Human VH4-34 antibodies derived from B1 cells are more frequently autoreactive than VH4-34 antibodies derived from memory cells.

Human B1 cells produce natural antibodies characterized by overutilization of heavy chain variable region VH4-34 in comparison to other B cell populations. VH4-34-containing antibodies have been reported to be autoreactive and to be associated with lupus and other autoimmune dyscrasias. However, it has been unclear to what extent VH4-34 antibodies manifest autoreactivity in B1 cells or other B cell populations-in other words, are VH4-34 containing antibodies autoreactive wherever found, or mainly within the B1 cell population? To address this issue we sort purified single human B1 and memory B cells and then amplified, sequenced, cloned and expressed VH4-34-containing antibodies from 76 individual B cells. Each of these antibodies was tested for autoreactivity by HEp-2 IFA and autoantigen ELISA. Antibodies were scored as autoreactive if positive by either assay. We found VH4-34 antibodies rescued from B1 cells were much more frequently autoreactive (14/48) than VH4-34 antibodies rescued from memory B cells (2/28). Among B1 cell antibodies, 4 were HEp-2+, 6 were dsDNA+ and 4 were positive for both. Considering only HEp-2+ antibodies, again these were found more frequently among B1 cell VH4-34 antibodies (8/48) than memory B cell VH4-34 antibodies (1/28). We found autoreactivity was associated with greater CDR3 length, as expected; however, we found no association between autoreactivity and a previously described FR1 "hydrophobic patch". Our results indicate that autoreactive VH4-34-containing antibodies tend to reside within the human B1 cell population.

A comparison of the effect of X and gamma irradiation on red cell storage quality.

BACKGROUND AND OBJECTIVES: Irradiation of red cell components is indicated for recipients at risk of transfusion-associated graft vs. host disease. Current technologies available comprise of a gamma (γ) or an x source of radiation. The benefits of x vs. γ include non-radioactivity and hence no decay of the source. We aimed to compare the effect of the two technologies on red cell component storage quality post-irradiation. MATERIALS AND METHODS: Paired units of red cell concentrates (RCC), neonatal red cell splits (RCS), red cells for intra-uterine transfusion (IUT) or neonatal exchange transfusion (ExTx) were either γ- or x-irradiated. Units were sampled and tested for five storage parameters until the end of shelf life. Equivalence analysis of storage quality parameters was performed for pairs of the same components (RCC, RCS, IUT or ExTx) that were either γ- or x-irradiated. RESULTS: Nearly all component comparisons studied showed equivalence between γ and x irradiation for haemolysis, ATP, 2,3-DPG, potassium release and lactate production. The exceptions found that were deemed non-equivalent were higher haemolysis with x irradiation for ExTx, lower 2,3-DPG with x irradiation for RCS irradiated early and higher ATP with x irradiation for IUT. However, these differences were considered not clinically significant. CONCLUSION: This study has demonstrated that a range of red cell components for use in different age groups are of acceptable quality following x irradiation, with only small differences deemed clinically insignificant in a few of the measured parameters.

Tear osmolarity is sensitive to exercise-induced fluid loss but is not associated with common hydration measures in a field setting.

This investigation (i) examined changes in tear osmolarity in response to fluid loss that occurs with exercise in a field setting, and (ii) compared tear osmolarity with common field and laboratory hydration measures.  Sixty-three participants [age 27.8 ± 8.4 years, body mass 72.15 ± 10.61 kg] completed a self-paced 10 km run outside on a predetermined course. Body mass, tear fluid, venous blood and urine samples were collected immediately before and after exercise.  Significant (p < 0.001) reductions in body mass (1.71 ± 0.44%) and increases in tear osmolarity (8 ± 15 mOsm.L-1), plasma osmolality (7 ± 8 mOsm.kg-1), and urine specific gravity (0.0014 ± 0.0042 g.mL-1; p = 0.008) were observed following exercise. Pre- to post-exercise change in tear osmolarity was not significantly correlated (all p > 0.05) with plasma osmolality (rs = 0.24), urine osmolality (rs = 0.14), urine specific gravity (rs = 0.13) or relative body mass loss (r = 0.20).  Tear osmolarity is responsive to exercise-induced fluid loss but does not correlate with the changes observed using other common measures of hydration status in the field setting. Practitioners shouldn't directly compare or replace other common hydration measures with tear osmolarity in the field. ABBREVIATIONS: BML: Body Mass Loss; CV: Coefficient of Variation; Posm: Plasma osmolality; SD: Standard Deviation; Tosm: Tear Osmolarity; Uosm: Urine Osmolality; USG: Urine Specific Gravity; WBGT: Wet bulb globe thermometer.

