Statins Prevent the Deleterious Consequences of Placental Chemerin Upregulation in Preeclampsia.

BACKGROUND: Chemerin, an inflammatory adipokine, is upregulated in preeclampsia, and its placental overexpression results in preeclampsia-like symptoms in mice. Statins may lower chemerin. METHODS: Chemerin was determined in a prospective cohort study in women suspected of preeclampsia and evaluated as a predictor versus the sFlt-1 (soluble fms-like tyrosine kinase-1)/PlGF (placental growth factor) ratio. Chemerin release was studied in perfused placentas and placental explants with or without the statins pravastatin and fluvastatin. We also addressed statin placental passage and the effects of chemerin in chorionic plate arteries. RESULTS: Serum chemerin was elevated in women with preeclampsia, and its addition to a predictive model yielded significant effects on top of the sFlt-1/PlGF ratio to predict preeclampsia and its fetal complications. Perfused placentas and explants of preeclamptic women released more chemerin and sFlt-1 and less PlGF than those of healthy pregnant women. Statins reversed this. Both statins entered the fetal compartment, and the fetal/maternal concentration ratio of pravastatin was twice that of fluvastatin. Chemerin constricted plate arteries, and this was blocked by a chemerin receptor antagonist and pravastatin. Chemerin did not potentiate endothelin-1 in chorionic plate arteries. In explants, statins upregulated low-density lipoprotein receptor expression, which relies on the same transcription factor as chemerin, and NO release. CONCLUSIONS: Chemerin is a biomarker for preeclampsia, and statins both prevent its placental upregulation and effects, in an NO and low-density lipoprotein receptor-dependent manner. Combined with their capacity to improve the sFlt-1/PlGF ratio, this offers an attractive mechanism by which statins may prevent or treat preeclampsia.

P-glycoprotein, FK-binding Protein-12, and the Intracellular Tacrolimus Concentration in T-lymphocytes and Monocytes of Kidney Transplant Recipients.

BACKGROUND: . Transplant recipients may develop rejection despite having adequate tacrolimus whole blood predose concentrations (C 0 ). The intra-immune cellular concentration is potentially a better target than C 0 . However, little is known regarding intracellular tacrolimus concentration in T-lymphocytes and monocytes. We investigated the tacrolimus concentrations in both cell types and their relation with the expression and activity of FK-binding protein (FKBP)-12 and P-glycoprotein (P-gp). METHODS: . T-lymphocytes and monocytes were isolated from kidney transplant recipients followed by intracellular tacrolimus concentration measurement. FKBP-12 and P-gp were quantified with Western blot, flow cytometry, and the Rhodamine-123 assay. Interleukin-2 and interferon-γ in T-lymphocytes were measured to quantify the effect of tacrolimus. RESULTS: . Tacrolimus concentration in T-lymphocytes was lower than in monocytes (15.3 [8.5-33.4] versus 131.0 [73.5-225.1] pg/million cells; P < 0.001). The activity of P-gp (measured by Rhodamine-123 assay) was higher in T-lymphocytes than in monocytes. Flow cytometry demonstrated a higher expression of P-gp (normalized mean fluorescence intensity 1.5 [1.2-1.7] versus 1.2 [1.1-1.4]; P = 0.012) and a lower expression of FKBP-12 (normalized mean fluorescence intensity 1.3 [1.2-1.7] versus 1.5 [1.4-2.0]; P = 0.011) in T-lymphocytes than monocytes. Western blot confirmed these observations. The addition of verapamil, a P-gp inhibitor, resulted in a 2-fold higher intra-T-cell tacrolimus concentration. This was accompanied by a significantly fewer cytokine-producing cells. CONCLUSIONS: . T-lymphocytes have a higher activity of P-gp and lower concentration of the FKBP-12 compared with monocytes. This explains the relatively lower tacrolimus concentration in T-lymphocytes. The addition of verapamil prevents loss of intracellular tacrolimus during the cell isolation process and is required to ensure adequate intracellular concentration measurement.

The use of freeze-dried blood samples affects the results of a dried blood spot analysis.

