Properties of Glycophorin A and its Deglycosylated Derivatives; Phospholipid Vesicle Formation and the Incorporation of Integral Membrane Proteins

The physical properties of native and deglycosylated glycophorin A and of membrane protein reconstituted vesicles were investigated. The goals of these studies were twofold. First, solutionscontaining glycophorin A were examined prior to and following removal of covalently attached carbohydrate from the protein to determine the contribution of carbohydrate to the glycoprotein amphiphilic properties. Second, the properties of glycophorin reconstituted vesicles were studied to address the question: Does the incorporation of a transmembrane protein into a membrane vesicle significantly increase bilayer premeability to ions? Glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, is approximately 31,000 daltons: 55% by weight is carbohydrate and 45% is protein. Neuraminidase was used to remove 99% of the sialic acid producing asialoglycoprotein and endo-a-N-acetylgalactosaminidase was used to remove 90% of the serine and threonine linked carbohydrate chains producing apoglycophorin A. Removal of carbohydrate results in increased binding of sodium dodecyl sulfate (SDS) to the polypeptide chain at saturating SDS concentrations. This increased binding indicates that sialic acid residues exhibit little or no SDS binding and actually inhibit SDS binding to the peptide chain. In detergent free solutions, native, asialo, and apoglycophorin are heterogeneously aggregated. SDS associates with aggregated glycophorin below its critical micelle concentration indicating the presence of noncooperative monomer binding sites; however, as with other membrane proteins, the association of SDS to glycophorin occurs principally as a cooperative transition above the critical micelle concentration. The additional binding of SDS to the deglycosylated forms does not produce significant changes in the peptide conformation as measured by circular dichroism. Removal of carbohydrate also produces corresponding changes in molecular weight, electrophoretic mobility on SDS gels, and distribution coefficients on gel chromatographic columns. At saturating levels of SDS binding, glycophorin and its deglycosylated derivatives were determined to be predominantly monomeric by sedimentation equilibrium. The relative mobility of the three forms on SDS gels increases with increasing carbohydrate removal. Relative mobility depends upon ionic strength, SDS concentration, and protein concentration. Native glycophorin and apoglycophorin in solution as a 1:1 molar mixture behave as noninteracting species by sedimentation equilibrium and gel filtration. Glycophorin incorporated vesicles were prepared by removal of detergent from solutions containing mixed micelles of egg phosphatidylcholine, octyl glucoside, and purified glycophorin. Glycophorin was not uniquely oriented in the membrane as shown by trypsin and neuraminidase treatment of vesicles. Vesicles that were formed in the presence or absence of glycophorin were similar in size and morphology (2300 ± 400 A in diameter) as imaged in the electron microscope. The permeability of these large unilamellar vesicles to Cl-, Na+, and Rb+ followed first order kinetics with rate constants of 2.1 x -5 -7 -7 10 , 2.6 x 10 , and 9.0 x 10 , respectively. The presence of glycophorin in the membrane at levels up to 220 copies per vesicle increased the permeability by less than a factor of five. This demonstrated that incorporation of membrane protein into vesicles does not necessarily lead to dramatic increases in membrane ion permeability, and therefore; this methodology may provide a useful control in the reconstitution of ion pumps into vesicles. To determine whether some biological activities can be maintained or restored under these conditions two integral membrane proteins, dopamine-8- hydroxylase and hepatic asialoglycoprotein receptor,were incorporated using similar procedures. These proteins were found to either retain or regain activity upon incorporation into vesicles. Both proteins are oriented in these vesicles such that all substrate binding sites are exposed on the vesicle exterior

Therapeutic Drug Monitoring: Performance of a FRET-Based Point-Of-Care Immunoassay for the Quantitation of Infliximab and Adalimumab in Blood

Two fast (<5 min), time-resolved fluorescence resonance energy transfer (FRET)-based immunoassays (Procise IFX™ and Procise ADL™) were developed for the quantitative detection of infliximab (IFX), adalimumab (ADL), and their respective biosimilars for use in therapeutic drug monitoring (TDM) using 20 µL of finger prick whole blood at the point-of-care or whole blood/serum in a central lab. Studies were performed to characterize analytical performance of the Procise IFX and the Procise ADL assays on the ProciseDx™ analyzer. The Procise IFX and Procise ADL assays both showed good analytical performance with respect to sensitivity, specificity, linearity, and precision suitable for routine clinical use as well as excellent correlation to current commercial ELISA IFX and ADL measurement methods. Results indicated that the Procise IFX and Procise ADL assays are sensitive, specific, and precise yielding results in less than 5 minutes from either whole blood or serum. This indicates the Procise IFX and Procise ADL assays are useful for obtaining fast and accurate IFX or ADL quantitation, thus avoiding delays inherent to current methods and enabling immediate drug level dosing decisions to be made during a single patient visit.</p

