HE4 Predicts Progressive Fibrosis and Cardiovascular Events in Patients With Dilated Cardiomyopathy.

Background Cardiac fibrosis plays a crucial role in the pathogenesis of dilated cardiomyopathy (DCM). HE4 (human epididymis protein 4) is a secretory protein expressed in activated fibroblasts that exacerbates tissue fibrosis. In the present study, we investigated the clinical utility of HE4 measurement in patients with DCM and its pathophysiological role in preclinical experiments in vivo and in vitro. Methods and Results We measured serum HE4 levels of 87 patients with DCM. Endomyocardial biopsy expressed severe fibrosis only in the high HE4 group (P<0.0001). Echocardiography showed that left ventricular end-diastolic diameter tends to decrease over time (58±7.3 to 51±6.6 mm; P<0.0001) in the low HE4 group (<59.65 pmol/L [median value]). HE4 was significantly associated with risk reduction of mortality and cardiovascular hospitalization in multivariate Cox model. In vivo, HE4 was highly expressed in kidney and lung tissue of mouse, and scarcely expressed in heart. In genetically induced DCM mouse model, HE4 expression increased in kidney but not in heart and lung. In vitro, supernatant from HE4-transfected human embryonic kidney 293T cells enhanced transdifferentiation of rat neonatal fibroblasts and increased expression of fibrosis-related genes, and this was accompanied by the activation of extracellular signal-regulated kinase signaling in cardiac fibroblasts. Treatment with an inhibitor of upstream signal of extracellular signal-regulated kinase or a neutralizing HE4 antibody canceled the profibrotic properties of HE4. Conclusions HE4 functions as a secretory factor, activating cardiac fibroblasts, thereby inducing cardiac interstitial fibrosis. HE4 could be a promising biomarker for assessing ongoing fibrosis and a novel therapeutic target in DCM. Registration URL: https://upload.umin.ac.jp/cgi-open-bin/ctr; Unique identifier: UMIN000043062.

Substrate-Induced Growth of Micro/Nanostructured Zn(OH)F Arrays for Highly Sensitive Microfluidic Fluorescence Assays.

To date, ZnO array-based microfluidic fluorescence assays have been widely investigated and have exhibited excellent performance in the detection of cancer biomarkers. However, the requirements of highly sensitive detection necessitate further improvement of current Zn-based fluorescence detection devices. Here, a rhombus-like Zn(OH)F array-based microfluidic fluorescence detection device is proposed. Construction of Zn(OH)F arrays on the inner wall of a microchannel is carried out via a microfluidic chemical method. A substrate-induced growth strategy for Zn(OH)F arrays is proposed, and various micro/nanostructured Zn(OH)F arrays are successfully obtained. Zn(OH)F nanorod arrays with a high aspect ratio can be constructed on the columnar ZnO nanorod arrays, and the results indicate that the fluorescence enhancement factor (EF) of the Zn(OH)F arrays toward Cy3 is approximately 4-fold that of the ZnO nanorod arrays, which can be attributed to the higher excitation light absorption and evanescent electric field. In human epididymis-specific protein 4 (HE4) detection, the limit of detection (LOD) reaches 9.3 fM, and the dynamic linear range is 10 fM to 100 pM. It has been demonstrated that Zn(OH)F nanorod array-based microfluidic devices are excellent fluorescence assay platforms that also provide a new design and construction strategy for fluorescence enhancement substrates for the detection of biomarkers.

Coupling metal-organic framework nanosphere and nanobody for boosted photoelectrochemical immunoassay of Human Epididymis Protein 4.

A "signal-on" photoelectrochemical (PEC) immunosensor for highly sensitive detection of Human Epididymis Protein 4 (HE4), a new serum biomarker of ovarian cancer with small molecular weight, was fabricated by coupling the porphyrin-based metal-organic framework (MOF) nanosphere (nPCN-224) and Nanobody (Nb). To label the Nb, the nPCN-224 with an average size of 160-200 nm was prepared by solvothermal method. The mechanism for the photocurrent generation of nPCN-224 was systematically investigated, showing that the dissolved O2 in aqueous solution participated in the charge separation and transport during the photoelectric conversion by generating O2˙-, which resulted in a 6-fold enhanced photocurrent by using ascorbic acid as the O2 ˙- scavenger. Moreover, the inherent structural porosity of nPCN-224 demonstrated advantage for reactant accessibility. Due to the superior properties of nPCN-224, and the high specificity and affinity of Nbs, the immunosensor exhibited a broad detection range from 1.00 pg mL-1 to 10.0 ng mL-1 and a detection limit of 0.560 pg mL-1, lower than most methods reported before. The immunosensor could clearly distinguish ovarian cancer patients in different stages from healthy individuals, and the as-obtained results matched well with those by traditional electrochemiluminescence immunoassay method from the hospital. This work would open a new avenue for PEC immunosensors in clinical diagnostics and evaluation of potential clinical efficacy.

Human epididymis protein 4 antigen-autoantibody complexes complement cancer antigen 125 for detecting early-stage ovarian cancer.

