Branched DNA Signal Amplification Research Articles

Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis

Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of EV inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed, and high-throughput assays. We captured four subpopulations of EVs from colorectal cancer (CRC) cell lines HT29 and SW403 based on EpCAM, CD9, CD63, and CD81 expression, and quantified the co-expression of eight outer [integrins (ITGs) and tetraspanins] and four inner (heat shock, endosomal, and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

Identification of Circulating Tumor Cell Phenotype in Differentiated Thyroid Carcinoma

Objective: Circulating tumor cells (CTCs) have been considered as the origin of tumor metastasis and recurrence, which always indicate a poor prognosis. There are three phenotypes of CTCs according on different epithelial-to-mesenchymal transition (EMT) markers, including epithelial, mesenchymal, and epithelial/mesenchymal (mixed phenotypic) CTCs. We intended to explore the relationship among CTC phenotypes and the clinicopathological characteristics of patients with differentiated thyroid carcinoma (DTC). Methods: Peripheral blood samples from 58 patients with DTC were collected, and CTCs were isolated by cell sizes. To identify phenotypes of CTCs, branched DNA signal amplification technology was adopted to capture and amplify target sequences, and then multiplex RNA-in situ hybridization (RNA-ISH) assay was used to identify CTC phenotypes depended on epithelial-mesenchymal transition (EMT) markers. Results: The positive rate of CTCs was 77.59% in 58 DTC patients. Totally, 488 CTCs with detective phenotype were found. Among them, there were 121 (24.80%) epithelial CTCs, 67 (13.72%) mesenchymal CTCs, and 300 (61.48%) mixed phenotypic CTCs. An obvious increased epithelial CTCs was observed in male patients compared with female. Notably, CTCs were more prevailing in younger male patients with ETI and bilateral focus. Conclusions: The CTCs are common in DTC patients, and mixed phenotypic is the major phenotype, indicating that EMT is prevalent in DTC even though its prognosis was better than other epithelial tumors. Detection of CTC and its phenotypes might independently predict the prognosis of DTC.

Niche formed by bone morphogenetic protein antagonists gremlin 1 and gremlin 2 in human hair follicles.

Niche formed by bone morphogenetic protein antagonists gremlin 1 and gremlin 2 in human hair follicles.

Abstract PD10-04: Prognostic potential of mesenchymal circulating tumor cell-associated white blood cell clusters in patients with HER2-negative metastatic breast cancer

Abstract Background:Circulating tumor cells (CTCs), potentially involved in the metastatic cascade, may have direct interactions with immune cells during cancer dissemination, and correlate with the inflammatory state in the target organ of metastatic extravasation. Although positive CTC status has been validated as a prognostic marker in breast cancer, the interaction between immune cells and CTCs during the progress of Epithelial-mesenchymal transition (EMT), and the clinical implications of CTC-associated white blood cell clusters (CTC-WBC clusters) for metastatic breast cancer are largely uncharacterized. We investigated the predictive value of different phenotypes of CTC-WBC clusters as a prognostic biomarker in a prospective study for metastatic breast cancer. Methods:The prospective study enrolled 134 patients with HER2-negative metastatic breast cancer who underwent docetaxel plus capecitabine as first-line chemotherapy and subsequently used capecitabine as maintenance therapy from Nov 2013 to Sep 2017. Serial peripheral blood samples were collected dynamically until disease progression for CTC detection using CanPatrol CTC enrichment technique. CTCs were isolated using a filter-based method combined with an RNA in situ hybridization method based on the branched DNA signal amplification technology according to EMT markers. Follow-up data collection was conducted until May 2019 and data analysis was performed in June 2019. Kaplan-Meier analysis and Cox proportional hazards regression analysis were performed to investigate the prognostic value of CTC-WBC clusters. Results:Median age of the 134 female patients was 51.0 years (range,21 to 73 years), and 119 (88.8%) of them were estrogen receptor-and/or progesterone receptor-positive. Median progression-free survival (PFS) was 9.9 months (95% CI, 8.1 to 11.6 months). 116 (86.6%) of the 134 patients had detectable CTCs at baseline with a mean number of 8 CTCs per 5 ml of blood, including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs and mesenchymal CTCs. Among them, CTC-WBC clusters were detectable in 9 patients, of whom 5 patients had mesenchymal CTCs contained in the CTC-WBC clusters. The patients with at least one detectable mesenchymal CTC-WBC clusters were characterized by significantly worse PFS, when compared with the patients with no mesenchymal CTC-WBC clusters (10.2 months vs 6.3 months, P=0.046), as well as the patients with at least one CTC per 5 ml blood (10.7 months vs 6.3 months, P=0.040). However, the presence of total CTC-WBC cluster regardless of CTC phenotypes was correlated with no significant survival difference in the cohort (10.0 months vs 8.6 months, P=0.913). Conclusions: Our data provide evidence that the emergence of mesenchymal CTC-WBC clusters before treatment is associated with significantly poorer PFS in HER2-negative metastatic breast cancer patients underwent first-line chemotherapy, which may be used as a parameter to predict the clinical outcomes for chemotherapy. This study validated the prognostic value of CTC-WBC cluster underwent mesenchymal transformation in a prospective cohort constituted by relatively homogeneous participants who were all HER2-negative metastatic breast cancer underwent docetaxel plus capecitabine as first-line chemotherapy. The results could provide evidence for mesenchymal CTC-WBC cluster to serve as a potential biomarker for individual therapy. However, limited sample size influenced the statistical power to draw firm conclusions. Thus, further large-scale external validation containing data of overall survival is warranted. Citation Format: Xiuwen Guan, Chunxiao Li, Xiaoying Sun, Lixi Li, Zongbi Yi, Binliang Liu, Jiasen Xu, Binghe Xu, Fei Ma. Prognostic potential of mesenchymal circulating tumor cell-associated white blood cell clusters in patients with HER2-negative metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD10-04.

