Pulmonary Embolism in Pregnancy.

Pulmonary embolism during and after pregnancy remains a significant contributor to maternal morbidity and mortality. Symptoms that would be a clear indicator of a pulmonary embolus in the nonpregnant population can be masked by pregnancy and its routine pregnancy-related symptoms. To affect a reduction in this severe maternal mortality indicator, physicians need to maintain a high degree of suspicion coupled with expedient testing.

Adapting an organ-on-chip device to study the effect of fetal sex and maternal race/ethnicity on preterm birth related intraamniotic inflammation leading to fetal neuroinflammation.

PROBLEM: Fetal neuroinflammation has been linked to preterm birth-related intraamniotic infection and inflammation; However, the contribution of fetal sex and maternal race/ethnicity is unknown. To determine if fetal sex and maternal race/ethnicity influence neuroinflammation, an organ-on-chip (OOC) model were established under normal or pathologic conditions utilizing amniotic fluid. METHOD OF STUDY: OOC is composed of two-cell culture chambers connected by Type IV collagen-coated microchannels. Human fetal astroglia (SVGp12) and microglia (HMC3) were co-cultured at an 80:20 ratio in the inner chamber. The outer chamber contained amniotic fluid (AF) from male and female fetuses of White Hispanic (WH) and African-American (AA) pregnant women with or without lipopolysaccharide (LPS-100 ng/ml) and incubated for 48 h. Glial migration (brightfield microscopy), activation (Immunocytochemistry), and cytokine production (Luminex assays) were quantified and compared (N = 4 for each category of sex and race/ethnicity). RESULTS: In a pooled analysis, AF+LPS did not induce glial activation or inflammatory changes compared to AF alone. When stratified by sex, male AF+LPS promoted significant glial activation (high CD11b:p < 0.05; low Iba1:p < 0.01) compared to male AF without LPS; however, this was not associated with changes in pro-inflammatory cytokines. When stratified by race/ethnicity, AF+LPS induced glial activation in both groups, but a differential increase in pro-inflammatory cytokines was seen between WH and AA AF (WH-interleukin-1ß: p < 0.05; AA-interleukin-8: p < 0.01). CONCLUSION: This OOC model of fetal neuroinflammation has determined that race/ethnicity differences do exist for perinatal brain injury. The fetal sex of neonates was not a determining factor of susceptibility to intraamniotic inflammation leading to neuroinflammation.

Disulfide disruption reverses mucus dysfunction in allergic airway disease.

Airway mucus is essential for lung defense, but excessive mucus in asthma obstructs airflow, leading to severe and potentially fatal outcomes. Current asthma treatments have minimal effects on mucus, and the lack of therapeutic options stems from a poor understanding of mucus function and dysfunction at a molecular level and in vivo. Biophysical properties of mucus are controlled by mucin glycoproteins that polymerize covalently via disulfide bonds. Once secreted, mucin glycopolymers can aggregate, form plugs, and block airflow. Here we show that reducing mucin disulfide bonds disrupts mucus in human asthmatics and reverses pathological effects of mucus hypersecretion in a mouse allergic asthma model. In mice, inhaled mucolytic treatment loosens mucus mesh, enhances mucociliary clearance, and abolishes airway hyperreactivity (AHR) to the bronchoprovocative agent methacholine. AHR reversal is directly related to reduced mucus plugging. These findings establish grounds for developing treatments to inhibit effects of mucus hypersecretion in asthma.

A rapid and reliable method for the quantitation of hydroxychloroquine in serum using turbulent flow liquid chromatography-tandem mass spectrometry.

