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Aims Long-COVID occurs after SARS-CoV-2 infection and results in diverse, prolonged symptoms. The present study aims to determine the underlying mechanisms, and to inform prognosis and treatment. Methods Plasma proteome from Long-COVID outpatients was analyzed in comparison to acutely ill COVID-19 (mild and severe) inpatients and healthy control subjects. The expression of approximately 3000 protein biomarkers was determined with proximity extension assays and then deconvoluted with multiple bioinformatics tools into both cell types and signaling mechanisms, as well as organ specificity. Results Compared to age- and sex-matched acutely ill COVID-19 inpatients and healthy control subjects, Long-COVID outpatients showed natural killer cells with a resting phenotype, as opposed to active, and neutrophils that formed extracellular traps. This resetting of cell phenotypes was reflected in vascular events mediated by both angiopoietin-1 (ANGPT1) and vascular-endothelial growth factor-A (VEGFA). Levels of ANGPT1 and VEGFA were validated by serological methods in different patient cohorts. Silent signaling of transforming growth factor-β1 with elevated EP300 favored not only vascular inflammation, but also tumor necrosis factor-α driven pathways. In addition, a vascular proliferative state associated with hypoxia inducible factor 1 pathway was predicted that progressed from COVID-19 to Long-COVID. The vasculo-proliferative process identified in Long-COVID was associated with significant changes in the organ-specific proteome reflective of neurological and cardiometabolic dysfunction. Conclusions Taken together, our study uncovered a vasculo-proliferative process in Long-COVID initiated by prior hypoxia, and identified potential organ-specific prognostic biomarkers and therapeutic targets.

Point of Care Testing (POCT) refers to clinical laboratory testing performed outside the central laboratory, nearer to the patient and sometimes at the patient bedside. The testing is usually performed by clinical staff, such as physicians or nurses, who are not laboratory trained. This document was developed by the POCT Interest group of the Canadian Society of Clinical Chemists (CSCC) as practical guidance for quality assurance practices related to POCT performed in hospital and outside hospital environments. The aspects of quality assurance addressed in this document include: (1) device selection, (2) initial device verification, (3) ongoing device verification, (4) ongoing quality assurance including reagent and quality control (QC) lot changes, and (5) quality management including operator and document management.

BackgroundDetection of viral RNA by nucleic acid amplification testing (NAAT) remains the gold standard for diagnosis of SARS-CoV-2 infection but is limited by high cost and other factors. Whether serology-based assays can be effectively incorporated into a diagnostic algorithm remains to be determined. Herein we describe the development of a serology-based testing algorithm for SARS-CoV-2 infection.Patients and MethodsBetween July 2020 and February 2021, we included symptomatic unvaccinated patients evaluated in the Emergency Department of our institution for suspected SARS-CoV-2. All patients had testing by real-time Reverse Transcription Polymerase Chain Reaction. The performance characteristics of five commercial enzymatic serology assays testing for different antibody isotypes were evaluated in a derivation cohort and the assay with the best performance was further tested on a validation cohort. Optimal cut-off points were determined using receiver operating characteristic (ROC) curves and further tested using logistic regression.ResultsThe derivation and validations cohorts included 72 and 319 patients, respectively. Based on its initial performance, the Elecsys Anti-SARS-CoV-2 assay (Roche Diagnostics) was further tested in the validation cohort. Using ROC curve analysis, we estimated the diagnostic performance for different cut-off points assuming a prevalence of positive tests of 5%. At any given cut-off point the NPV was over 97%.DiscussionThis study suggests that an initial diagnostic strategy using the Elecsys Anti-SARS-CoV-2 serology test in symptomatic unvaccinated patients could help to rule out an acute SARS-CoV2 infection and potentially lead to appropriately tailored infection control measures or rational guidance for further testing with a potential cost reduction and increased availability.

