Inflammation and intracellular exposure of dolutegravir, darunavir, tenofovir and emtricitabine in people living with HIV.

AIMS: Antiretroviral (ARV) therapy reduces inflammation and immune activation in people with HIV, but not down to the levels observed in people without HIV. Limited drug penetration within tissues has been argued as a potential mechanism of persistent inflammation. Data on the inflammation role on ARV plasma/intracellular (IC) pharmacokinetics (PK) through to expression of cytochrome P450 3A/membrane transporters are limited. The aim of this study was to investigate the correlation between inflammation markers (IM) and plasma/IC PK of ARV regimen in HIV-positive patients. METHODS: We included ART-experienced patients switching to three different ARV regimens. Plasma and IC ARV drug concentration means at the end of dosing interval (T0 ), IM on samples concomitantly with ARV PK determination: sCD14, CRP, IL-6 and LPS were analysed. RESULTS: Plasma and IC drug concentrations were measured in 60 samples. No significative differences between CRP, sCD14, IL-6 and LPS values in the three arms were observed. A significant inverse correlation between tenofovir plasma concentration and sCD14 (rho = -0.79, P < .001), and between DRV IC/plasma ratio and Log10 IL-6 concentrations (rho = -0.36, P = .040), and a borderline statistically significant positive trend between DRV plasma concentration and sCD14 (rho = 0.31, P = .070) were suggested. Furthermore, a borderline statistically significant inverse trend between DTG IC concentrations and sCD14 (rho = -0.34, P = .090) was observed in 24 patients on DTG-based triple therapy. CONCLUSIONS: Our preliminary data support the hypothesis of lower DRV and DTG IC concentrations and lower TFV plasma exposure in patients with higher plasma IM suggesting an interplay between HIV drug penetration and persistent inflammation in cART-treated HIV-positive patients.

Plasma soluble suppression of tumorigenesis 2 measured in the emergency department for diagnosis and outcome prediction of sepsis: A single-center prospective study.

BACKGROUND AND AIMS: The diagnostic and prognostic performance of soluble Suppression of Tumorigenicity 2 (sST2) in suspected septic patients presenting to the Emergency Department (ED) is largely unknown. MATERIALS AND METHODS: Patients were included in this prospective study if there was high suspicion of sepsis. The plasma level of sST2 was measured during initial ED evaluation. Outcomes were the evaluation of (1) sST2 diagnostic performance (alone and in combination with procalcitonin [PCT]), and (2) sST2 ability to predict 30-day and 90-day all-cause mortality. RESULTS: Among 569 patients included, 481 (84.5 %) had sepsis or septic shock. Plasma sST2 levels were more elevated in septic patients (159 [71-331] vs 50 [31-103] ng/mL, P < 0.001). The AUC of sST2 for sepsis diagnosis was lower than the AUC of PCT (0.76 vs 0.85, P = 0.03). The best cut-off for sST2 was 61.7 ng/mL, with a sensitivity of 79.9 % and a specificity of 70.6 %. sST2 was able to correctly reclassify septic patients with PCT <0.5 (NRI 28.9 % [P = 0.02]). sST2 level was an independent predictor of 30-day mortality in a model including clinical variables (aHR 2.03 [1.24-3.33], C-index 0.69). CONCLUSION: sST2 could be a useful adjunct in diagnosing sepsis and in all-cause mortality prediction.

COVID-19: A Possible Contribution of the MAPK Pathway.

