Serologic Response to Vaccine for COVID-19 in Patients with Hematologic Malignancy: A Prospective Cohort Study.

BACKGROUND: Patients with hematological cancers have increased COVID-19 morbidity and mortality, and these patients show attenuated vaccine responses. This study aimed to characterize the longitudinal humoral immune responses to COVID-19 vaccination in patients with hematological malignancies. PATIENTS AND METHODS: We conducted a prospective cohort study, collecting samples from March 2021 to July 2022, from patients seen at a cancer treatment center in London, Ontario, Canada, who met the following eligibility criteria: age ≥18 years, diagnosed with a hematological malignancy, recipient of a COVID-19 vaccine during the study period, and able to provide informed consent. RESULTS: Median anti-S titers (MST) were 0.0, 64.0, and 680.5 U/mL following first (V1), second (V2), and third (V3) vaccine doses, respectively. Patients with lymphoid malignancies' response to vaccination was attenuated compared to myeloid malignancy patients after V2 and V3 (P < .001, P < .01). Active treatment was associated with lower antibody titers (MST 10) compared to treatment 12-24 months (MST 465, P = .04367) and >24 months (MST 1660.5, P = .0025) prior to vaccination. V3 significantly increased antibody titers compared to V2 for patients less than 3 months from treatment. Increasing age was associated with smaller antibody response following V2 (P < .05), but not following V3. Patients receiving anti-CD20 therapy did not demonstrate increased antibody titer levels after V3 (V2 MST 0, V3 MST 0; P > .05). CONCLUSION: We report an attenuated serologic response to COVID-19 vaccination in our study population of patients with hematological malignancy. The immune response to vaccination was affected by patient age, diagnosis, treatment, and timing of treatment exposure.

Plasma proteome of Long-COVID patients indicates HIF-mediated vasculo-proliferative disease with impact on brain and heart function.

AIMS: Long-COVID occurs after SARS-CoV-2 infection and results in diverse, prolonged symptoms. The present study aimed to unveil potential mechanisms, and to inform prognosis and treatment. METHODS: Plasma proteome from Long-COVID outpatients was analyzed in comparison to matched acutely ill COVID-19 (mild and severe) inpatients and healthy control subjects. The expression of 3072 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 cell redistribution with a dominant resting phenotype, as opposed to active, and neutrophils that formed extracellular traps. This potential resetting of cell phenotypes was reflected in prospective vascular events mediated by both angiopoietin-1 (ANGPT1) and vascular-endothelial growth factor-A (VEGFA). Several markers (ANGPT1, VEGFA, CCR7, CD56, citrullinated histone 3, elastase) were validated by serological methods in additional patient cohorts. Signaling of transforming growth factor-ß1 with probable connections to elevated EP/p300 suggested vascular inflammation and tumor necrosis factor-α driven pathways. In addition, a vascular proliferative state associated with hypoxia inducible factor 1 pathway suggested progression from acute COVID-19 to Long-COVID. The vasculo-proliferative process predicted in Long-COVID might contribute to changes in the organ-specific proteome reflective of neurologic and cardiometabolic dysfunction. CONCLUSIONS: Taken together, our findings point to a vasculo-proliferative process in Long-COVID that is likely initiated either prior hypoxia (localized or systemic) and/or stimulatory factors (i.e., cytokines, chemokines, growth factors, angiotensin, etc). Analyses of the plasma proteome, used as a surrogate for cellular signaling, unveiled potential organ-specific prognostic biomarkers and therapeutic targets.

Investigation of N,N,N-Trimethyl-L-alanyl-L-proline Betaine (TMAP) as a Biomarker of Kidney Function.