Dried blood spot (DBS) microsampling has several advantages over venous blood sampling. In a clinical validation study of tacrolimus microsampling it was noted that tacrolimus DBS concentrations ([Tac]DBS) were systematically higher than tacrolimus whole-blood concentrations ([Tac]WB). This observation was explored by investigating the effect of using freeze-dried standards (STFD) for [Tac]DBS measurement. For all experiments, both non-frozen whole-blood samples and whole-blood samples that were frozen and thawed (to simulate freeze-drying) of 10 patients were analyzed. Multiple tacrolimus concentrations were measured: 1) [Tac]WB, 2) [Tac]DBS, where 15 µL was volumetrically applied to a pre-punched DBS disk, and 3) [Tac]DBS, where 50 µL was applied before a 6 mm DBS disk was punched from the card. All tacrolimus concentrations were determined independently using STFD and standards made of non-frozen blood spiked with tacrolimus (STSP). In both non-frozen and frozen and thawed whole-blood samples, [Tac]WB measured with STFD appeared similar to [Tac]WB measured with STSP (Ratios 1.061 and 1.077, respectively). In non-frozen samples, the median ratio between the [Tac]DBS measured with STFD, and [Tac]WB measured with STFD (the reference method), was 1.396. When blood was volumetrically applied to the DBS card (to eliminate the effect of the spreading over the filter paper), this ratio was 1.009. In conclusion, when using DBS microsampling to quantify concentrations of analytes, one should be aware that using the commercially available freeze-dried blood samples for the preparation of standards may affect the spreading of blood on the filter-paper, leading to a systematic error in the results.

Association Between the Intracellular Tacrolimus Concentration in CD3 + T Lymphocytes and CD14 + Monocytes and Acute Kidney Transplant Rejection.

BACKGROUND: Intracellular tacrolimus concentration in peripheral blood mononuclear cells (PBMCs) (TAC [PBMC] ) has been proposed to better represent its active concentration than its whole blood concentration. As tacrolimus acts on T lymphocytes and other white blood cells, including monocytes, we investigated the association of tacrolimus concentration in CD3 + T lymphocytes (TAC [CD3] ) and CD14 + monocytes (TAC [CD14] ) with acute rejection after kidney transplantation. METHODS: From a total of 61 samples in this case-control study, 28 samples were obtained during biopsy-proven acute rejection (rejection group), and 33 samples were obtained in the absence of rejection (control group). PBMCs were collected from both cryopreserved (retrospectively) and freshly obtained (prospectively) samples. CD3 + T lymphocytes and CD14 + monocytes were isolated from PBMCs, and their intracellular tacrolimus concentrations were measured. RESULTS: The correlation between tacrolimus whole-blood and intracellular concentrations was poor. TAC [CD3] was significantly lower than TAC [CD14] (median 12.8 versus 81.6 pg/million cells; P < 0.001). No difference in TAC [PBMC] (48.5 versus 44.4 pg/million cells; P = 0.82), TAC [CD3] (13.4 versus 12.5 pg/million cells; P = 0.28), and TAC [CD14] (90.0 versus 72.8 pg/million cells; P = 0.27) was found between the rejection and control groups. However, freshly isolated PBMCs showed significantly higher TAC [PBMC] than PBMCs from cryopreserved samples. Subgroup analysis of intracellular tacrolimus concentrations from freshly isolated cells did not show a difference between rejectors and nonrejectors. CONCLUSIONS: Differences in TAC [CD3] and TAC [CD14] between patients with and without rejection could not be demonstrated. However, further optimization of the cell isolation process is required because a difference in TAC [PBMC] between fresh and cryopreserved cells was observed. These results need to be confirmed in a study with a larger number of patients.

Monitoring intracellular tacrolimus concentrations and its relationship with rejection in the early phase after renal transplantation.

INTRODUCTION: After kidney transplantation, rejection and drug-related toxicity occur despite tacrolimus whole-blood pre-dose concentrations ([Tac]blood) being within the target range. The tacrolimus concentration within peripheral blood mononuclear cells ([Tac]cells) might correlate better with clinical outcomes. The aim of this study was to investigate the correlation between [Tac]blood and [Tac]cells, the evolution of [Tac]cells and the [Tac]cells/[Tac]blood ratio, and to assess the relationship between tacrolimus concentrations and the occurrence of rejection. METHODS: In this prospective study, samples for the measurement of [Tac]blood and [Tac]cells were collected on days 3 and 10 after kidney transplantation, and on the morning of a for-cause kidney transplant biopsy. Biopsies were reviewed according to the Banff 2019 update. RESULTS: Eighty-three [Tac]cells samples were measured of 44 kidney transplant recipients. The correlation between [Tac]cells and [Tac]blood was poor (Pearson's r = 0.56 (day 3); r = 0.20 (day 10)). Both the dose-corrected [Tac]cells and the [Tac]cells/[Tac]blood ratio were not significantly different between days 3 and 10, and the median inter-occasion variability of the dose-corrected [Tac]cells and the [Tac]cells/[Tac]blood ratio were 19.4% and 23.4%, respectively (n = 24). Neither [Tac]cells, [Tac]blood, nor the [Tac]cells/[Tac]blood ratio were significantly different between patients with biopsy-proven acute rejection (n = 4) and patients with acute tubular necrosis (n = 4) or a cancelled biopsy (n = 9; p > 0.05). CONCLUSION: Tacrolimus exposure and distribution appeared stable in the early phase after transplantation. [Tac]cells was not significantly associated with the occurrence of rejection. A possible explanation for these results might be related to the low number of patients included in this study and also due to the fact that PBMCs are not a specific enough matrix to monitor tacrolimus concentrations.

