Deficiency of Adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disorder presenting with a broad spectrum of clinical manifestations, including immunodeficiency, vasculopathy and hematologic disease. Biallelic mutations in ADA2 gene have been associated with a decreased ADA2 activity, leading to reduction in deamination of adenosine and deoxyadenosine into inosine and deoxyinosine and subsequent accumulation of extracellular adenosine. In the early reports, the pivotal role of innate immunity in DADA2 pathogenic mechanism has been underlined, showing a skewed polarization from the M2 macrophage subtype to the proinflammatory M1 subtype, with an increased production of inflammatory cytokines such as TNF-α. Subsequently, a dysregulation of NETosis, triggered by the excess of extracellular Adenosine, has been implicated in the pathogenesis of DADA2. In the last few years, evidence is piling up that adaptive immunity is profoundly altered in DADA2 patients, encompassing both T and B branches, with a disrupted homeostasis in T-cell subsets and a B-cell skewing defect. Type I/type II IFN pathway upregulation has been proposed as a possible core signature in DADA2 T cells and monocytes but also an increased IFN-β secretion directly from endothelial cells has been described. So far, a unifying clear pathophysiological explanation for the coexistence of systemic inflammation, immunedysregulation and hematological defects is lacking. In this review, we will explore thoroughly the latest understanding regarding DADA2 pathophysiological process, with a particular focus on dysregulation of both innate and adaptive immunity and their interacting role in the development of the disease.

Cannabidiol (CBD) is a promising therapeutic agent with analgesic, myorelaxant, and anti-epileptic actions. Recently, a purified form of CBD (Epidiolex®) has been approved by the European Medicines Agency (EMA) for the treatment of two highly-refractory childhood-onset epilepsies (Dravet and Lennox-Gastaut syndrome). Given the interindividual response and the relationship between the dose administered and CBD blood levels, therapeutic drug monitoring (TDM) is a valuable support in the clinical management of patients. We herein report for the first time a newly developed and validated method using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS) to evaluate CBD and its metabolites (i.e., cannabidiol-7-oic acid (7-COOH-CBD), 7-hydroxycannabidiol (7-OH-CBD), 6-α-hydroxycannabidiol (6-α–OH–CBD) and 6-β-hydroxycannabidiol (6-β–OH–CBD)) in serum samples. The method reached the sensitivity needed to detect minimal amounts of analytes under investigation with limits of quantification ranging from 0.5 to 20 ng/mL. The validation results indicated in this method were accurate (average inter/intra-day error, <15%), precise (inter/intra-day imprecision, <15%), and fast (8 min run time). The method resulted to be linear in the range of 1–10,000 ng/mL for CBD-COOH, 1–500 ng/mL for 7-OH-CBD and CBD and 1–25 ng/mL for 6-α–OH–CBD and 6-β–OH–CBD. Serum levels of CBD (88.20–396.31 and 13.19–170.63 ng/mL) as well as of 7-OH-CBD (27.11–313.63 and 14.01–77.52 ng/mL) and 7-COOH-CBD (380.32–10,112.23 and 300.57–2851.82 ng/mL) were significantly higher (p < 0.05) in patients treated with GW pharma CBD compared to those of patients treated with galenic preparations. 6-α–OH–CBD and 6-β–OH–CBD were detected in the first group and were undetectable in the second group. 7-COOH-CBD was confirmed as the most abundant metabolite in serum (5–10 fold higher than CBD) followed by 7-OH-CBD. A significant correlation (p < 0.05) between the dose administrated and a higher bioavailability was confirmed in patients treated with a GW pharma CBD preparation.

The role of therapeutic drug monitoring (TDM) of valaciclovir (VA)/aciclovir (A) and valganciclovir/ganciclovir (VG/G) in critically ill patients is still a matter of debate. More data on the dose–concentration relationship might therefore be useful, especially in pediatrics where clinical practice is not adequately supported by robust PK studies. We developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) micro-method to simultaneously quantify A and G from plasma and dried plasma spots (DPS). The method was based on rapid organic extraction from DPS and separation on a reversed-phase C-18 UHPLC column after addition of deuterated internal standards. Accurate analyte quantification using SRM detection was then obtained using a Thermo Fisher Quantiva triple-quadrupole MS coupled to an Ultimate 3000 UHPLC. It was validated following international (EMA) guidelines for bioanalytical method validation and was tested on samples from pediatric patients treated with A, VG, or G for cytomegalovirus infection following solid organ or hematopoietic stem cell transplantation. Concentrations obtained from plasma and DPS were compared using Passing–Bablok and Bland–Altman statistical tests. The assay was linear over wide concentration ranges (0.01–20 mg/L) in both plasma and DPS for A and G, suitable for the expected therapeutic ranges for both Cmin and Cmax, accurate, and reproducible in the absence of matrix effects. The results obtained from plasma and DPS were comparable. Using an LC-MS/MS method allowed us to obtain a very specific, sensitive, and rapid quantification of these antiviral drugs starting from very low volumes (50 μL) of plasma samples and DPS. The stability of analytes for at least 30 days allows for cost-effective shipment and storage at room temperature. Our method is suitable for TDM and could be helpful for improving knowledge on PK/PD targets of antivirals in critically ill pediatric patients.

Cannabidiol (CBD) exhibits anti-inflammatory, anxiolytic, antiseizure, and neuroprotective proprieties without addictive or psychotropic side effects, as opposed to Δ9-tetrahydrocannabinol (THC). While recreational cannabis contains higher THC and lower CBD concentrations, medical cannabis contains THC and CBD in different ratios, along with minor phytocannabinoids, terpenes, flavonoids and other chemicals. A volumetric absorptive microsampling (VAMS) method combined with ultra-high-performance liquid chromatography coupled with mass spectrometry in tandem for quantification of CBD, THC and their respective metabolites: cannabidiol-7-oic acid (7-COOH-CBD); 7-hydroxy-cannabidiol (7-OH-CBD); 6-alpha-hydroxy-cannabidiol (6-α-OH-CBD); and 6-beta-hydroxycannabidiol (6-β-OH-CBD); 11- Hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH). After overnight enzymatic glucuronide hydrolysis at 37°C, samples underwent acidic along with basic liquid-liquid extraction with hexane: ethyl acetate (9:1, v/v). Chromatographic separation was carried out on a C18 column, with the mass spectrometer operated in multiple reaction monitoring mode and negative electrospray ionization. Seven patients with intractable epilepsy were dosed with various CBD-containing formulations and blood collected just before their daily morning administration. The method was validated following international guidelines in toxicology. Linear ranges were (ng/ml) 0.5–25 THC, 11-OH-THC, THCCOOH, 6-α-OH-CBD and 6-β-OH-CBD; 10–500 CBD and 7-OH-CBD; and 20–5000 7-COOH-CBD. 7-COOH-CBD was present in the highest concentrations, followed by 7-OH-CBD and CBD. This analytical method is useful for investigating CBD, THC and their major metabolites in epilepsy patients treated with CBD preparations employing a minimally invasive microsampling technique requiring only 30 µL blood.