Novel Assay to Detect RNA Polymerase I Activity In Vivo.

This report develops an analytically validated chromogenic in situ hybridization (CISH) assay using branched DNA signal amplification (RNAscope) for detecting the expression of the 5' external transcribed spacer (ETS) of the 45S ribosomal (r) RNA precursor in formalin-fixed and paraffin-embedded (FFPE) human tissues. 5'ETS/45S CISH was performed on standard clinical specimens and tissue microarrays (TMA) from untreated prostate carcinomas, high-grade prostatic intraepithelial neoplasia (PIN), and matched benign prostatic tissues. Signals were quantified using image analysis software. The 5'ETS rRNA signal was restricted to the nucleolus. The signal was markedly attenuated in cell lines and in prostate tissue slices after pharmacologic inhibition of RNA polymerase I (Pol I) using BMH-21 or actinomycin D, and by RNAi depletion of Pol I, demonstrating validity as a measure of Pol I activity. Clinical human prostate FFPE tissue sections and TMAs showed a marked increase in the signal in the presumptive precursor lesion (high-grade PIN) and invasive adenocarcinoma lesions (P = 0.0001 and P = 0.0001, respectively) compared with non-neoplastic luminal epithelium. The increase in 5'ETS rRNA signal was present throughout all Gleason scores and pathologic stages at radical prostatectomy, with no marked difference among these. This precursor rRNA assay has potential utility for detection of increased rRNA production in various tumor types and as a novel companion diagnostic for clinical trials involving Pol I inhibition.Implications: Increased rRNA production, a possible therapeutic target for multiple cancers, can be detected with a new, validated assay that also serves as a pharmacodynamic marker for Pol I inhibitors. Mol Cancer Res; 15(5); 577-84. ©2017 AACR.

Abstract 361: Single cell detection of protein and RNA: Development of a novel method combining immunocytochemistry and in situ hybridization in breast cancer cells

Abstract Immunocytochemistry (ICC) utilizes antibodies to visualize the localization of specific proteins in cells while in situ hybridization (ISH) is a useful research tool for examining the presence and changes in levels of gene expression in cells. The ability to simultaneously visualize the co-localization of protein and RNA species in individual cells is necessary to address important biological questions. Additionally, studying both protein and RNA can be useful in cases where expression levels vary between mRNA and protein and to detect post-translational modifications. However, a combined technique has proved challenging due to the incompatibility of ICC and ISH protocols. Furthermore, traditional ISH methods have lacked sensitivity to detect low abundance RNA species and have limited multiplexing capability. Here, we describe a novel technique combining ICC staining with RNA ISH (based on the ViewRNA Assay), which utilizes branched DNA signal amplification technology allowing for single copy mRNA sensitivity. We demonstrate the utility and versatility of this technique using a variety of antibody and probe combinations relevant for cancer research. We compared expression of protein and mRNA of HER2, estrogen receptor alpha, and progesterone receptor in several breast cancer cell lines (MCF7, SKBR3, MDA-MB-175, MDA-MB-231, and BT474) known to express differential levels of these targets. We also compared expression of several microRNAs (let-7a, miR-17, miR-30d, miR-107, and miR-93) known to play a role in cancer development in combination with staining for proteins involved in proliferation (Ki-67), anti-apoptosis (survivin), and cell structure (tubulin). We show successful detection of low abundance RNA species together with various protein markers. This novel technique is a valuable tool not only for cancer researchers, but also for investigators across numerous fields in biological research. Citation Format: Lisa J. Ma, Richard T. Frank, Xiaoxiao Li, Steve Lai, Jeanette Schmidt, Peggy Just, Sara Becker-Catania. Single cell detection of protein and RNA: Development of a novel method combining immunocytochemistry and in situ hybridization in breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 361.