Significant Closure of the Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Preseroconversion Detection Windows with a Transcription-Mediated-Amplification-Driven Assay

While the present generation of serology-based assays has significantly decreased the number of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections acquired by transfusion, the possibility of infected donations escaping detection still exists. The average seronegative viremic window duration during which immunological assays are unable to detect the virus is estimated to be between 16 and 22 days for HIV-1 and approximately 70 days for HCV. Significant reduction of detection window duration was demonstrated using a nucleic acid amplification assay, the Procleix HIV-1/HCV Assay, which utilizes transcription-mediated amplification technology to simultaneously detect HIV-1 and HCV RNAs. For 26 commercially available HIV-1 seroconversion panels tested, specimens were reactive in the HIV-1/HCV assay at the same time as or earlier than in serological assays. Overall, the HIV-1/HCV assay was able to reduce the detection window duration by an average of 14 days and 6 days compared to tests relying on recognition of HIV-1 antibody and p24 antigen, respectively. For 24 commercially available HCV seroconversion panels tested, the specimens were reactive in the HIV-1/HCV assay at an earlier blood sampling date than in serological assays, reducing the detection window duration by an average of 26 days. Similar results were obtained in testing the HIV-1 and HCV seroconversion panels in the virus-specific HIV-1- and HCV-discriminatory assays, respectively. In conclusion, the HIV-1/HCV assay and corresponding discriminatory assays significantly reduced detection window durations compared to immunoassays

Analytical Performance of a FRET-Based Point-of-Care Immunoassay for the Quantitation of C-Reactive Protein in Serum and Finger Prick Capillary Whole Blood

Time-resolved fluorescence resonance energy transfer (FRET) provides a useful technology for immunoassay measurement of proteins, This has been used to develop an immunoassay for C-reactive protein (Procise CRP™) that provides point-of-care measurement of CRP in less than 4 minutes using 20 µL of either whole blood or serum. Analytical studies of the assay on the ProciseDx™ analyzer were performed to characterize its measurement capabilities.Sensitivity, specificity, linearity, and precision - including reproducibility of finger prick blood collection and testing, suitable for routine clinical use in a point-of-care setting were demonstrated for the CRP assay. It also showed excellent analytical agreement with a current commercial CRP test. These results indicate the Procise CRP assay is useful for obtaining fast and accurate CRP quantitation in a point of care setting that can aid in the immediate assessment of patients’ inflammatory status. </div

Performance of a FRET-Based Point-of-Care Immunoassay for the Quantitation of Fecal Calprotectin

A fast (~5 min), time-resolved fluorescence resonance energy transfer based immunoassay (Procise FCP™) was developed for the point-of-care quantitative detection of fecal calprotectin (FCP) using 15 mg of fecal specimen eluted in collection fluid from the Procise Stool Collection Device™. Studies were performed to characterize analytical performance of the Procise FCP assay on the ProciseDx™ analyzer. The Procise FCP assay showed good analytical performance with respect to sensitivity, specificity, linearity, and precision suitable for routine clinical use in a point-of-care setting as well as excellent analytical agreement with a current commercial FCP measurement method. Results indicate that the Procise FCP assay is sensitive, specific, and precise yielding results in less than 5 minutes. This indicates the Procise FCP assay is useful for obtaining fast and accurate FCP quantitation, thus avoiding delays inherent to current methods and enabling immediate clinical assessment to be made during a single patient visit.</p

Use of Four Next-Generation Sequencing Platforms to Determine HIV-1 Coreceptor Tropism

HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage

Serum Concentrations of 7α-hydroxy-4-cholesten-3-one Are Associated With Bile Acid Diarrhea in Patients With Crohn's Disease