A LC-MS/MS Method for Quantifying Adenosine, Guanosine and Inosine Nucleotides in Human Cells.

PURPOSE: To develop and validate a method for the simultaneous measurement of adenosine, guanosine, and inosine derived from mono (MP) and triphosphate (TP) forms in peripheral blood mononuclear cells (PBMCs), red blood cells (RBCs) and dried blood spots (DBS). METHODS: Solid phase extraction of cell lysates followed by dephosphorylation to molar equivalent nucleoside and LC-MS/MS quantification. RESULTS: The assay was linear for each of the three quantification ranges: 10-2000, 1.0-200 and 0.25-50 pmol/sample for adenosine, guanosine, and inosine, respectively. Intraassay (n = 6) and interassay (n = 18) precision (%CV) were within 1.7 to 16% while accuracy (%deviation) was within -11.5 to 14.7% for all three analytes. Nucleotide monophosphates were less concentrated than triphosphates (except for inosine) and levels in PBMCs were higher than RBCs for all three nucleotides (10, 55, and 5.6 fold for ATP, GTP and ITP, respectively). DBS samples had an average (SD) of -26% (22.6%) lower TP and 184% (173%) higher MP levels compared to paired RBC lysates, suggesting hydrolysis of the TP in DBS. CONCLUSION: This method was accurate and precise for physiologically relevant concentrations of adenosine, guanosine and inosine nucleotides in mono- and triphosphate forms, providing a bioanalytical tool for quantitation of nucleotides for clinical studies.

Plasma and intracellular ribavirin concentrations are not significantly altered by abacavir in hepatitis C virus-infected patients.

OBJECTIVES: The objective of this study was to evaluate the effects of abacavir on intracellular ribavirin triphosphate and plasma ribavirin trough concentrations. METHODS: Hepatitis C virus-infected subjects who had been cured or failed prior treatment were randomized to 8 weeks of ribavirin alone (N = 14; weight-based dosing) or weight-based ribavirin + abacavir (N = 14; 300 mg orally every 12 h). Ribavirin trough concentrations were measured on days 14, 28, 42 and 56; PBMCs for ribavirin triphosphate determination were sampled on days 28 and 56, pre-dose and at 6 and 12 h post-dose. ClinicalTrials.gov: NCT01052701. RESULTS: Twenty-six subjects completed the study (24 males, 17 Caucasians, median age 52 years); 2 were excluded for missed pharmacokinetic visits. Fourteen subjects received ribavirin + abacavir and 12 received ribavirin alone. Mean ±â€ŠSD plasma ribavirin trough concentrations (µg/mL) on days 14, 28, 42 and 56, respectively, were not significantly different with coadministration of abacavir (1.54 ±â€Š0.60, 1.93 ±â€Š0.54, 2.14 ±â€Š0.73 and 2.54 ±â€Š1.05) compared with ribavirin alone (1.48 ±â€Š0.32, 2.08 ±â€Š0.41, 2.32 ±â€Š0.47 and 2.60 ±â€Š0.62) (P > 0.40). Mean ribavirin triphosphate intracellular concentrations (pmol/10(6) cells) on days 28 and 56, respectively, did not differ statistically between abacavir users (11.98 ±â€Š9.86 and 15.87 ±â€Š12.52) and non-users (15.91 ±â€Š15.58 and 15.93 ±â€Š12.69) (P > 0.4). Adverse events were mild or moderate, except for three grade 3 occurrences of transaminitis, cholecystitis and low absolute neutrophil count that resolved and were judged not attributable to study medications. CONCLUSIONS: Abacavir did not significantly alter ribavirin or ribavirin triphosphate concentrations.