BACKGROUND. The role of humoral immunity in COVID-19 is not fully understood, owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome. METHODS. Using SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients’ plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution. RESULTS. We identified linear epitopes from the spike (S) and nucleocapsid (N) proteins and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S-811–825, S-881–895, and N-156–170 epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes. CONCLUSION. Epitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, but it also may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern in both the peptide array and latex agglutination formats. FUNDING. Ontario Research Fund (ORF) COVID-19 Rapid Research Fund, Toronto COVID-19 Action Fund, Western University, Lawson Health Research Institute, London Health Sciences Foundation, and Academic Medical Organization of Southwestern Ontario (AMOSO) Innovation Fund.

We have developed a rapid, accurate, and cost-effective serologic test for SARS-CoV-2 virus, which caused the COVID-19 pandemic, on the basis of antibody-dependent agglutination of antigen-coated latex particles. When validated using plasma samples that are positive or negative for SARS-CoV-2, the agglutination assay detected antibodies against the receptor-binding domain of the spike (S-RBD) or the nucleocapsid protein of SARS-CoV-2 with 100% specificity and ∼98% sensitivity. Furthermore, we found that the strength of the S-RBD antibody response measured by the agglutination assay correlated with the efficiency of the plasma in blocking RBD binding to the angiotensin-converting enzyme 2 in a surrogate neutralization assay, suggesting that the agglutination assay might be used to identify individuals with virus-neutralizing antibodies. Intriguingly, we found that >92% of patients had detectable antibodies on the day of a positive viral RNA test, suggesting that the agglutination antibody test might complement RNA testing for the diagnosis of SARS-CoV-2 infection., Graphical abstract, Motivation As COVID-19 continues to spread around the world, there is urgent need for a rapid yet accurate antibody test to detect individuals' humoral immune responses to the SARS-CoV-2 virus and emerging variants of concern. A simple and reliable antibody test would also allow longitudinal analysis of antibody responses to inform vaccination strategies. We addressed this pressing need by developing a simple, quick, and sensitive antibody test based on latex particle agglutination., Esmail et al. develop and validate a rapid and cost-effective antibody-dependent agglutination (aggregation) test by using SARS-CoV-2 antigen-coated latex particles. The agglutination assay detects SARS-CoV-2 antibodies with 100% specificity and ∼98% sensitivity. They further show that the agglutination assay might be used to identify neutralizing antibodies and monitor dynamic changes in antibody responses over time.

Objectives A consensus guidance is provided for testing, utility and verification of SARS-CoV-2 point-of-care test (POCT) performance and implementation of a quality management program, focusing on nucleic acid and antigen targeted technologies. Design and Methods The recommendations are based on current literature and expert opinion from the members of Canadian Society of Clinical Chemists (CSCC), and are intended for use inside or outside of healthcare settings that have varied levels of expertise and experience with POCT. Results and Conclusions Here we discuss sampling requirements, biosafety, SARS-CoV-2 point-of-care testing methodologies (with focus on Health Canada approved tests), test performance and limitations, test selection, testing utility, development and implementation of quality management systems, quality improvement, and medical and scientific oversight.