BACKGROUND: COVID-19 is characterized by an uncontrolled inflammatory response with high pro-inflammatory cytokine production through the activation of intracellular pathways, such as mitogen-activated protein kinase (MAPK). Viruses are able to exploit the MAPK pathway to their advantage; this pathway relevance to severe COVID-19 is poorly described. The aim of this study was to quantify biomarkers involved in the MAPK pathway and to clarify its possible role in affecting some COVID-19-related clinical features. METHODS: H-RAS, C-RAF, MAPK1, MAPK2, and ERK were quantified through ELISA, and genetic polymorphisms were evaluated through real-time PCR. RESULTS: We prospectively recruited 201 individuals (158 positive and 43 negative for SARS-CoV-2): 35 were male, and their median age was 65 years. MAPK-related biomarker levels were increased in SARS-CoV-2-positive participants (n = 89) compared to negative ones (n = 29). Dyspnea was reported by 48%; this symptom was associated with PBMC C-RAF levels in positive participants (p = 0.022) and type of ventilation (p = 0.031). The highest degree of ventilation was used by 8% for invasive ventilation and 41% for continuous positive airway pressure (CPAP). CONCLUSIONS: This is the first study that showed a possible contribution of MAPK-related biomarkers in affecting COVID-19 clinical features, and this may be relevant for identifying COVID-19 positive participants at risk of serious complications.

Pregnancy and COVID-19: The Possible Contribution of Vitamin D.

BACKGROUND: Vitamin D deficiency has been associated with the severity of COVID-19. The role of vitamin D in pregnant women with COVID-19 has been poorly investigated to date. The aim of this study was to evaluate the influence of vitamin D in affecting some clinical features in pregnancy between SARS-CoV-2 positive and negative patients. METHODS: Vitamin D pathway related polymorphisms and 25-hydroxyvitamin D levels were quantified in pregnant women followed from the first to the third trimester of pregnancy. Vitamin D deficiency was considered with values ≤ 30 ng/mL. RESULTS: In total, 160 women were enrolled: 23 resulted positive for at least one SARS-CoV-2 related test (molecular swab or antibody tests). Vitamin D-associated polymorphisms were able to affect vitamin D levels in SARS-CoV-2 negative and positive subjects: remarkably, all the VDR TaqICC genotype patients were negative for SARS-CoV-2. In a sub-population (118 patients), vitamin D levels correlated with pregnancy-related factors, such as alpha-fetoprotein levels. Third-trimester vitamin D levels were lower in preterm births compared to full-term pregnancy: this trend was highlighted for SARS-CoV-2 positive patients. CONCLUSIONS: This is the first study demonstrating a role of vitamin D in affecting the clinical characteristics of pregnant women during the COVID-19 era. Further studies in larger and different cohorts of patients are required to confirm these findings.

Possible Impact of Vitamin D Status and Supplementation on SARS-CoV-2 Infection Risk and COVID-19 Symptoms in a Cohort of Patients with Inflammatory Bowel Disease.

The coronavirus disease (COVID-19) pandemic represents a global health challenge, particularly considering concomitant diseases. Patients with inflammatory bowel diseases (IBD) can be considered a population at risk. On the other hand, the risk of developing IBD and COVID-19 have both been described as modulated by vitamin D (VD) levels. In this work, a cohort of 106 adult patients affected by IBD was prospectively enrolled, during the second wave of the pandemic in Italy. In these patients, VD plasma levels, demographic, and clinical characteristics were tested for a correlation/an association with the risk of infection with SARS-CoV-2 in the study period (anti-spike IgG positivity) and the severity of COVID-19 symptoms. By multivariate logistic regression analysis, VD supplementation (Odds Ratio; OR 0.116, p = 0.002), therapy with monoclonal antibodies (OR 0.227, p = 0.007), and the use of mesalazine (OR 2.968, p = 0.046) were found to be independent predictors of SARS-CoV-2 positivity. Moreover, hypertension was associated with severe disease (p = 0.019), while a VD level higher than 30 ng/mL (p = 0.031, OR 0.078) was associated with asymptomatic infection. No interplay between IBD activity and COVID-19 risk of infection or symptoms was observed. These results confirm the importance of VD levels in defining the risk of COVID-19 and give encouraging data about the safety of maintaining immunomodulatory treatments for IBD during the COVID-19 pandemic.

Development and Validation of an Up-to-Date Highly Sensitive UHPLC-MS/MS Method for the Simultaneous Quantification of Current Anti-HIV Nucleoside Analogues in Human Plasma.