Measurement of intracellular ribavirin mono-, di- and triphosphate using solid phase extraction and LC-MS/MS quantification.

Ribavirin (RBV) is a nucleoside analog used to treat a variety of DNA and RNA viruses. RBV undergoes intracellular phosphorylation to a mono- (MP), di- (DP), and triphosphate (TP). The phosphorylated forms have been associated with the mechanisms of antiviral effect observed in vitro, but the intracellular pharmacology of the drug has not been well characterized in vivo. A highly sensitive LC-MS/MS method was developed and validated for the determination of intracellular RBV MP, DP, and TP in multiple cell matrix types. For this method, the individual MP, DP, and TP fractions were isolated from lysed intracellular matrix using strong anion exchange solid phase extraction, dephosphorylated to parent RBV, desalted and concentrated and quantified using LC-MS/MS. The method utilized a stable labeled internal standard (RBV-(13)C5) which facilitated accuracy (% deviation within ±15%) and precision (coefficient of variation of ≤15%). The quantifiable linear range for the assay was 0.50 to 200 pmol/sample. The method was applied to the measurement of RBV MP, DP, and TP in human peripheral blood mononuclear cells (PBMC), red blood cells (RBC), and dried blood spot (DBS) samples obtained from patients taking RBV for the treatment of chronic Hepatitis C virus infection.

Pharmacokinetic interaction between boceprevir and etravirine in HIV/HCV seronegative volunteers.

OBJECTIVE: The primary aim of this study was to determine the bioequivalence of boceprevir, an HCV protease inhibitor and etravirine, an HIV non-nucleoside reverse transcriptase inhibitor; area under the concentration time curve (AUC(0,τ)); maximum concentration (C(max)); and trough concentration (C(8) or C(min)) when administered in combination versus alone. DESIGN: Open-label crossover study in healthy volunteers. METHODS: Boceprevir, etravirine, and the combination were administered for 11-14 days with intensive sampling between days 11 and 14 of each sequence. Boceprevir and etravirine were quantified using validated liquid chromatography coupled with tandem mass spectrometry and high-performance liquid chromatography/ultraviolet assays, respectively and pharmacokinetics determined using noncompartmental methods. Geometric mean ratios (GMRs) and 90% confidence interval (CI) for the combination versus each drug alone were evaluated using 2 one-sided t tests. The hypothesis of equivalence was rejected if 90% GMR CI was not contained in the interval (0.8-1.25). RESULTS: Twenty subjects completed study. GMRs (90% CI) for etravirine AUC(o,τ), C(max), and C(min) were 0.77 (0.66 to 0.91), 0.76 (0.68 to 0.85), and 0.71 (0.54 to 0.95), respectively, in combination versus alone. Boceprevir GMRs (90% CI) for AUC(o,τ), C(max), and C(8) were 1.10 (0.94 to 1.28), 1.10 (0.94 to 1.29), and 0.88 (0.66 to 1.17), respectively, in combination versus alone. All adverse events (n = 112) were mild or moderate. Six subjects discontinued: 4 due to rash, 1 due to central nervous system effects, and 1 for a presumed viral illness. CONCLUSIONS: Etravirine AUC(o,τ), C(max), and C(min)decreased 23%, 24%, and 29%, respectively, with boceprevir. Boceprevir AUC(0,τ) and C(max) increased 10% and C(8) decreased 12% by etravirine. Additional research is needed to elucidate the mechanism(s) and therapeutic implications of the observed interaction.

Determination of nucleoside analog mono-, di-, and tri-phosphates in cellular matrix by solid phase extraction and ultra-sensitive LC-MS/MS detection.