e18780 Background: Patients with hematologic malignancies have a lower vaccine response and higher rates for SARS CoV-2 morbidity and mortality. We present preliminary data focusing on humoral vaccine responses and correlates with disease subtype and treatment exposure. Methods: We analyzed data from 332 patients with a hematologic malignancy from May 1, 2021 – Jan 31, 2022 who received SARS-CoV-2 vaccination and performed a prospective cohort serologic study with the Elecsys® Anti-SARS-CoV-2-S test. Patients received homologous or heterologous vaccine combination of BNT162b2, mRNA1273, ChAdOx1 nCoV-19, and/or Ad26.COV2.S. Blood samples were obtained before any vaccination, 2-6 weeks after the second vaccine (2V), before third vaccine (3V), and 2-6 weeks after 3V. Results: The median age was 67 years (range 18-91years) with 41.9% female. At 2V, 11.5% and at 3V, 23.8% received heterologous vaccines. Treatment status at first vaccine dose significantly affected peak 2V antibody response (p < 0.05). Seropositive rate and median antibody titer after 2V for previously untreated patients were higher compared to patients on active therapy or had previously been treated. Treatment naïve (n = 60; seropositivity 85.1%; median titer 1306 U/mL; [Q1-Q3:11.4- > 2499]); first-line (1L) active therapy (n = 127;65.4%; 41.25 U/mL; [ < 0.8-592.5]); second-line and beyond (2L+) active therapy (n = 56; 60.7%; 2.6U/mL; [ < 0.8-154]); previous treatment with 1L (n = 66;64.8%; 118 U/mL; [ < 0.8- > 2499]); previous treatment with 2L+ (n = 23; 59.1%; 4U/mL; [ < 0.8-229.5]). Of 61 patients that were seronegative at 2V, 17 (27.9%) seroconverted after 3V. Anti-CD20 monoclonal antibody (mAb) containing therapy as the most recent treatment from 2V had the greatest impact on humoral response. Exposure to anti-CD20 mAb based regimens or as monotherapy revealed low antibody responses (n = 84; seropositivity 22.6%; median titer < 0.8 U/mL; Q1-Q3 [ < 0.8- < 0.8]). On analysis of indolent B-cell Non-Hodgkin Lymphomas whereby antiCD-20 mAb are often incorporated, treatment proximity to 2V impacted responses: < 3 months (n = 33; 22%; < 0.8 U/mL; [ < 0.8- < 0.8]) vs. 12-24 months (n = 4; 60%; 228 U/mL; [ < 0.8-232]). In contrast, tyrosine kinase inhibitor (n = 38; 100%; 858 U/mL; [221- > 2499]), proteosome inhibitor monotherapy (n = 4;100%; median titer 1520 U/mL; [462- > 2499]) were among the subgroups with the highest numerical responses, however, the addition of corticosteroids impacted vaccine response as seen in proteosome inhibitor with corticosteroids (n = 7; 85.7%; 6.6 U/mL; [1.8-115.2]). Conclusions: The humoral response from our single institution cohort identifies diminished responses depending on treatment status and the type of treatment including the proximity of treatment exposure to receipt of vaccination. Furthermore, vaccine boosters can induce antibody responses in patients who were previously seronegative.

BACKGROUNDThe role of humoral immunity in the coronavirus disease 2019 (COVID-19) is not fully understood owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome.METHODSUsing SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution.RESULTSWe identified 54 linear epitopes from the Spike (S) and Nucleocapsid (N) protein and showed that epitopes enabled higher resolution antibody profiling than protein antigens. Specifically, we found that antibody responses to the S(811-825), S(881-895) and N(156-170) epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant B.1.1.7 altered the specificity of the corresponding epitopes.CONCLUSIONSEpitope-resolved antibody testing not only offers a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, it may also be used as predictor of clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants in both the peptide array and latex agglutination formats.FUNDINGOntario Research Fund (ORF)-COVID-19 Rapid Research Fund, the Toronto COVID-19 Action Fund, Western University, the Lawson Health Research Institute, the London Health Sciences Foundation, and the AMOSO Innovation Fund.

Key Messages • Consider safety precautions and infection control processes, particularly in remote testing locations when using rapid SARS-CoV-2 devices. • Seek oversight and partnership with an accredited clinical laboratory for guidance on setting up a quality assurance framework. • Rapid-testing for SARS-CoV-2 requires method verification prior to clinical implementation.

The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has grown into worst public health crisis since the 1918 influenza pandemic. As COVID-19 continues to spread around the world, there is urgent need for a rapid, yet accurate antibody test to identify infected individuals in populations to inform health decisions. We have developed a rapid, accurate and cost-effective serologic test based on antibody-dependent agglutination of antigen-coated latex particles, which uses ∼5 µl plasma and takes 92% of patients had detectable antibodies on the day of positive viral RNA test, suggesting that seroconversion may occur earlier than previously thought and that the agglutination antibody test may complement RNA testing for POC diagnosis of SARS-CoV-2 infection.