Therapeutic options to treat HIV infection have widened in the past years, improving both effectiveness and tolerability, but nucleoside reverse transcriptase inhibitors (NRTIs) are still considered the standard backbone of the combination regimens. Therapeutic drug monitoring (TDM) can be useful for these drugs, due to concentration-effect relationship, with risk of ineffectiveness, toxicity or adherence concerns: in this scenario, robust and multiplexed methods are needed for an effective TDM activity. In this work, the first validated ultra-high spectrometry liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method is described for the high-sensitive simultaneous quantification of all the currently used NRTIs in human plasma, including tenofovir alafenamide (TAF), following FDA and EMA guidelines. The automated sample preparation consisted in the addition of an internal standard (IS) working solution, containing stable-isotope-linked drugs, protein precipitation and drying. Dry extracts were reconstituted with water, then, these underwent reversed phase chromatographic separation: compounds were detected through electrospray ionization and multiple reaction monitoring. Accuracy, precision, recovery and IS-normalized matrix effect fulfilled guidelines' requirements. The application of this method on samples from people living with HIV (PLWH) showed satisfactory performance, being capable of quantifying the very low concentrations of tenofovir (TFV) in patients treated with TAF.

Seasonal Variation of Antiretroviral Drug Exposure during the Year: The Experience of 10 Years of Therapeutic Drug Monitoring.

Although studies show an annual trend for immunosuppressive drugs, particularly during different seasons, no data are available for antiretroviral drugs exposures in different periods of the year. For this reason, the aim of this study was to investigate an association between seasonality and antiretroviral drugs plasma concentrations. Antiretroviral drugs exposures were measured with liquid chromatography validated methods. A total of 4148 human samples were analysed. Lopinavir, etravirine and maraviroc levels showed seasonal fluctuation. In detail, maraviroc and etravirine concentrations decreased further in summer than in winter. In contrast, lopinavir concentrations had an opposite trend, increasing more in summer than in winter. The etravirine efficacy cut-off value of 300 ng/mL seems to be affected by seasonality: 77.1% and 22.9% of samples achieved this therapeutic target, respectively, in winter and summer, whereas 30% in winter and 70% in summer did not reach this value. Finally, age over 50 years and summer remained in the final multivariate regression model as predictors of the etravirine efficacy cut-off. This study highlights the seasonal variation in antiretroviral drugs plasma concentrations during the year, leading to a better understanding of inter-individual variability in drug exposures. Studies are required in order to confirm these data, clarifying which aspects may be involved.

Prospective diagnostic accuracy study of plasma soluble ST2 for diagnosis of acute aortic syndromes.

Acute aortic syndromes (AASs) are difficult to diagnose emergencies. Plasma soluble ST2 (sST2), a prognostic biomarker for heart failure, has been proposed as a diagnostic biomarker of AASs outperforming D-dimer, the current diagnostic standard. We performed a prospective diagnostic accuracy study of sST2 for AASs in the Emergency Department (ED). In 2017-2018, patients were enrolled if they had ≥1 red-flag symptoms (chest/abdominal/back pain, syncope, perfusion deficit) and a clinical suspicion of AAS. sST2 was detected with the Presage® assay. Adjudication was based on computed tomography angiography (CTA) or on diagnostic outcome inclusive of 30-day follow-up. 297 patients were enrolled, including 88 with AASs. The median age was 67 years. In 162 patients with CTA, the median sST2 level was 41.7 ng/mL (IQR 29.4-103.2) in AASs and 34.6 ng/mL (IQR 21.4-51.5) in alternative diagnoses (P = 0.005). In ROC analysis, the AUC of sST2 was 0.63, as compared to 0.82 of D-dimer (P < 0.001). Sensitivity and specificity values of sST2 associated with different cutoffs were: 95.5% and 10.8% (≥12 ng/mL), 84.1% and 29.7% (≥23.7 ng/mL), 35.2% and 85.1% (≥66.5 ng/mL). Results were similar in the full cohort. In conclusion, in patients from a European ED, plasma sST2 provided modest accuracy for diagnosis of AASs.