Development and Validation of Hematocrit Level Measurement in Dried Blood Spots Using Near-Infrared Spectroscopy.

BACKGROUND: Dried blood spots (DBSs) have gained recent popularity as a sampling method for therapeutic drug monitoring. For patients, DBS sampling has several advantages over venous blood sampling. However, technical issues primarily influenced by hematocrit levels, interfere with the implementation of this method in daily clinical practice. The results of concentration measurements of drugs that are influenced by hematocrit should be corrected for hematocrit levels. In this article, we developed a fast, nondestructive, near-infrared (NIR)-based method for measuring the hematocrit in DBSs. METHOD: Using a partial least squares algorithm, an NIR-based quantification method was developed for measuring hematocrit levels of 0.19-0.49 L/L. Residual venous blood of 522 patients was used to build this partial least squares model. The validity of the method was evaluated using 40 patient samples. DBSs were created by adding a small amount (50 µL) of blood on a Whatman filter paper and drying for 24 hours in a desiccator cabinet. The robustness was evaluated by measuring 24 additional samples with a high hemolysis, icterus, and lipemia (HIL) index. The hematocrit values obtained using a Sysmex XN hemocytometry analyzer were used as reference. RESULTS: The difference between hematocrit measurements obtained with NIR spectroscopy and a hemocytometry analyzer was <15% for the 40 samples. The accuracy (≤9%) and precision (≤7%) for all the quality control samples were within the acceptance criteria of <15%. The intraassay and interassay coefficient of variability was ≤3% and ≤6%, respectively, for the different quality control levels. There were no deviations in the measurements for the samples with high HIL indices. The stability of hematocrit in DBS was up to 14 days for all levels. CONCLUSIONS: We developed and validated a hematocrit model using NIR spectroscopy. This nondestructive, accurate, and reproducible method has a short analysis time (51 seconds), and can be used to analyze DBS samples stored for up to 2 weeks in a desiccator cabinet.

Simultaneous quantification of ambrisentan, macitentan and sitaxentan in human plasma using UPLC-MS/MS.

Endothelin receptor antagonists (ERAs) such as, ambrisentan, macitentan and sitaxentan are primarily used for the treatment of pulmonary arterial hypertension. Considering the rise in endothelin in pre-eclampsia, ERAs may also be useful in its treatment. To evaluate the pharmacokinetics of ERAs, a rapid ultra-performance liquid chromatography tandem mass spectrometry method was developed and validated to determine the concentration of ambrisentan, macitentan and sitaxentan in human plasma. Plasma samples were treated with methanol to induce protein precipitation. A chromatographic separation was performed on a C18 column using a gradient of methanol-water containing 0.1% formic acid and 0.013% ammonium acetate and a flow rate of 0.5 ml/min. Multiple reaction monitoring was used for quantification. This method was validated in a linear range of 20.28-2028 µg/l for ambrisentan, 4.052-405.2 µg/l for macitentan and 205.4-10 270 µg/l for sitaxentan. The method was successfully validated according to US Food and Drug Administration guidelines to determine the concentrations of macitentan, ambrisentan and sitaxentan in human plasma. This method is now being used for study samples and clinical patient samples.

Prosody perception and production by children with cochlear implants.

The perception and production of emotional and linguistic (focus) prosody were compared in children with cochlear implants (CI) and normally hearing (NH) peers. Thirteen CI and thirteen hearing-age-matched school-aged NH children were tested, as baseline, on non-verbal emotion understanding, non-word repetition, and stimulus identification and naming. Main tests were verbal emotion discrimination, verbal focus position discrimination, acted emotion production, and focus production. Productions were evaluated by NH adult Dutch listeners. All scores between groups were comparable, except a lower score for the CI group for non-word repetition. Emotional prosody perception and production scores correlated weakly for CI children but were uncorrelated for NH children. In general, hearing age weakly predicted emotion production but not perception. Non-verbal emotional (but not linguistic) understanding predicted CI children's (but not controls') emotion perception and production. In conclusion, increasing time in sound might facilitate vocal emotional expression, possibly requiring independently maturing emotion perception skills.

Basic Measures of Prosody in Spontaneous Speech of Children With Early and Late Cochlear Implantation.