Organic anion transporting polypeptide 2B1 (OATP2B1) is a widely expressed membrane transporter with diverse substrate specificity. In vitro and clinical studies suggest a role for intestinal OATP2B1 in the oral absorption of medications. Moreover, OATP2B1 is highly expressed in hepatocytes where it is thought to promote liver drug clearance. But until now, a shortcoming of studies implicating OATP2B1 in drug disposition has been a lack of in vivo models. Here, we report the development of a mouse model with targeted, global disruption of the Slco2b1 gene (KO) for examining the disposition of two confirmed mOATP2B1 substrates, namely fexofenadine and rosuvastatin. The plasma pharmacokinetics of intravenously administered fexofenadine was not different between KO and wildtype (WT) mice. However, after oral fexofenadine administration, KO mice had 70% and 41% lower maximal plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC0-last) than WT mice, respectively. In WT mice, co-administration of fexofenadine with grapefruit juice (GFJ) or apple juice (AJ) was associated with reduced Cmax by 80% and 88%, respectively, while the AUC0-last was lower by 35% and 70%, respectively. In KO mice, AJ co-administration reduced oral fexofenadine Cmax and AUC0-last by 67% and 59%, respectively, while GFJ had no effects. Intravenous and oral rosuvastatin pharmacokinetics were similar among WT and KO mice. We conclude that intestinal OATP2B1 is a determinant of oral fexofenadine absorption, as well as a target for fruit juice interactions. OATP2B1 does not significantly influence rosuvastatin disposition in mice. SIGNIFICANCE STATEMENT A novel mouse model with targeted disruption of the Slco2b1 gene revealed that OATP2B1 is a determinant of the oral absorption but not systemic disposition of fexofenadine, as well as a target of fruit juice interactions. Rosuvastatin oral and intravenous pharmacokinetics were not dependent on OATP2B1. These findings support the utility of the Slco2b1 knockout mice model for defining mechanisms of drug disposition at the intersection of in vitro and clinical pharmacology.

Dual antiplatelet therapy with clopidogrel and aspirin has become the mainstay of therapy for patients with acute coronary syndrome (ACS)6 undergoing percutaneous coronary interventions (PCI). Many pharmacokinetic and pharmacodynamic studies have demonstrated substantial interindividual variation in antiplatelet response with clopidogrel, a significant proportion of which is explained by the variation in plasma concentrations of the clopidogrel active metabolite. Clopidogrel is a prodrug that requires bioactivation by the highly polymorphic enzyme CYP2C19 to form the active metabolite. The growing body of literature has implicated the loss-of-function cytochrome P450, family 2, subfamily C, polypeptide 19 variant ( CYP2C19 * 2 )7 variant with an increased risk of major cardiovascular events. This evidence prompted the US Food and Drug Administration (FDA) to implement a boxed warning on the clopidogrel label describing the relationship between CYP2C19 pharmacogenetics and drug response, emphasizing the diminished effectiveness in CYP2C19 poor metabolizers. CYP2C19 pharmacogenetic testing is currently available to guide antiplatelet therapy; however, there are challenges with implementing pharmacogenetic-guided therapy in clinical practice. In this Q&A, 3 experts discuss the current state, challenges, and future direction of CYP2C19 pharmacogenetic testing for clopidogrel therapy. Can you briefly summarize the latest developments and evidence for pharmacogenetic testing in patients on clopidogrel therapy? Jean Hulot: Clopidogrel is a prodrug that requires hepatic bioactivation to generate an active metabolite with antiplatelet properties. The CYP2C19 enzyme is directly involved in this bioactivation process but its activity is genetically determined. A common loss-of-function genetic variant (named CYP2C19 * 2 ; c.681G>A; rs4244285) is associated with reduced formation of the clopidogrel active metabolite and reduced pharmacodynamic response to the drug, thus resulting in high on-treatment platelet reactivity and more frequent adverse cardiovascular events. This effect is particularly observed in homozygous carriers of the mutated allele (so-called poor metabolizers), who represent 3%–4% of Caucasian patients. …