An improved strategy to detect the epithelial-mesenchymal transition process in circulating tumor cells in hepatocellular carcinoma patients.

We adopted a new strategy to explore the relationship between the EMT process of CTCs and hepatocellular carcinoma (HCC). Furthermore, we intend to illustrate the potential diagnostic value of CTCs of distinct phenotypes in HCC. The clinical data of 33 HCC patients and 10 healthy volunteers were collected retrospectively. By using the optimized CanPatrol CTC enrichment technique, patient blood samples of about 5ml were collected, and CTCs were identified and characterized. The first step of this detection process was to isolate CTCs via a filter-based method; then, an RNA in situ hybridization (RNA-ISH) technique based on the branched DNA signal amplification technology was used to classify the CTCs according to EMT markers. The relationships between HCC CTCs and clinical characteristics were analyzed. The number of epithelial CTCs was related to tumor size (r=0.456, p=0.008), epithelial-mesenchymal-mixed CTCs were related to tumor number (r=0.421, p=0.015), and mesenchymal CTC was associated with metastasis (r=0.375, p=0.032). There was no significant correlation between CTC number and other clinicopathological factors, such as age, serum AFP level or cirrhosis. Epithelial-mesenchymal-mixed CTCs seem to play an important role in EMT transition in HCC, mixed CTCs might be a vital factor for intrahepatic metastasis, and mesenchymal CTCs had the potential to be a predictor of extrahepatic metastasis.

QuantiGene Plex Represents a Promising Diagnostic Tool for Cell-of-Origin Subtyping of Diffuse Large B-Cell Lymphoma

Emerging therapies targeting the molecularly distinct GCB and non-GCB/ABC subtypes of diffuse large B-cell lymphoma (DLBCL) have created the need to develop an accurate subtyping assay for routine use. We investigated the potential of QuantiGene Plex (QGP)-branched DNA signal amplification assay-for DLBCL subtyping. We performed in silico analysis of public DLBCL datasets to develop and validate a naïve Bayes classifier, and migrated the resulting 21-gene classifier to QGP and real-time quantitative PCR (qPCR) assays. Forty DLBCL formalin-fixed, paraffin-embedded tumors of known subtype (20 per subtype by gene expression profiling of paired fresh-frozen tissues) were reclassified, and results for QGP (on 38/40 for 21/21 targets) and qPCR (on 40/40 samples for 19/21 targets) compared for recapitulation of microarray data and classification accuracy. The 21-gene bayesian classifier achieved mean area under the curve values >0.9 on independent validation. QGP showed a higher correlation with microarray data (mean R(2) = 0.66 ± 0.05 versus 0.34 ± 0.07; P < 0.0001) and classification accuracy (92.1% versus 78.9%). The proportion of validated targets was also higher for QGP (85.7% versus 47.4%). The QGP protocol was rapid and simple to perform, at a cost similar to qPCR. These promising preliminary results strongly support ongoing work to develop a QGP companion diagnostic assay for DLBCL subtyping.

Classification of circulating tumor cells by epithelial-mesenchymal transition markers.