Purpose: Relative to normally hearing (NH) peers, the speech of children with cochlear implants (CIs) has been found to have deviations such as a high fundamental frequency, elevated jitter and shimmer, and inadequate intonation. However, two important dimensions of prosody (temporal and spectral) have not been systematically investigated. Given that, in general, the resolution in CI hearing is best for the temporal dimension and worst for the spectral dimension, we expected this hierarchy to be reflected in the amount of CI speech's deviation from NH speech. Deviations, however, were expected to diminish with increasing device experience. Method: Of 9 Dutch early- and late-implanted (division at 2 years of age) children and 12 hearing age-matched NH controls, spontaneous speech was recorded at 18, 24, and 30 months after implantation (CI) or birth (NH). Six spectral and temporal outcome measures were compared between groups, sessions, and genders. Results: On most measures, interactions of Group and/or Gender with Session were significant. For CI recipients as compared with controls, performance on temporal measures was not in general more deviant than spectral measures, although differences were found for individual measures. The late-implanted group had a tendency to be closer to the NH group than the early-implanted group. Groups converged over time. Conclusions: Results did not support the phonetic dimension hierarchy hypothesis, suggesting that the appropriateness of the production of basic prosodic measures does not depend on auditory resolution. Rather, it seems to depend on the amount of control necessary for speech production.

The perception of emotion and focus prosody with varying acoustic cues in cochlear implant simulations with varying filter slopes.

This study aimed to find the optimal filter slope for cochlear implant simulations (vocoding) by testing the effect of a wide range of slopes on the discrimination of emotional and linguistic (focus) prosody, with varying availability of F0 and duration cues. Forty normally hearing participants judged if (non-)vocoded sentences were pronounced with happy or sad emotion, or with adjectival or nominal focus. Sentences were recorded as natural stimuli and manipulated to contain only emotion- or focus-relevant segmental duration or F0 information or both, and then noise-vocoded with 5, 20, 80, 120, and 160 dB/octave filter slopes. Performance increased with steeper slopes, but only up to 120 dB/octave, with bigger effects for emotion than for focus perception. For emotion, results with both cues most closely resembled results with F0, while for focus results with both cues most closely resembled those with duration, showing emotion perception relies primarily on F0, and focus perception on duration. This suggests that filter slopes affect focus perception less than emotion perception because for emotion, F0 is both more informative and more affected. The performance increase until extreme filter slope values suggests that much performance improvement in prosody perception is still to be gained for CI users.

The effect of spectral smearing on the identification of pure F0 intonation contours in vocoder simulations of cochlear implants.

OBJECTIVES: Performance of cochlear implant (CI) users on linguistic intonation recognition is poorer than that of normal-hearing listeners, due to the limited spectral detail provided by the implant. A higher spectral resolution is provided by narrow rather than by broad filter slopes. The corresponding effect of the filter slope on the identification of linguistic intonation conveyed by pitch movements alone was tested using vocoder simulations. METHODS: Re-synthesized intonation variants of naturally produced phrases were processed by a 15-channel noise vocoder using a narrow (40 dB/octave) and a broad (20 dB/octave) filter slope. There were three different intonation patterns (rise/fall/rise-fall), differentiated purely by pitch and each associated to a different meaning. In both slope conditions as well as a condition with unprocessed stimuli, 24 normally hearing Dutch adults listened to a phrase, indicating which of two meanings was associated to it (i.e. a counterbalanced selection of two of the three contours). RESULTS: As expected, performance for the unprocessed stimuli was better than for the vocoded stimuli. No overall difference between the filter conditions was found. DISCUSSION AND CONCLUSIONS: These results are taken to indicate that neither the narrow (20 dB/octave) nor the shallow (40 dB/octave) slope provide enough spectral detail to identify pure F(0) intonation contours. For users of a certain class of CIs, results could imply that their intonation perception would not benefit from steeper slopes. For them, perception of pitch movements in language requires more extreme filter slopes, more electrodes, and/or additional (phonetic/contextual) cues.

The nature of hemispheric specialization for linguistic and emotional prosodic perception: a meta-analysis of the lesion literature.

It is unclear whether there is hemispheric specialization for prosodic perception and, if so, what the nature of this hemispheric asymmetry is. Using the lesion-approach, many studies have attempted to test whether there is hemispheric specialization for emotional and linguistic prosodic perception by examining the impact of left vs. right hemispheric damage on prosodic perception task performance. However, so far no consensus has been reached. In an attempt to find a consistent pattern of lateralization for prosodic perception, a meta-analysis was performed on 38 lesion studies (including 450 left hemisphere damaged patients, 534 right hemisphere damaged patients and 491 controls) of prosodic perception. It was found that both left and right hemispheric damage compromise emotional and linguistic prosodic perception task performance. Furthermore, right hemispheric damage degraded emotional prosodic perception more than left hemispheric damage (trimmed g=-0.37, 95% CI [-0.66; -0.09], N=620 patients). It is concluded that prosodic perception is under bihemispheric control with relative specialization of the right hemisphere for emotional prosodic perception.