Platelet-Derived Extracellular Vesicles as Lipid Carriers in Severe Allergic Inflammation.

The resolution of inflammation is a complex process that is critical for removing inflammatory cells and restoring tissue function. The dysregulation of these mechanisms leads to chronic inflammatory disorders. Platelets, essential cells for preserving homeostasis, are thought to play a role in inflammation as they are a source of immunomodulatory factors. Our aim was to identify key metabolites carried by platelet-derived extracellular vesicles (PL-EVs) in a model of allergic inflammation. PL-EVs were isolated by serial ultracentrifugation using platelet-rich plasma samples obtained from platelet apheresis from severely (n = 6) and mildly (n = 6) allergic patients and non-allergic individuals used as controls (n = 8). PL-EVs were analysed by a multiplatform approach using liquid and gas chromatography coupled to mass spectrometry (LC-MS and GC-MS, respectively). PL-EVs obtained from severely and mildly allergic patients and control individuals presented comparable particle concentrations and sizes with similar protein concentrations. Strikingly, PL-EVs differed in their lipid and metabolic content according to the severity of inflammation. L-carnitine, ceramide (Cer (d18:0/24:0)), and several triglycerides, all of which seem to be involved in apoptosis and regulatory T functions, were higher in PL-EVs from patients with mild allergic inflammation than in those with severe inflammation. In contrast, PL-EVs obtained from patients with severe allergic inflammation showed an alteration in the arachidonic acid pathway. This study demonstrates that PL-EVs carry specific lipids and metabolites according to the degree of inflammation in allergic patients and propose novel perspectives for characterising the progression of allergic inflammation.

A method based on plateletpheresis to obtain functional platelet, CD3+ and CD14+ matched populations for research immunological studies.

BACKGROUND: In previous studies with peripheral blood cells, platelet factors were found to be associated with severe allergic phenotypes. A reliable method yielding highly concentrated and pure platelet samples is usually not available for immunological studies. Plateletpheresis is widely used in the clinics for donation purposes. In this study, we designed a protocol based on plateletpheresis to obtain Platelet-Rich Plasma (PRP), Platelet-Poor Plasma (PPP) as well as CD3+ and CD14+ cells matched samples from a waste plateletpheresis product for immunological studies. METHODS: Twenty-seven subjects were voluntarily subjected to plateletpheresis. PRP, PPP and blood cell concentrate contained in a leukocyte reduction system chamber (LRSC) were obtained in this process. CD3+ and CD14+ cells were isolated from the LRSC by density-gradient centrifugation and positive magnetic bead isolation. RNA was isolated from PRP, CD3+ and CD14+ cell samples and used for transcriptomic studies by Affymetrix. PRP and PPP samples were used for platelet protein quantification by multiplex assays. RESULTS: A reliable high yield method to obtain matched samples of PRP, PPP, CD3+ and CD14+ from a single donor for RNA and protein analyses has been designed. The RNA quality indicators (RQI) routinely used for other cell types were not suitable for platelet RNA characterization. Despite this, the platelet RNA was valid for transcriptomic studies by Affymetrix, as platelet transcripts obtained in our previous studies were confirmed in PRP samples. Platelet samples were enriched in platelet factors as determined in protein multiplex analysis. CONCLUSIONS: We have developed a method that yields not only high content and pure platelet samples from a single donor but also CD3+ and CD14+ matched samples that can be used for RNA and protein analyses in immunological studies.

A paired trial comparing mononuclear cell collection in two machines for further inactivation through an inline or offline extracorporeal photopheresis procedure.

BACKGROUND: Extracorporeal photopheresis (ECP) is an effective treatment. However, protocols differ widely, and some questions, such as the number of cells to be collected or the number of ECP treatment days per treatment cycle, are still unsolved. The aim of this study was to compare a multistep (offline) (Spectra Optia and Macogenic G2) against an integrated (inline) ECP system (Therakos Cellex system) with respect to mononuclear cell (MNC) collection. STUDY DESIGN AND METHODS: The number and quality parameters of the MNC products collected were evaluated together with some machine parameters, such as collection time. Comparisons were made through paired sample analysis with the t test. RESULTS: Fourteen patients underwent 15 double-paired procedures using both ECP protocols. The average MNC collected in the multistep procedure was 77.4 × 108 , four times more than in the integrated procedure (18.5 × 108 ). MNC purity (84.4% vs. 63.8%) and enrichment (27.9 vs. 5.9) in the product collected were also higher in the multistep procedure. The whole ECP time was higher in the multistep than in the integrated procedure (272 vs. 106 min), but the calculated time to collect 25 × 108 MNCs in the multistep was shorter compared with the one-step procedure (77.8 vs. 172 min). All these differences between the two protocols were statistically significant. CONCLUSIONS: These two ECP protocols are different with respect to MNC collection and length of procedure. Some unresolved questions, such as the better MNC dose to inactivate or the number of consecutive days that ECP should be performed for optimal clinical efficacy, require further review.

Understanding Platelets in Infectious and Allergic Lung Diseases.