Introduction We assessed the pharmacokinetic and pharmacodynamic impact of converting stable simultaneous pancreas-kidney (SPK) recipients from standard tacrolimus (Prograf) to long-acting tacrolimus (Advagraf). Methods In a randomized prospective crossover study, stable SPK recipients on Prograf were assigned to Prograf with 1:1 conversion to Advagraf or vice versa. Demographics, tacrolimus, mycophenolic acid levels, and Cylex CD4 + ATP levels were taken at specified intervals in addition to standard blood work. Results Twenty-one patients, who were a minimum of 1 year post-transplant, were entered into the study. No difference in tacrolimus or mycophenolic acid levels was noted between patients who were first assigned to Prograf or Advagraf. Additionally, Cylex levels as well as serum creatinine, lipase, and blood sugar levels were unchanged. There were no episodes of rejection during the 6-month study. Conclusions It is safe to convert between Prograf and Advagraf 1:1, without major impact on pharmacokinetics or pharmacodynamics in SPK recipients.

Background: Therapeutic drug monitoring of immunosuppressant drugs are used to monitor drug efficacy and toxicity and to prevent organ transplantation rejection. This study evaluates the analytical performance of semi-automated electrochemiluminescence immunoassays (ECLIA) for cyclosporine (CSA), tacrolimus (TAC) and sirolimus (SRL) on the Roche cobas e 411 analyzer at a major transplant hospital to identify method suitability and limitations.Methods:Residual whole blood samples from patients undergoing immunosuppressant therapy were used for evaluation. Experiments included imprecision, linearity, functional sensitivity, method comparisons and lot-to-lot assessments.Results:Total imprecision ranged from 3.3 to 7.1% for CSA, 3.9 to 9.4% for TAC, and 4.6 to 8.2% for SRL. Linearity was verified from 30.0 to 960.9 μg/L for CSA, from 1.1 to 27.1 μg/L for TAC, and from 0.5 to 32.3 µg/L for SRL. The functional sensitivity met the manufacturer’s claims and was determined to be rof 0.985 for CSA, 0.938 (95%CI: 0.895-0.981) androf 0.974 for TAC, and 0.842 (0.810-1.110) androf 0.982 for SRL. Deming regression analysis of comparisons with the LC–MS/MS method yielded slopes of 1.331 (95%CI: 1.167-1.496) androf 0.969 for CSA, 0.924 (95%CI: 0.843-1.005) androf 0.984 for TAC, and 0.971 (95%CI: 0.913-1.030) androf 0.993 for SRL.Conclusions:The cobas e 411 ECLIA for CSA, TAC, and SRL have acceptable precision, linearity, and functional sensitivity. The method comparisons correlated well with the ARCHITECT immunoassay and LC–MS/MS and is fit for therapeutic drug monitoring.

Human organic anion transporting polypeptide 2B1 (OATP2B1) is a membrane transporter that facilitates the cellular uptake of a number of endogenous compounds and drugs. OATP2B1 is widely expressed in tissues including the small intestine, liver, kidney, placenta, heart, skeletal muscle, and platelets. It was recently shown that differential promoter usage in tissues results in expression of five OATP2B1 transcriptional start site variants that use distinct first exons but share common subsequent exons. These variants are expected to encode either a full-length (OATP2B1-FL) or shortened protein lacking 22 N terminus amino acids (OATP2B1-Short). Little is known regarding the transport activity and regulation of OATP2B1 variants with N terminus truncation. Here, using absolute quantitative polymerase chain reaction, we find the full-length variant is the major form expressed in duodenum but the short variant predominates in liver. Using a transient heterologous cell expression system, we find that the transport activities of the short OATP2B1 variant toward substrates estrone sulfate and rosuvastatin are similar to the well characterized full-length variant. Transcriptional activity screening of the liver-enriched OATP2B1 variant promoter identified hepatocyte nuclear factor 4 α (HNF4 α ) as a novel transacting factor. With a combination of in silico screening, promoter mutation in cell-based reporter assays, small interfering RNA knockdown, and chromatin immunoprecipitation (ChIP) studies, we identified a functional HNF4 α binding site close to the transcription start site (−17 to −4 bp). We conclude that the major OATP2B1 protein form in liver is transport competent and its hepatic expression is regulated by HNF4 α .