In cancer, epithelial-mesenchymal transition (EMT) is associated with metastasis. Characterizing EMT phenotypes in circulating tumor cells (CTCs) has been challenging because epithelial marker-based methods have typically been used for the isolation and detection of CTCs from blood samples. The aim of this study was to use the optimized CanPatrol CTC enrichment technique to classify CTCs using EMT markers in different types of cancers. The first step of this technique was to isolate CTCs via a filter-based method; then, an RNA in situ hybridization (RNA-ISH) method based on the branched DNA signal amplification technology was used to classify the CTCs according to EMT markers. Our results indicated that the efficiency of tumor cell recovery with this technique was at least 80%. When compared with the non-optimized method, the new method was more sensitive and more CTCs were detected in the 5-ml blood samples. To further validate the new method, 164 blood samples from patients with liver, nasopharyngeal, breast, colon, gastric cancer, or non-small-cell lung cancer (NSCLC) were collected for CTC isolation and characterization. CTCs were detected in 107(65%) of 164 blood samples, and three CTC subpopulations were identified using EMT markers, including epithelial CTCs, biophenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. Compared with the earlier stages of cancer, mesenchymal CTCs were more commonly found in patients in the metastatic stages of the disease in different types of cancers. Circulating tumor microemboli (CTM) with a mesenchymal phenotype were also detected in the metastatic stages of cancer. Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulation and provides useful evidence for determining an appropriate clinical approach. This method is suitable for a broad range of carcinomas.

Recurrent amplification of MYC and TNFRSF11B in 8q24 is associated with poor survival in patients with gastric cancer.

Gastric cancer (GC) is an aggressive malignancy whose mechanisms of development and progression are poorly understood. The identification of prognosis-related genomic loci and genes may suffer from the relatively small case numbers and a lack of systematic validation in previous studies. Array-based comparative genomic hybridization (aCGH) coupled with patient clinical information was applied to identify prognosis-related loci and genes with high-frequency recurrent gains in 129 GC patients. The candidate loci and genes were then validated using an independent cohort of 384 patients through branched DNA signal amplification analysis (QuantiGene assays). In the 129 patients, a copy number gain of three chromosome regions-namely, 8q22 (including ESRP1 and CCNE2), 8q24 (including MYC and TNFRSF11B), and 20q11-q13 (including SRC, MMP9, and CSE1L)--conferred poor survival for patients. In addition, the correlation between the branched DNA signal amplification analysis results and the aCGH results was analyzed in 73 of these 129 patients, and MYC, TNFRSF11B, ESRP1, CSE1L, and MMP9 were found to be well correlated. Further validation using an independent cohort (n = 384) verified that only MYC and TNFRSF11B within 8q24 are related to survival. Patients with gains in both MYC and TNFRSF11B had poorer survival than those with no gains, particularly those with noncardia GC. Gains in both of these genes were also a significant independent prognostic indicator. Our results revealed that copy number gains in MYC and TNFRSF11B located at 8q24 are associated with survival in GC, particularly noncardia GC.

Study of serum cell free DNA in patients with gastric cancer by branched DNA

Objective To quantify cell free DNA (cf-DNA) in blood of patients with gastric cancer by a branched DNA (bDNA) -based Alu assay and analyze the relationship between serum cf-DNA levels and clinical and pathological features as well as the applicational value of the aided diagnosis of cf-DNA in GC. Methods A case-control study was conducted.Ninety-nine patients with GC diagnosed by pathological diagnosis and 100 normal controls presenting to the Nantong tumor hospital from September 2011 to February 2012 were recruited.Thirty-two patients with gastric benign tumor presenting to the Affiliated Hospital of Nantong University from March 2010 to February 2012 were also recruited.The concentration of cf-DNA was detected by bDNA and the concetrations of carcinoembryonic antigen (CEA),carcinoembryonic antigen 19-9 (CA19-9),carcinoembryonic antigen 50 (CA50),and carcinoembryonic antigen 72-4 (CA72-4) were assayed by chemiluminescence.The Dunn′s test was used to compare the difference of cf-DNA between patients with GC and control groups (gastric benign tumor and healthy individuals).The diagnostic value of the five indicators was evaluated by drawing the Receiver Operating Characteristic curves (ROC).The Mann-Whitney test was used to analyze the difference of cf-DNA levels of the patient′s gender and age.Kruskal-Wallis test was used to compare cf-DNA correlation with the patient′s tumor site,tumor differentiation,American Joint Committee on Cancer. Results The levels of cf-DNA in patients with GC,patients with gastric benign tumor and healthy individuals were 2316.0 (1028.7-3742.1),423.3 (256.3-519.2),317.5 (172.5-499.7) μg/L,respectively.There were significant differences between GC patients and gastric benign tumor patients (q=7.612,P 0.05).The optimal diagnostic thresholds of the five indicators were 973.6μg/L,4.0μg/L,32.8 kU/L,12.8 kU/L,5.1 kU/L,respectively.The areas under the ROC curves and sensitivity assessing cf-DNA level[0.94,76.8% (76/99)]were significantly larger than traditional indicators of serum CEA[0.66,31.3% (31/99)],CA19-9[0.63,16.2% (16/99)],CA50[0.83,44.4% (44/99)],CA72-4[0.67,35.4% (35/99)] for GC patients.Specificity of cf-DNA[96% (96/100)]was closer to traditional indicators of serum CEA[98% (98/100)],CA19-9[78% (78/100)],CA50[97% (97/100)],CA72-4[98% (98/100)]. Conclusions cf-DNA levels detected by bDNA are higher specificity and better sensitivity than traditional tumor markers (CEA,CA19-9,CA50,CA72-4) for GC patients.Certain guiding significance and application value was existed by detecting cf-DNA level in the serum of GC patients.(Chin J Lab Med,2013,36:699-703) Key words: Stomach neoplasms; DNA; Branched DNA signal amplification assay