Glomerular filtration rate (GFR) is the most widely used tool for the measurement of kidney function, but endogenous biomarkers such as cystatin C and creatinine have limitations. A previous metabolomic study revealed N,N,N-trimethyl-L-alanyl-L-proline betaine (TMAP) to be reflective of kidney function. In this study, we developed a quantitative LCMS assay for the measurement of TMAP and evaluated TMAP as a biomarker of GFR. An assay to measure TMAP was developed using liquid chromatography-mass spectrometry. After validation of the method, we applied it to plasma samples from three distinct kidney disease patient cohorts: nondialysis chronic kidney disease (CKD) patients, patients receiving peritoneal and hemodialysis, and living kidney donors. We investigated whether TMAP was conserved in other mammalian and nonmammalian species, by analyzing plasma samples from Wistar rats with diet-induced CKD and searching for putative matches to the m/z for TMAP and its known fragments in the raw sample data repository "Metabolomics Workbench". The assay can measure plasma TMAP at a lower limit of quantitation (100 ng/mL) with an interday precision and accuracy of 12.8 and 12.1%, respectively. In all three patient cohorts, TMAP concentrations are significantly higher in patients with CKD than in controls with a normal GFR. Further, TMAP concentrations are also elevated in rats with CKD and TMAP is present in the sap produced from Acer saccharum trees. TMAP concentration is inversely related to GFR suggesting that it is a marker of kidney function. TMAP is present in nonmammalian species suggesting that it is part of a biologically conserved process.

Elevated vascular transformation blood biomarkers in Long-COVID indicate angiogenesis as a key pathophysiological mechanism.

BACKGROUND: Long-COVID is characterized by prolonged, diffuse symptoms months after acute COVID-19. Accurate diagnosis and targeted therapies for Long-COVID are lacking. We investigated vascular transformation biomarkers in Long-COVID patients. METHODS: A case-control study utilizing Long-COVID patients, one to six months (median 98.5 days) post-infection, with multiplex immunoassay measurement of sixteen blood biomarkers of vascular transformation, including ANG-1, P-SEL, MMP-1, VE-Cad, Syn-1, Endoglin, PECAM-1, VEGF-A, ICAM-1, VLA-4, E-SEL, thrombomodulin, VEGF-R2, VEGF-R3, VCAM-1 and VEGF-D. RESULTS: Fourteen vasculature transformation blood biomarkers were significantly elevated in Long-COVID outpatients, versus acutely ill COVID-19 inpatients and healthy controls subjects (P < 0.05). A unique two biomarker profile consisting of ANG-1/P-SEL was developed with machine learning, providing a classification accuracy for Long-COVID status of 96%. Individually, ANG-1 and P-SEL had excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, P < 0.0001; validated in a secondary cohort). Specific to Long-COVID, ANG-1 levels were associated with female sex and a lack of disease interventions at follow-up (P < 0.05). CONCLUSIONS: Long-COVID patients suffer prolonged, diffuse symptoms and poorer health. Vascular transformation blood biomarkers were significantly elevated in Long-COVID, with angiogenesis markers (ANG-1/P-SEL) providing classification accuracy of 96%. Vascular transformation blood biomarkers hold potential for diagnostics, and modulators of angiogenesis may have therapeutic efficacy.

Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern.

BACKGROUNDThe 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.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 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.CONCLUSIONEpitope-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.FUNDINGOntario 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.

Fexofenadine and Rosuvastatin Pharmacokinetics in Mice with Targeted Disruption of Organic Anion Transporting Polypeptide 2B1.

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. However, 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 knockout (KO) mouse model with targeted, global disruption of the Slco2b1 gene to examine the disposition of two confirmed mOATP2B1 substrates, namely, fexofenadine and rosuvastatin. The plasma pharmacokinetics of intravenously administered fexofenadine was not different between KO and wild-type (WT) mice. However, after oral fexofenadine administration, KO mice had 70% and 41% lower maximal plasma concentration (C max) and area under the plasma concentration-time curve (AUC0-last) than WT mice, respectively. In WT mice, coadministration of fexofenadine with grapefruit juice (GFJ) or apple juice (AJ) was associated with reduced C max by 80% and 88%, respectively, while the AUC0-last values were lower by 35% and 70%, respectively. In KO mice, AJ coadministration reduced oral fexofenadine C max and AUC0-last values 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 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 KO mouse model for defining mechanisms of drug disposition at the intersection of in vitro and clinical pharmacology.

Transport function and transcriptional regulation of a liver-enriched human organic anion transporting polypeptide 2B1 transcriptional start site variant.

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 (HNF4α) 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 HNF4α 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 HNF4α.

Human skeletal muscle drug transporters determine local exposure and toxicity of statins.

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.

The human proton-coupled folate transporter (hPCFT): modulation of intestinal expression and function by drugs.

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 micromol/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.