AimsIt is thought that clopidogrel bioactivation and antiplatelet response are related to cytochrome P450 2C19 (CYP2C19). However, a recent study challenged this notion by proposing CYP2C19 as wholly irrelevant, while identifying paraoxonase-1 (PON1) and its Q192R polymorphism as the major driver of clopidogrel bioactivation and efficacy. The aim of this study was to systematically elucidate the mechanism and relative contribution of PON1 in comparison to CYP2C19 to clopidogrel bioactivation and antiplatelet response.Methods and resultsFirst, the influence of CYP2C19 and PON1 polymorphisms and plasma paraoxonase activity on clopidogrel active metabolite (H4) levels and antiplatelet response was assessed in a cohort of healthy subjects (n = 21) after administration of a single 75 mg dose of clopidogrel. There was a remarkably good correlation between H4 AUC (0-8 h) and antiplatelet response (r2 = 0.78). Furthermore, CYP2C19 but not PON1 genotype was predictive of H4 levels and antiplatelet response. There was no correlation between plasma paraoxonase activity and H4 levels. Secondly, metabolic profiling of clopidogrel in vitro confirmed the role of CYP2C19 in bioactivating clopidogrel to H4. However, heterologous expression of PON1 in cell-based systems revealed that PON1 cannot generate H4, but mediates the formation of another thiol metabolite, termed Endo. Importantly, Endo plasma levels in humans are nearly 20-fold lower than H4 and was not associated with any antiplatelet response.ConclusionOur results demonstrate that PON1 does not mediate clopidogrel active metabolite formation or antiplatelet action, while CYP2C19 activity and genotype remains a predictor of clopidogrel pharmacokinetics and antiplatelet response. © 2012 The Author.

Rationale : The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins. Interindividual differences in the activities of hepatic membrane drug transporters and metabolic enzymes are known to influence statin plasma pharmacokinetics and risk for myopathy. Interestingly, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity. Objective : We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro. Methods and Results : We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance–associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1. Conclusions : These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.

Folic acid is a vitamin essential for thymidylate and purine synthesis. The human proton-coupled folate transporter (hPCFT) has recently been identified as a pH-dependent folic acid transporter, and mutations in this transporter have been linked to hereditary folic acid malabsorption. In this study, we assessed hPCFT-mediated transport activity in vitro, intersubject variability of intestinal expression in relation to blood folates, and the relationship of proton-pump inhibitor (PPI) therapy on hPCFT expression in vivo. We created a Madin-Darby canine kidney strain II (MDCKII) cell line stably expressing hPCFT to evaluate its drug substrates and inhibitors. Intestinal pinch biopsies (duodenum, ileum, colon) were collected from patients undergoing routine endoscopy procedures, and expressed levels of hPCFT were determined by RT-PCR. When assessed using MDCKII-hPCFT cells, folic acid and methotrexate were found to be high-affinity hPCFT substrates. Sulfasalazine and pyrimethamine were noted to inhibit hPCFT activity with Ki values of 42.3 and 161.7 μmol/l, respectively. hPCFT was localized to the brush-border membrane of enterocytes with highest expression in the duodenum and reduced levels in the ileum and colon. When we assessed hPCFT expression in a subset of patients who were receiving PPI therapy, a near 50% reduction in duodenal hPCFT mRNA expression was noted. These results suggest that hPCFT transporter activity can be modulated by many drugs in clinical use, and expression of this transporter in the gastrointestinal tract is higher in the duodenum than more distal sites (duodenum > ileum > colon). Importantly, we note that PPI drug use appears to be associated with reduced hPCFT expression in vivo.