Differential Regulation of Hepatic Organic Cation Transporter 1, Organic Anion-Transporting Polypeptide 1a4, Bile-Salt Export Pump, and Multidrug Resistance-Associated Protein 2 Transporter Expression in Lymphocyte-Deficient Mice Associates with Interleukin-6 Production

Cholestasis results from interrupted bile flow and is associated with immune-mediated liver diseases. It is unclear how inflammation contributes to cholestasis. The aim of this study was to determine whether T and B cells contribute to hepatic transporter expression under basal and inflammatory conditions. C57BL/6J wild-type mice or strains lacking T, B, or both T and B cells were exposed to lipopolysaccharide (LPS) or saline, and livers were collected 16 hours later. Branched DNA signal amplification was used to assess mRNA levels of organic anion-transporting polypeptides (Oatp) 1a1, 1a4, and 1b2; organic cation transporter (Oct) 1; canalicular bile-salt export pump (Bsep); multidrug resistance-associated proteins (Mrp) 2 and 3; and sodium-taurocholate cotransporting polypeptide (Ntcp). Real-time polymerase chain reaction analysis was used to correlate changes of transporter expression with interleukin-1b (IL-1b), IL-6, IL-17A, IL-17F, tumor necrosis factor-α (TNF-α), and interferon-γ expression in the liver. LPS treatment inhibited Bsep and Oct1 mRNA expression, and this was abrogated with a loss of T cells, but not B cells. In addition, the absence of T cells increased Mrp2 mRNA expression, whereas B cell deficiency attenuated Oatp1a4 mRNA in LPS-treated mice. Oatp1a1, Oatp1b2, Ntcp, and Mrp3 were largely unaffected by T or B cell deficiency. Lymphocyte deficiency altered basal and inflammatory IL-6, but not TNF-α or IL-1b, mRNA expression. Taken together, these data implicate lymphocytes as regulators of basal and inflammatory hepatic transporter expression and suggest that IL-6 signaling may play a critical role.

A new biodosimetric method: branched DNA-based quantitative detection of B1 DNA in mouse plasma

A simple and accurate method for measuring the biological effects of radiation is of increasing importance, especially in mass casualty scenarios. We have therefore developed a new biodosimetric technique targeting circulating B1 DNA in mouse plasma by branched DNA signal amplification for rapid quantification of plasma DNA. This technology targets repetitive elements of the B1 retrotransposon in the mouse genome, followed by signal amplification using Panomics Quantigene 2.0 reagents. Evaluation was conducted concerning precision, accuracy and linearity. Plasma samples were collected from mice 0-24 h after 0-10 Gy total body irradiation (TBI). The average inter- and intra-assay coefficients of variance were 8.7% and 12.3%, respectively. The average recovery rate of spiked DNA into plasma was 89.5%. This assay revealed that when BALB/c and NIH Swiss mice were exposed to 6 Gy TBI, plasma B1 DNA levels increased significantly at 3 h post-TBI, peaked at 9 h and gradually returned toward baseline levels in 24 h. A dose-dependent change in plasma DNA was observed at 9 h post-TBI; the dose-response relation was monotonic, exhibiting linearity for BALB/c mice from 3 to 6 Gy (r = 0.993) and NIH Swiss mice from 3 to 7 Gy (r = 0.98). This branched DNA-based assay is reliable, accurate and sensitive in detecting plasma B1 DNA quantitatively. A radiation dose-correlated increase in plasma B1 DNA was demonstrated in BALB/c and NIH Swiss mice in the dose range from 3 to 6 Gy, suggesting that plasma B1 DNA has potential as a biomarker for radiation biological effect.