BACKGROUND: Hydroxychloroquine is routinely used in managing systemic lupus erythematosus and rheumatoid arthritis. Whole blood levels are currently measured in the laboratory but at least one study suggests that serum levels may be equally useful. Moreover, serum samples are the preferred matrix type in the clinical laboratory as a result of their reduced complexity compared to whole blood. These observations suggest that the clinical utility of serum hydroxychloroquine levels needs to be reevaluated using larger studies and more robust assays. We report a turbulent flow LC-MS/MS method we developed for this purpose. METHODS: After protein precipitation from serum with 0.33 mol/l perchloric acid, hydroxychloroquine and its deuterated analog were injected onto a Cyclone turbulent flow column for sample cleanup. Analytical separation was accomplished on a HypersilGold C8 column with a gradient of water and methanol, each containing 0.1% formic acid and 10 mmol/l ammonium formate. Analytes were ionized and detected by electrospray ionization mass spectrometry with multiple reaction monitoring. RESULTS: Our method was linear from 15.7 to 2000 ng/ml. Total imprecision at multiple levels was <5% and accuracy was within ±15%. The method showed minimal carryover. Our extraction efficiency was 103% and the matrix factor was 101%. Comparison with a reference laboratory method identified constant bias but good correlation between the 2 methods. CONCLUSIONS: We present a novel turbulent flow liquid chromatography-tandem mass spectrometry method for quantification of hydroxychloroquine in serum. Our method has comparable sensitivity, selectivity, precision, accuracy, and linearity to previously reported methods. However, it offers simpler sample processing, shorter overall analysis time, and minimal carryover. These characteristics make our method well-suited for efficient analysis of the large number of samples necessary for studies on the clinical utility of serum HQ levels.

Alternative calibration strategies for the clinical laboratory: application to nortriptyline therapeutic drug monitoring.

BACKGROUND: The addition of a calibration curve with every run is both time-consuming and expensive for clinical mass spectrometry assays. We present alternative calibration strategies that eliminate the need for a calibration curve except as required by laboratory regulations. METHODS: We measured serum nortriptyline concentrations prospectively in 68 patients on 16 days over a 2-month period using a method employing calibration schemes that relied on the measurement of the response ratio (RR) corrected by the response factor (RF), i.e., a measurement of the RR for an equimolar mixture of the analyte and internal standard. Measurements were taken with contemporaneous RF (cRF) measurements as well as sporadic RF (sRF) measurements. The measurements with these alternative calibration schemes were compared against the clinical results obtained by interpolation on a calibration curve, and those differences were evaluated for analytical and clinical significance. RESULTS: The differences between the values measured by cRF and sRF calibration and interpolation on a calibration curve were not significant (P = 0.088 and P = 0.091, respectively). Both the cRF- and sRF-based calibration results demonstrated a low mean bias against the calibration curve interpolations of 3.69% (95% CI, -15.8% to 23.2%) and 3.11% (95% CI, -16.4% to 22.6%), respectively. When these results were classified as subtherapeutic, therapeutic, or supratherapeutic, there was categorical agreement in 95.6% of the cRF calibration results and 94.1% of the sRF results. CONCLUSIONS: cRF and sRF calibration in a clinically validated liquid chromatography-tandem mass spectrometry assay yields results that are analytically and clinically commensurate to those produced by interpolation with a calibration curve.

Rapid quantification of the aminoglycoside arbekacin in serum using high performance liquid chromatography-tandem mass spectrometry.

BACKGROUND: This project entails the development and validation of a method for quantification of the aminoglycoside antibiotic arbekacin in serum using liquid chromatography tandem mass spectrometry (LC-MS/MS) for therapeutic drug monitoring in future clinical trials. METHODS: Following a protein precipitation with 0.3 mol/l perchloric acid containing internal standard dibekacin at a concentration of 0.6 µg/ml, human serum samples containing arbekacin were analyzed using a Hypersil Gold PFP column and a liquid chromatography system. Elution occurred with a gradient of water and acetonitrile, each containing 0.05% (v/v) trifluoroacetic acid and 0.1% (v/v) formic acid. Analytes were detected over a 3.25 minute run time using a tandem mass spectrometer with a heated electrospray-ionization (HESI) source in positive ionization mode with selected reaction monitoring (SRM). Matrix effects, carryover, linearity, recovery, precision, and limit of quantification were carefully evaluated. RESULTS: The limit of quantification for arbekacin was 0.1 µg/ml. All simple and total precision CV's were less than 6.2%. The method was linear from 0.1 µg/ml to 45.9 µg/ml (slope of 0.973). The mean recovery ranged from 94.7 to 103.8%. No matrix effects were detected. CONCLUSIONS: This developed and validated LC-MS/MS method allows for the quantification of arbekacin in serum following protein precipitation.