An ultra-sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of nucleotide analogs from lysed intracellular matrix. The method utilized a strong anion exchange isolation of mono-(MP), di-(DP), and tri-phosphates (TP) from intracellular matrix. Each fraction was then dephosphorylated to the parent moiety yielding a molar equivalent to the original nucleotide analog intracellular concentration. The analytical portion of the methodology was optimized in specific nucleoside analog centric modes (i.e. tenofovir (TFV) centric, zidovudine (ZDV) centric), which included desalting/concentration by solid phase extraction and detection by LC-MS/MS. Nucleotide analog MP-, DP-, and TP-determined on the TFV centric mode of analysis include TFV, lamivudine (3TC), and emtricitibine (FTC). The quantifiable linear range for TFV was 2.5-2000 fmol/sample, and that for 3TC/FTC was 0.1 200 pmol/sample. Nucleoside analog MP-, DP-, and TP-determined on the ZDV centric mode of analysis included 3TC and ZDV. The quantifiable linear range for 3TC was 0.1 100 pmol/sample, and 5-2000 fmol/sample for ZDV. Stable labeled isotopic internal standards facilitated accuracy and precision in alternative cell matrices, which supported the intended use of the method for MP, DP, and TP determinations in various cell types. The method was successfully applied to clinical research samples generating novel intracellular information for TFV, FTC, ZDV, and 3TC nucleotides. This document outlines method development, validation, and application to clinical research.

Effect of antacids on the pharmacokinetics of raltegravir in human immunodeficiency virus-seronegative volunteers.

Raltegravir's divalent metal ion chelating motif may predispose the drug to interactions with divalent cations. We determined whether a divalent cation-containing antacid interacted with raltegravir. Twelve HIV-1-seronegative subjects were enrolled in this randomized, prospective, crossover study of single-dose raltegravir (400 mg) with and without an antacid. Subjects underwent two intensive pharmacokinetic visits in the fasted state separated by a 5- to 12-day washout period. With simultaneous antacid administration, time to peak raltegravir concentration occurred 2 h sooner (P = 0.002) and there was a 67% lower raltegravir concentration at 12 h postdose (P < 0.0001) than with administration of raltegravir alone. The raltegravir area under the-concentration-time curve from 0 to 12 h and maximum concentration were unchanged with the addition of an antacid. Studies are needed to determine the clinical relevance of this interaction, whether it remains after multiple dosing to steady state, whether it is mitigated by temporal separation, and whether raltegravir interacts with divalent cation-containing vitamins, supplements, or foods.

Atazanavir pharmacokinetics in genetically determined CYP3A5 expressors versus non-expressors.

OBJECTIVES: The objective of this study was to compare atazanavir pharmacokinetics in genetically determined CYP3A5 expressors versus non-expressors. METHODS: HIV-negative adult volunteers were pre-screened for CYP3A5 *3, *6 and *7 polymorphisms and enrollment was balanced for CYP3A5 expressor status, gender and race (African-American versus non-African-American). Participants received atazanavir 400 mg daily for 7 days followed by atazanavir/ritonavir 300 mg/100 mg daily for 7 days with pharmacokinetic studies on days 7 and 14. Other measures collected were bilirubin, UGT1A1 *28, and ABCB1 1236C > T, 2677G > T/A and 3435C > T genotypes. Data analyses utilized least squares regression. RESULTS: Fifteen CYP3A5 expressors and 16 non-expressors participated. The day 7 atazanavir oral clearance (CL/F) was 1.39-fold faster (0.25 versus 0.18 L/h/kg; P = 0.045) and the C(min) was half (87 versus 171 ng/mL; P = 0.044) in CYP3A5 expressors versus non-expressors. Non-African-American CYP3A5 expressor males had 2.1-fold faster CL/F (P = 0.003) and <20% the C(min) (P = 0.0001) compared with non-African-American non-expressor males. No overall CYP3A5 expressor effects were observed during the ritonavir phase. One or two copies of wild-type ABCB1 haplotype (1236C/2677G/3435C) was predictive of slower atazanavir and ritonavir CL/F compared with zero copies (P < 0.06). Indirect bilirubin increased 1.6- to 2.8-fold more in subjects with UGT1A1 *28/*28 versus *1/*28 or *1/*1. CONCLUSIONS: CYP3A5 expressors had faster atazanavir CL/F and lower C(min) than non-expressors. The effect was most pronounced in non-African-American men. Ritonavir lessened CYP3A5 expressor effects. The wild-type ABCB1 CGC haplotype was associated with slower CL/F and the UGT1A1 *28 genotype was associated with increased bilirubin. Thus, CYP3A5, ABCB1 and UGT1A1 polymorphisms are associated with atazanavir pharmacokinetics and pharmacodynamics in vivo.