Indoleamine 2,3-Dioxygenase Tissue Distribution and Cellular Localization in Mice: Implications for Its Biological Functions

Earlier studies have suggested that indoleamine 2,3-dioxygenase (IDO) has a wide tissue distribution in mammals. However, detailed information on its cellular localization and also the levels of expression in various tissues is still scarce. In the present study, we sought to determine the cellular localization of IDO and also to quantify the level of its expression in various mouse tissues by using the branched DNA signal amplification assay, Western blotting, and immunohistochemical staining. The highest levels of constitutive IDO expression were found to be selectively present in the caput of epididymis, except for its initial segment. IDO expression was also detected inside the luminal compartment and even in the stereocilia within this region. In the prostate, high levels of IDO were selectively expressed in the capsular cells. In addition, high levels of IDO expression were also selectively detected in certain types of cells in the placenta, spleen, thymus, lung, and digestive tract. Notably, the morphological features of most of the positively stained cells in these organs closely resembled those of antigen-presenting cells. Based on the tissue distribution and cellular localization characteristics of IDO, it is hypothesized that its expression may serve two main functions: one is to deplete tryptophan in an enclosed microenvironment (such as in the epididymal duct lumen) to prevent bacterial or viral infection, and the other is to produce bioactive tryptophan catabolites that would serve to suppress T-cell-mediated immune responses against self-antigens, fetal antigens, or allogeneic antigens, in different situations.

Comparison of Clinical Application of the Abbott HBV PCR Kit and the Versant HBV DNA 3.0 Test to Measure Serum Hepatitis B Virus DNA in Taiwanese Patients

With an estimated 350-400 million people worldwide chronically infected with hepatitis B virus (HBV), and the subsequent serious complications caused by liver damage including cirrhosis, liver failure, and hepatocellular carcinoma, HBV infection remains a global health issue, particularly in Taiwan, an HBV-hyperendemic area. Sensitive and accurate quantification of HBV DNA is necessary to monitor patients with chronic hepatitis B who are receiving antiviral therapy to determine treatment response and adapt therapy. We evaluated and compared the clinical performance of two HBV DNA assays based on different technologies: the RealArt HBV PCR Kit (Abbott HBV DNA PCR kit, real-time polymerase chain reaction assay, detection limit: 27 IU/mL) and the VERSANT bDNA 3.0 assay (Bayer, branched DNA signal amplification assay, detection limit: 357 IU/mL). Serum levels of HBV DNA in 173 chronic HBV carriers were determined using both the RealArt HBV PCR Kit and the VERSANT bDNA 3.0 test. Of the 173 samples analyzed for baseline viral load detection, HBV DNA was quantifiable in 147 patients (82.1%) by the RealArt HBV PCR Kit, which was significantly higher than the 92 (53.2%) samples quantified by the VERSANT bDNA 3.0 assay. A total of 86 (49.7%) samples were quantifiable by both assays, whereas 25 (14.5%) were below the detection limit of both assays. The HBV DNA quantification values measured by the RealArt HBV PCR Kit and the VERSANT bDNA 3.0 assay were positively correlated (Spearman's rank correlation coefficient r = 0.932, p < 0.001). On average, the results derived from the RealArt HBV PCR Kit were 0.67 log lower than those of the VERSANT bDNA 3.0 assay. HBV DNA concentrations were significantly higher in 63 HBV e antigen (HBeAg)-seropositive patients than in 110 HBeAg-seronegative patients (5.42 +/- 2.34 logs vs. 3.21 +/- 2.27 logs, p < 0.001). The RealArt HBV PCR Kit is more sensitive and has a wider dynamic range than the VERSANT bDNA 3.0 assay in the clinical setting of chronic hepatitis B patients. The sensitivity and wide dynamic range of the PCR assay allow optimal monitoring and timely adaptation of antiviral therapy. Nevertheless, the HBV DNA values measured by the RealArt HBV PCR Kit and the VERSANT bDNA 3.0 assay were significantly correlated.

Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of diagnoses ranging from simple fatty liver (SFL), to non-alcoholic steatohepatitis (NASH). This study aimed to determine the effect of moderate and severe NAFLD on hepatic transporter expression and function in vivo. Rats were fed a high-fat diet (SFL model) or a methionine-choline-deficient diet (NASH model) for eight weeks. Hepatic uptake transporter function was determined by bromosulfophthalein (BSP) disposition. Transporter expression was determined by branched DNA signal amplification assay and western blotting; inflammation was identified by immunostaining of liver slices for interleukin 1 beta (IL-1β). MC− rats showed significant retention of BSP in the plasma when compared to control rats. Hepatic NTCP, OATP1a1, 1a4, 1b2 and 2b1; and OAT 2 and 3 mRNA levels were significantly decreased in high-fat and MC− diet rats when compared to control. Protein expression of OATP1a1 was significantly decreased in high-fat animals, while OATP1a1 and OATP1b2 expressions were significantly lower in MC− rats when compared to control. Liver tissue from high-fat and MC− rats stained positive for IL-1β, a pro-inflammatory cytokine known to decrease expression of NTCP, OATP and OAT transporters, suggesting a plausible mechanism for the observed transporter alterations. These data suggest that different stages of NAFLD result in altered hepatic uptake transporter expression that can lead to a functional impairment of xenobiotic uptake from the blood. Furthermore, NAFLD may alter the plasma retention time of clinically relevant drugs that are reliant on these transporters and may increase the potential drug toxicity.

Effect of allyl alcohol on hepatic transporter expression: Zonal patterns of expression and role of Kupffer cell function

During APAP toxicity, activation of Kupffer cells is critical for protection from hepatotoxicity and up-regulation of multidrug resistance-associated protein 4 (Mrp4) in centrilobular hepatocytes. The present study was performed to determine the expression profile of uptake and efflux transporters in mouse liver following treatment with allyl alcohol (AlOH), a periportal hepatotoxicant. This study also investigated the role of Kupffer cells in AlOH hepatotoxicity, and whether changes in transport protein expression by AlOH are dependent on the presence of Kupffer cells. C57BL/6J mice received 0.1 ml clodronate liposomes to deplete Kupffer cells or empty liposomes 48 h prior to dosing with 60 mg/kg AlOH, i.p. Hepatotoxicity was assessed by plasma ALT and histopathology. Hepatic transporter mRNA and protein expression were determined by branched DNA signal amplification assay and Western blotting, respectively. Depletion of Kupffer cells by liposomal clodronate treatment resulted in heightened susceptibility to AlOH toxicity. Exposure to AlOH increased mRNA levels of several Mrp genes, while decreasing organic anion transporting polypeptides (Oatps) mRNA expression. Protein analysis mirrored many of these mRNA changes. The presence of Kupffer cells was not required for the observed changes in uptake and efflux transporters induced by AlOH. Immunofluorescent analysis revealed enhanced Mrp4 staining exclusively in centrilobular hepatocytes of AlOH treated mice. These findings demonstrate that Kupffer cells are protective from AlOH toxicity and that induction of Mrp4 occurs in liver regions away from areas of AlOH damage independent of Kupffer cell function. These results suggest that Kupffer cell mediators do not play a role in mediating centrilobular Mrp4 induction in response to periportal damage by AlOH.

Tissue Distribution, Ontogeny, and Regulation of Aldehyde Dehydrogenase (Aldh) Enzymes mRNA by Prototypical Microsomal Enzyme Inducers in Mice

Aldehyde dehydrogenases (Aldhs) are a group of nicotinamide adenine dinucleotide phosphate-dependent enzymes that catalyze the oxidation of a wide spectrum of aldehydes to carboxylic acids. Tissue distribution and developmental changes in the expression of the messenger RNA (mRNA) of 15 Aldh enzymes were quantified in male and female mice tissues using the branched DNA signal amplification assay. Furthermore, the regulation of the mRNA expression of Aldhs by 15 typical microsomal enzyme inducers (MEIs) was studied. Aldh1a1 mRNA expression was highest in ovary; 1a2 in testis; 1a3 in placenta; 1a7 in lung; 1b1 in small intestine; 2 in liver; 3a1 in stomach; 3a2 and 3b1 expression was ubiquitous; 4a1, 6a1, 7a1, and 8a1 in liver and kidney; 9a1 in liver, kidney, and small intestine; and 18a1 in ovary and small intestine. mRNAs of different Aldh enzymes were detected at lower levels in fetuses than adult mice and gradually increased after birth to reach adult levels between 15 and 45 days of age, when the gender difference began to appear. Aromatic hydrocarbon receptor (AhR) ligands induced the liver mRNA expression of Aldh1a7, 1b1, and 3a1, constitutive androstane receptor (CAR) activators induced Aldh1a1 and 1a7, whereas pregnane X receptor (PXR) ligands and NF-E2 related factor 2 (Nrf2) activators induced Aldh1a1, 1a7, and 1b1. Peroxisome proliferator activator receptor alpha (PPAR alpha) ligands induced the mRNA expression in liver of almost all Aldhs. The Aldh organ-specific distribution may be important in elucidating their role in metabolism, elimination, and organ-specific toxicity of xenobiotics. Finally, in contrast to other phase-I metabolic enzymes such as CYP450 enzymes, Aldh mRNA expression seems to be generally insensitive to typical microsomal inducers except PPAR alpha ligands.