Background & Aims: Patients with Crohn's disease (CD) often have bile acid diarrhea (BAD), due to bile acid malabsorption following ileal resection (IR). Bile acid malabsorption increases production of 7α-hydroxy-4-cholesten-3-one (C4), a bile acid precursor. We investigated relationships between serum concentrations of C4 and BAD in patients with CD. Methods: We collected demographic data, serum samples, and information on the presence of diarrhea (>3 liquid bowel movements/day), as well as clinical, endoscopic, and histologic scores from 26 patients with CD and IR, 21 patients with CD without IR, and 37 patients with ulcerative colitis (UC). We compared serum concentrations of C4 and fibroblast growth factor 19 (FGF19) between groups. We performed area under the receiver operating characteristic curve (AUROC) analysis to identify the optimal cutoff C4 concentrations for the diagnosis of diarrhea attributable to bile acid malabsorption (BAD), defined as diarrhea and a serum concentration of FGF19 <60 pg/mL. Results: Patients with UC had a median serum C4 concentration of 11.8 ng/mL, whereas patients with CD and IR with ileitis (documented endoscopically) had a median concentration of 100.0 ng/mL (P compared to UC <.0001) and patients with CD and IR without ileitis had a median concentration of 51.6 ng/mL (P compared to UC <.001). Patients with CD without IR did not have a significantly higher median concentration of C4 than patients with UC (P =.71), regardless of ileitis (P =.34). When endoscopic findings were confirmed histologically, similar results were found to analyses using endoscopic findings alone. A higher proportion of patients with active UC had diarrhea (72.0% vs 0 patients with inactive UC; P <.001), but their median concentrations of C4 did not differ significantly from that of patients with inactive UC (12.1 ng/mL vs 9.7 ng/mL; P =.3). A cutoff concentration of C4 of 48.3 ng/mL or greater identified patients with diarrhea attributable to bile acid malabsorption with 90.9% sensitivity, 84.4% specificity, and an AUROC 0.94. A significantly higher proportion of patients with concentrations of C4 above this cutoff had BAD (50.0%) than below this cutoff (1.8%) (P <.001). When we analyzed only patients with diarrhea, a C4 cutoff of 48.3 ng/mL identified those with low FGF19 concentrations (<60 pg/mL) with 91% sensitivity and 95.5% specificity (AUROC, 0.99). Above this cutoff, 83.3% of patients had a serum concentration of FGF19 <60 pg/mL compared to 4.5% below this threshold (P <.0001). C4 concentrations correlated with the number of daily bowel movements (r = 0.41; P =.004) and correlated inversely with FGF19 concentrations (r = –0.72; P<.0001). Conclusion: We observed significantly increased serum concentrations of C4 in patients with CD with IR, compared to patients with UC. A cutoff concentration of C4 above 48.3 ng/mL identifies patients with diarrhea likely attributable to bile acid malabsorption (BAD) with an AUROC value of 0.94. Increased serum levels of bile acid precursors identify patients with diarrhea and a low serum concentration of FGF19, and concentrations of C4 correlate with daily liquid bowel movements and correlate inversely with FGF19 concentrations. C4 may be a biomarker to identify patients with diarrhea attributable to bile acid malabsorption

Validation of Serum Test for Advanced Liver Fibrosis in Patients With Nonalcoholic Steatohepatitis

Background &amp; aimsWe analyzed markers of fibrosis in serum samples from patients with nonalcoholic fatty liver disease (NAFLD), assessed by liver biopsy. We used serum levels of markers to develop an algorithm to discriminate patients with advanced fibrosis from those with mild or moderate fibrosis and validated its performance in 2 independent cohorts of patients with NAFLD.MethodsWe performed a retrospective analysis of serum samples from 396 patients with NAFLD and different stages of fibrosis (F0-F4), collected from 2007 through 2017 on the day of liver biopsy (training cohort 1). We measured serum concentrations of alpha-2 macroglobulin (A2M), hyaluronic acid (HA), and TIMP metallopeptidase inhibitor 1 (TIMP1), and used measurements to develop an algorithm that could discriminate patients with NAFLD with advanced fibrosis (F3-F4; 24.1% of cohort) from those with mild or moderate fibrosis (F0-F2; 79.5% of cohort). We validated the algorithm using serum samples collected from a separate 396 patients from the same time period and location (validation cohort 1), as well as 244 patients with NAFLD evaluated at a separate location, from 2011 through 2017, within a median of 11 days of liver biopsy (cohort 2).ResultsThe algorithm identified patients with advanced fibrosis vs mild or moderate fibrosis in training cohort 1 with an area under the receiver operating characteristic (AUROC) curve of 0.867 (95% CI, 0.827-0.907), 84.8% sensitivity (95% CI, 75.5%-91.0%), and 72.3% specificity (95% CI, 66.9%-77.3%), at a cutoff score of 17. The AUROC for the combined validation cohorts 1 and 2 (n=640) was 0.856 (95% CI, 0.820-0.892), identifying patients with 79.7% sensitivity (95% CI, 71.9%-86.2%) and 75.7% specificity (95% CI, 71.8%-79.4%) at the predetermined cutoff score of 17. The algorithm had negative predictive values that ranged from 92.5% to 94.7% in the validation cohorts; it correctly classified 90.0% of F0 samples, 75.0% of F1 samples, 77.4% of F3 samples, and 94.4% of F4 samples.ConclusionWe developed an algorithm that identifies patients with advanced fibrosis from those with mild to moderate fibrosis in patients with NAFLD with an AUROC value of approximately 0.86, based on levels of serum biomarkers. We validated the findings in 2 separate sets of patients with biopsy-proven NAFLD. The algorithm can be used non-invasively to determine risk of advanced fibrosis in patients with NAFLD