Liquid chromatography-tandem mass spectrometric determination of tenofovir-diphosphate in human peripheral blood mononuclear cells.

To facilitate the evaluation of drug safety, virologic activity, and pharmacokinetics, an anion exchange isolation of tenofovir-diphosphate (TFV-DP) from human peripheral blood mononuclear cells (hPBMCs), coupled with dephosphorylation, desaltation, and detection by LC-MS-MS was validated. hPBMCs were harvested from whole blood, lysed, and a suspension of intracellular tenofovir moieties was produced. TFV-DP was isolated from TFV-monophosphate (TFV-MP) and tenofovir (TFV), dephosphorylated with acid phosphatase to form TFV and then desalted and concentrated, making it possible for tandem mass spectral detection. An LC-MS-MS methodology was developed and validated for the determination of TFV concentrations, which directly correspond with the intra-hPBMC TFV-DP concentration. The assay was linear in the range of 50-10,000 fmol per sample. The lower limit of quantitation (LLOQ) of the method is 10 fmol per million cells with 5 million hPBMCs used. This paper outlines the development and validation of the determination of TFV-DP concentrations in femtomoles per million hPBMCs.

Quantitation of zidovudine triphosphate concentrations from human peripheral blood mononuclear cells by anion exchange solid phase extraction and liquid chromatography-tandem mass spectroscopy; an indirect quantitation methodology.

To facilitate the assessment of drug safety and determination of phamacokinetics, an anion exchange isolation of zidovudine triphosphate (ZDV-TP) from human peripheral blood mononuclear cells (hPBMC), coupled with dephosphorylation, desaltation, and detection by liquid chromatography-tandem mass spectroscopy (LC-MS-MS) was validated. hPBMCs were harvested from whole blood, lysed, and a suspension of intracellular ZDV-TP was produced. ZDV-TP was isolated from ZDV, ZDV-monophosphate (ZDV-MP), and ZDV-diphosphate (ZDV-DP), which were all present in the cell lysate, by performing a salt gradient anion exchange SPE. Isolated ZDV-TP was dephosphorylated with acid phosphatase to its parent drug form, ZDV. ZDV was then desalted and concentrated for tandem mass spectral detection. An LC-MS-MS methodology was developed and validated for the determination of molar ZDV directly corresponding to the intra-hPBMC molar ZDV-TP concentration. ZDV-TP concentrations were determined in femtomoles per million hPBMCs (fmol/10(6)cells). The assay was able to determine ZDV-TP concentrations accurately and precisely within the range of 5-640 fmol/10(6)cells with 10 million cells per sample analyzed. Inter- and intra-day accuracy and precision data for back calculated standards and quality controls fell within 15% of nominal. The assay correlated well with a previous ELISA method developed and validated in our laboratory, and has been successfully used to quantitate ZDV-TP concentrations in patients being routinely monitored and treated with ZDV.

Can textbook covers be used to increase poison center utilization?

An education program was implemented at a regional poison center to increase use of a new nationwide 800 number (800/222-1222) in counties in our region that had low rates of utilization. We identified 10 counties with the lowest utilization rates and provided textbook covers to the elementary and secondary schools in these areas. The covers contained the poison help logo and information about what to do if a poisoning occurs. Changes in utilization rate for these counties were compared to similar counties over the course of a year. Utilization rates increased in both sets of counties over the study period, but there was no significant difference (p = 0.84) between the 2 groups. Use of textbook covers to increase awareness and utilization made little impact beyond our normal efforts and was not cost-effective.