BACKGROUND: Early detection of ovarian cancer could significantly improve patient outcomes. Cancer antigen 125 (CA 125) is elevated in sera from approximately 60% of patients with early-stage (I/II) disease. Sensitivity might be improved through the combination of CA 125 with other biomarkers. Among potential biomarkers, antigen-autoantibody (Ag-AAb) complexes have received relatively little attention. METHODS: Luminex-based immunoassays were used to measure human epididymis protein 4 (HE4), anti-HE4 autoantibody, and HE4 Ag-AAb complexes in sera from patients with early- (n = 73) and late-stage ovarian cancers (n = 49) at the time of diagnosis and from asymptomatic women with (n = 15) or without ovarian cancer (n = 212) enrolled in the Normal Risk Ovarian Cancer Screening Study. RESULTS: At 98% specificity for healthy, asymptomatic women, 7% of patients with early-stage (I/II) ovarian cancer and 4% of patients with late-stage (III/IV) disease had elevated levels of HE4 autoantibody, whereas elevated levels of HE4 Ag-AAb complexes were detected in sera from 38% of early-stage cases and 31% of late-stage cases. Complementarity was observed in receiver operating characteristic (ROC) curves between HE4 Ag-AAb complexes and CA 125 levels in early-stage ovarian cancer (P < .001). CA 125 detected 63% of cases, and a combination of CA 125 and HE4 Ag-AAb complexes detected 81%. Complementarity was also observed in ROC curves for an independent validation set with 69 early-stage patients (P = .039). HE4 Ag-AAb complexes were detected in serial preclinical serum samples from women destined to develop ovarian cancer: they correlated with CA 125 but did not provide a lead time. CONCLUSIONS: HE4 Ag-AAb complexes could complement CA 125 in detecting a higher fraction of early-stage ovarian cancers.

Ovarian carcinomas express HE4 epitopes independently of each other.

BACKGROUND: The gene encoding HE4 undergoes alternative splicing to yield multiple protein isoforms. We investigated anti-HE4 mAbs which recognize epitopes on the C (2H5 and 3D8) or N (12A2 and 14E2) terminals. METHODS: A Luminex assay was applied to determine mAb affinity. Binding of mAbs to sections from formaline fixed ovarian carcinomas was determined by immunohistology, binding to cultured ovarian carcinoma cells was tested by flow cytometry and immunocytochemistry, and HE4 secretion to patient serum or supernatants of cultured ovarian carcinoma was assayed by ELISA. RESULTS: mAb 12A2 bound to formalin-fixed sections from 18 of 19 ovarian carcinomas, while 14E2, 2H5 and 3D8 bound to sections from 14, 7 and 4 patients, respectively. The mAbs bound independently of each other, i.e. a sample bound one mAb most strongly while another sample with similar affinity strongest bound another mAb. The intensity of binding to sections did not significantly correlate with the serum level of HE4 except for mAb 14E2, but there was a significant correlation between HE4 expression and its detection in supernatants of cultured ovarian carcinomas. CONCLUSIONS: HE4 epitopes are expressed independently of each other and their expression of HE4 by cultured ovarian carcinoma cells correlates with the release of HE4 into culture supernatants. he epitope recognized by mAb 14E2 was significantly more expressed by platinum resistant tumors. IMPACT: Expression of HE4 by most ovarian carcinomas makes it an excellent biomarker.. Further studies are needed to investigate the clinical relevance of overexpression of a particular epitope.

Ultrasensitive Electrochemical Immunoassay Based on Cargo Release from Nanosized PbS Colloidosomes.

Colloidosome is a novel nanostructure composed of millions of colloid particles. In this work, nanosized PbS colloidosomes were initially prepared and applied as nanoprobes for an ultrasensitive immunoassay. The colloidosomes were simply prepared in mild conditions by assembling the elementary approximately 8 nm PbS nanoparticles at the water-in-oil interface of emulsion droplets. To enhance the rigidity and biocompatibility of the colloidosomes, interfacial polymer was introduced by utilizing self-polymerization of dopamine. By treating with dilute nitric acid, a bursting release of lead ions from the colloidosomes occurred and the lead ions can be detected easily by anodic stripping voltammetry. In this way, a colloidosome-based electrochemical immunoassay was developed by using the nanosized PbS colloidosomes as electroactive labels. The proposed method featured a linear calibration range from 10 fg·mL-1 to 100 ng·mL-1 with a low detection limit of 3.4 fg·mL-1 for the detection of human epididymis protein 4. This work introduced a new member for the family of colloidosomes and offered a novel perspective for the rational implementation of various colloidosomes for novel low-abundance cancer biomarkers analysis.

Neutral Charged Immunosensor Platform for Protein-based Biomarker Analysis with Enhanced Sensitivity.

A noninvasive, highly sensitive universal immunosensor platform for protein-based biomarker detection is described in this Article. A neutral charged sensing environment is constructed by an antibody with an oppositely charged amino acid as surface charge neutralizer. By adjusting the pH condition of the testing environment, this neutral charged immunosensor (NCI) directly utilizes the electrostatic charges of the analyte for quantification of circulating protein markers, achieving a wide dynamic range covering through the whole picomole level. Comparing with previous studies on electrostatic charges characterization, this NCI demonstrates its capability to analyze not only the negatively charged biomolecules but also positively charged analytes. We applied this NCI for the detection of HE4 antigen with a detection limit at 2.5 pM and Tau antigen with a detection limit at 0.968 pM, demonstrating the high-sensitivity property of this platform. Furthermore, this NCI possesses a simple fabrication method (less than 2 h) and a short testing turnaround time (less than 30 min), providing an excellent potential for further clinical point-of-care applications.