Lack of concordance between EMIT assay and LC-MS/MS for Therapeutic Drug Monitoring of Mycophenolic Acid: Potential increased risk for graft rejection?

Therapeutic drug monitoring (TDM) of immunosuppressive drugs is crucial in organ-transplanted patients to prevent rejection or toxic effects due to inadequate dosage. Mycophenolic acid (MPA) is a commonly used immunosuppressant in this setting. Nowadays, MPA concentrations are monitored by Enzyme Multiplied Immunoassay Technology (EMIT), and Liquid Chromatography (LC)-based techniques, particularly coupled to Tandem Mass Spectrometry (LC-MS/MS). This study evaluates the concordance between TDM results for MPA obtained through CE-IVD EMIT and LC-MS/MS assays in plasma samples. LC-MS/MS quantification was based on a commercial kit and the analytical performance in terms of accuracy was tested through external proficiency tests and inter-laboratory comparison with a home-made HPLC-UV method. Both these evaluations confirmed the reliability of the LC-MS/MS method (1.6 % and 9.0 % of bias, respectively). Conversely, the comparison between EMIT and LC-MS/MS showed overestimation by EMIT of 33.5 %. This bias resulted concentration-dependent, ranging from 46.4 % in the concentration range of 1-2 mg/L, to 21.4 % over 4 mg/L. Considering the theoretical clinical impact of this overestimation, a fraction comprised between 12.4 % and 31.4 % of samples which resulted over three different minimum effective concentration values by EMIT (no indication for dose adjustment) had discordant indications by LC-MS/MS (dose adjustment needed). Concluding, this study highlights a clinically relevant systematic overestimation of MPA concentration by EMIT, supporting the switch to LC-MS/MS techniques for TDM purpose. However, further prospective studies are needed in order to evaluate the clinical impact of switching the TDM activity from EMIT to LC-MS/MS in a larger cohort in a long period.

Validation of a UHPLC-MS/MS Method to Quantify Twelve Antiretroviral Drugs within Peripheral Blood Mononuclear Cells from People Living with HIV.

Recently, anti-HIV treatment has achieved high efficacy and tolerability. Nevertheless, few data are available about the intracellular penetration of antiretrovirals, partly due to the technical challenges related to intracellular quantification. This work aimed to validate an ultra-high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method for the simultaneous quantification of maraviroc, nevirapine, rilpivirine, dolutegravir, raltegravir, cobicistat, darunavir, ritonavir, atazanavir, efavirenz, elvitegravir, and etravirine within peripheral blood mononuclear cells (PBMCs) and apply it to samples from patients. PBMCs were isolated by density gradient on cell preparation tubes (CPT). Samples were prepared by addition of internal standards (IS), sonication, centrifugation, and drying. Reconstituted extracts underwent chromatographic separation by reversed phase UHPLC and detection was performed by electrospray ionization and multiple reaction monitoring. Method validation followed FDA and EMA guidelines, showing acceptable accuracy, precision, recovery and IS-normalized matrix effect. The application to 56 samples from patients undergoing antiretroviral treatment provided description of intracellular penetration, showing method eligibility for future studies.

Extracellular nicotinate phosphoribosyltransferase binds Toll like receptor 4 and mediates inflammation.

Damage-associated molecular patterns (DAMPs) are molecules that can be actively or passively released by injured tissues and that activate the immune system. Here we show that nicotinate phosphoribosyltransferase (NAPRT), detected by antibody-mediated assays and mass spectrometry, is an extracellular ligand for Toll-like receptor 4 (TLR4) and a critical mediator of inflammation, acting as a DAMP. Exposure of human and mouse macrophages to NAPRT activates the inflammasome and NF-κB for secretion of inflammatory cytokines. Furthermore, NAPRT enhances monocyte differentiation into macrophages by inducing macrophage colony-stimulating factor. These NAPRT-induced effects are independent of NAD-biosynthetic activity, but rely on NAPRT binding to TLR4. In line with our finding that NAPRT mediates endotoxin tolerance in vitro and in vivo, sera from patients with sepsis contain the highest levels of NAPRT, compared to patients with other chronic inflammatory conditions. Together, these data identify NAPRT as a endogenous ligand for TLR4 and a mediator of inflammation.

