（ELISA）. The cost of TPO expressed in B. mori is about 1/4 that of in insect cells,and the cost of TPO purified from silkworm for ELISA is only 1/8 that of TPO produced from Sf9 cells. It indicates the Bm NPV-silkworm expression system is a cost-effectiv e platform for producing TPO with high antigen activity.

Association between serum levels of TSH and free T4 and per- and polyfluoroalkyl compounds concentrations in pregnant women

Many per-and polyfluoralkyl substances(PFASs)may disrupt maternal thyroid hormone homeostasis in pregnancy.Concerns should be raised regarding the PFASs exposure in pregnant women because thyroid hormones are involved in the early development of the fetus.In this study,we measured the concentrations of 13 PFASs,including five novel shortchain PFASs,in serum from 123 pregnant women in Beijing,China.Linear regression models were used to investigate the association between thyroid-stimulating hormone(TSH)or free thyroxine(FT4)levels and PFASs concentrations under consideration of the impacts of pregnancy-induced physiological factors.We found that perfluorobutanoic acid(PFBA)(β=0.189,95%CI=-0.039,0.417,p=0.10)and perfluorodecanoic acid(PFDA)(β=-0.554,95%CI=-1.16,0.049,p=0.071)were suggestive of significant association with TSH in thyroid peroxidase antibody(TPOAb)negative women.No association was observed between all PFASs and FT4 levels after controlling for these confounding factors,such as BMI,gestational weight gain and maternal age.These findings suggest that it should pay more attention to the association between thyroid hormone levels and short-chain PFASs concentrations.Future studies could consider a greater sample and the inclusion of other clinical indicators of thyroid function,such as free T3 and total T3.

Expression of ICAM-1,B7.1 and TPO on human thyrocytes induced by IFN-α

Objective To detect expression of intercellular adhesion molecule 1 (ICAM 1), B7.1 and thyroid peroxidase (TPO) on thyrocyte and study the possible mechanism of interferon alpha (IFN α) in the pathogenesis of autoimmune thyroid disease (AITD). Methods Thyrocytes were cultured from 6 normal persons. Antigen expression on thyrocytes induced by cytokines was examined using immunofluorescence staining with flow cytometer. Results IFN α significantly stimulated the expression of ICAM 1, B7.1 and TPO, as compared with those of control group. IFN γ markedly enhanced the expression of HLA DR and ICAM 1, but not B7.1. Prolactin (PRL) resulted in increased expression of ICAM 1, B7.1, as well as overexpression of TPO, which is more significant than that stimulated by IFN α. Conclusions Thyroid autoimmunity induced by IFN α is associated with the expression of ICAM 1, B7.1 and TPO. IFN γ could not induce the expression of B7.1, therefore it is not an initiator in AITD. In addition, we should pay more attention to PRL which possibly plays an important role in the initiation and perpetuation of postpartum thyroiditis.