Influence of Acetaminophen Vehicle on Regulation of Transporter Gene Expression During Hepatotoxicity

Researchers who study acetaminophen (APAP) hepatotoxicity use either a 50% propylene glycol solution or saline as a diluent. Previous studies demonstrated differential expression of hepatobiliary transporter mRNA in mice treated with a toxic dose of APAP dissolved in 50% propylene glycol. The purpose of this study was to determine whether using saline as a diluent for APAP alters regulation of transporter gene expression during hepatotoxicity. Male C57BL/6J mice received acetaminophen (APAP 400 mg/kg, ip in saline) or saline (20 ml/kg). Plasma and liver samples were collected at 24 and 48 h for assessment of alanine aminotransferase (ALT) activity and gene expression. It was determined that plasma ALT activity was elevated at 24 and 48 h after APAP administration. Using the branched DNA signal amplification assay, reductions in organic anion-transporting polypeptides Oatp1a1, Oatp1b2, sodium/taurocholate-cotransporting polypeptide (Ntcp), and bile salt export pump (Bsep) mRNA were observed in APAP-treated mice. In contrast, multidrug resistance-associated proteins Mrp1, Mrp2, Mrp3, and Mrp4, as well as multidrug resistance proteins Mdr1a and Mdr1b genes, were increased following APAP. No changes in Oatp1a4, Mdr2, or breast cancer resistance protein (Bcrp) mRNA were observed. Alterations in transporter gene expression in this study were similar to those reported previously using propylene glycol as diluent. With the exceptions of Oatp1a1, Ntcp, and Mrp1, these data mirror previous results suggesting that the solution used to dissolve APAP may alter the susceptibility of mice to hepatotoxicity, but only minimally change the regulation of transporter gene expression.

Gender Differences in mRNA Expression of ATP-Binding Cassette Efflux and Bile Acid Transporters in Kidney, Liver, and Intestine of 5/6 Nephrectomized Rats

ATP-binding cassette (ABC) transporters including multidrug resistance proteins (Mdr), multidrug resistance-associated proteins (Mrp), and breast cancer resistance protein (Bcrp/Abcg2) play major roles in tissue defense. Abcg5/g8 is essential in cholesterol efflux. The present study was aimed at elucidating alteration in expression of these transporters and bile-acid transporters during chronic kidney disease (CKD) and underlying molecular mechanisms. Seven weeks after 5/6 nephrectomy (Nx), mRNA expression of 16 aforementioned transporters in kidney, liver, jejunum, and large intestine of male and female Nx rats was quantified with the branched DNA signal amplification assay. In Nx males, intestinal expression of all the transporters remained unchanged; hepatic expression of most transporters was not altered, except increases in Mdr1a, Mrp3, and Abcg8. In male remnant kidneys, kidney-predominant transporter Abcg2 decreased and correlated with CKD severity, whereas Mdr1b, Mrp3, and ileal bile-acid transporter increased and correlated with CKD severity. Such changes were largely absent in Nx females. Renal alterations of these transporters correlated with increases of cytokines and/or decreases of nuclear receptors such as estrogen receptor alpha and glucocorticoid receptor. Renal protein expression of Mrp2 increased, whereas that of Mrp4 remained unchanged in both genders of Nx rats. Treatment of rat proximal tubule NRK-52E cells with interleukin (IL)-1beta and IL-6 increased Mrp3 mRNA expression. In conclusion, during CKD, renal expression of many ABC transporters was altered at the transcriptional level, whereas hepatic mRNA expression of most ABC transporters remained unchanged. Down-regulation of steroid hormone receptors and increase of inflammatory cytokines may contribute to alteration of transporter gene expression in kidney during CKD.