Prospective diagnostic and prognostic study of copeptin in suspected acute aortic syndromes.

Acute aortic syndromes (AAS) are cardiovascular emergencies with unmet diagnostic needs. Copeptin is released upon stress conditions and is approved for rule-out of myocardial infarction (MI). As MI and AAS share presenting symptoms, stress mechanisms and necessity for rapid diagnosis, copeptin appears as an attractive biomarker also for AAS. We thus performed a diagnostic and observational study in Emergency Department (ED) outpatients. Inclusion criteria were chest/abdominal/back pain, syncope and/or perfusion deficit, plus AAS in differential diagnosis. Blood samples were obtained in the ED. 313 patients were analyzed and 105 (33.5%) were diagnosed with AAS. Median copeptin was 38.91 pmol/L (interquartile range, IQR, 16.33-173.4) in AAS and 7.51 pmol/L (IQR 3.58-15.08) in alternative diagnoses (P < 0.001). Copeptin (≥10 pmol/L) had a sensitivity of 80.8% (95% confidence interval, CI, 72.2-87.2) and a specificity of 63.6% (CI 56.9-69.9) for AAS. Within 6 hours, the sensitivity and specificity were 88.7% (CI 79.3-94.2) and 52.4% (CI 42.9-61.8) respectively. Combination with D-dimer did not increase the diagnostic yield. Furthermore, copeptin ≥25 pmol/L predicted mortality in patients with alternative diagnoses but not with AAS. In conclusion, copeptin increases in most patients with AAS within the first hours, but the accuracy of copeptin for diagnosis AAS is suboptimal.

Effects of meal timing on changes in circulating epinephrine, norepinephrine, and acylated ghrelin concentrations: a pilot study.

BACKGROUND: Timing of food intake impacts on metabolic diseases. Few data are available about post-meal changes in epinephrine (E), norepinephrine (NE), and acylated ghrelin (AG) at different times of the day. SUBJECTS AND METHODS: This randomized cross-over trial investigated E/NE/AG concentrations after identical meals consumed at 0800 or 2000 hours in 20 healthy volunteers, by standardizing diet, exercise, duration of fast, and resting. Participants randomly received the test meal at 0800 or 2000 hours, and vice versa after 1 week. Blood samples were collected before and up to 180-min post-meal, every 30 min, with participants supine, motionless, but awake. RESULTS: Median E levels increased at 30-60 min, then declined and rose again at 150 min; values at 60 min (19.0 vs. 15.0 ng/l, p = 0.03) and 180 min (25.0 vs. 11.0 ng/l, p < 0.001) were higher after the morning meals. NE rose at 30-60 min and then progressively declined; median values at 60 min (235.3 vs. 206.3 ng/l, p = 0.02) and 120 min (208.8 vs. 142.0 ng/l, p = 0.04) increased more after morning meals. AG progressively declined to increase again at 90 min after meal; median AG area-under-the-curve (AUC) values were lower at morning (7206.8 vs. 8828.3 pg/mL×h). AG-AUC was inversely associated with diet-induced thermogenesis (ß = -121.6; 95% CI -201.0 to 42.2; p = 0.009 for each unit increase), while log NE-AUC was inversely associated with log-triglyceride AUC (ß = -0.57; 95% CI -0.98 to 0.16; p = 0.015) in a multiple regression model, after multiple adjustments. CONCLUSIONS: In conclusion, E/NE concentrations were higher after the morning meal, while AG showed an opposite behavior. These data, although requiring confirmation in larger samples, suggest an adjunctive possible mechanism explaining the unfavorable effects of evening eating on metabolic risk.