Emerging evidence suggests that platelets, cytoplasmic fragments derived from megakaryocytes, can no longer be considered just as mediators in hemostasis and coagulation processes, but as key modulators of immunity. Platelets have received increasing attention as the emergence of new methodologies has allowed the characterization of their components and functions in the immune continuum. Platelet activation in infectious and allergic lung diseases has been well documented and associated with bacterial infections reproduced in several animal models of pulmonary bacterial infections. Direct interactions between platelets and bacteria have been associated with increased pulmonary platelet accumulation, whereas bacterial-derived toxins have also been reported to modulate platelet function. Recently, platelets have been found extravascular in the lungs of patients with asthma, and in animal models of allergic lung inflammation. Their ability to interact with immune and endothelial cells and secrete immune mediators makes them one attractive target for biomarker identification that will help characterize their contribution to lung diseases. Here, we present an original review of the last advances in the platelet field with a focus on the contribution of platelets to respiratory infections and allergic-mediated diseases.

Evaluation of convalescent plasma versus standard of care for the treatment of COVID-19 in hospitalized patients: study protocol for a phase 2 randomized, open-label, controlled, multicenter trial.

BACKGROUND: COVID-19 is a respiratory disease caused by a novel coronavirus (SARS-CoV-2) and causes substantial morbidity and mortality. At the time this clinical trial was planned, there were no available vaccine or therapeutic agents with proven efficacy, but the severity of the condition prompted the use of several pharmacological and non-pharmacological interventions. It has long been hypothesized that the use of convalescent plasma (CP) from infected patients who have developed an effective immune response is likely to be an option for the treatment of patients with a variety of severe acute respiratory infections (SARI) of viral etiology. The aim of this study is to assess the efficacy and safety of convalescent plasma in adult patients with severe COVID-19 pneumonia. METHODS/DESIGN: The ConPlas-19 study is a multicenter, randomized, open-label controlled trial. The study has been planned to include 278 adult patients hospitalized with severe COVID-19 infection not requiring mechanical ventilation (invasive or non-invasive). Subjects are randomly assigned in a 1:1 ratio (139 per treatment arm), stratified by center, to receive intravenously administered CP (single infusion) plus SOC or SOC alone, and are to be followed for 30 days. The primary endpoint of the study is the proportion of patients that progress to category 5, 6, or 7 (on the 7-point ordinal scale proposed by the WHO) at day 15. Interim analyses for efficacy and/or futility will be conducted once 20%, 40%, and 60% of the planned sample size are enrolled and complete D15 assessment. DISCUSSION: This clinical trial is designed to evaluate the efficacy and safety of passive immunotherapy with convalescent plasma for the treatment of adult patients hospitalized with COVID-19. The results of this study are expected to contribute to establishing the potential place of CP in the therapeutics for a new viral disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT04345523 . Registered on 30 March, 2020. First posted date: April 14, 2020.

Biallelic ATM alterations detected at diagnosis identify a subset of treatment-naïve chronic lymphocytic leukemia patients with reduced overall survival similar to patients with p53 deletion.

The prognostic impact of biallelic ATM abnormalities (ATM mutation and concurrent 11q deletion) remains unknown. We studied ATM, BIRC3, SF3B1, and NOTCH1 genes in 118 treatment-naïve CLL patients at diagnosis. Patients with biallelic ATM alteration had a similar time to first treatment (TTFT) and shorter overall survival (OS) compared with patients with isolated 11q deletion and shorter TTFT and OS when compared to patients with wild-type ATM. Furthermore, biallelic ATM alteration (HR: 6.4; p ≤ 0.007) was significantly associated with an increased risk of death similar to p53 deletion (HR: 6.1; p ≤ 0.004), superior to 11q deletion alone (HR: 2.8; p ≤ 0.022) and independent of other significant parameters such as age, advanced clinical stage, and complex karyotype. Our results suggest the identification of ATM mutations in CLL patients with 11q deletion at diagnosis is clinically relevant and predicts disease progression, poor response to the treatment, and reduced OS independent of other molecular prognostic factors.

Von Willebrand Factor Deficiency Corrected by Lung Transplantation.

BACKGROUND: In experimental models with von Willebrand disease pigs, plasma von Willebrand factor (vWF) was significantly increased after lung transplantation because lung endothelial cells strongly express vWF. However, these findings have not been confirmed in human beings. METHODS: A 26-year-old man with mild vWF deficiency (FvW:antigen 39 IU/dL; FvW:ristocetin cofactor activity 44 IU/dL; factor VIII 99%; normal multimeric plasma vWF pattern) was referred to our institution and underwent bilateral lung transplantation for cystic fibrosis. The patient received factor VIII/vWF concentrate both during and after surgery without any relevant bleeding events. At hospital discharge, he was taking immunosuppression with oral tacrolimus, prednisone, and mycophenolate mofetil, which has continued until the present day (22 months after the procedure). RESULTS: Plasma vWF level increased during the postoperative days, presumably due to endothelial injuries and the infusion of vWF concentrate. Laboratory tests at 5, 11, 14, and 22 months after lung transplantation demonstrated sustained normalization of all parameters. CONCLUSIONS: To our knowledge, this is the first reported case of von Willebrand deficiency corrected through lung transplantation.

The efficacy of photochemical treatment with amotosalen HCl and ultraviolet A (INTERCEPT) for inactivation of Trypanosoma cruzi in pooled buffy-coat platelets.

BACKGROUND: This study evaluated the efficacy of photochemical treatment (PCT) with amotosalen and ultraviolet A (UVA) light to inactivate Trypanosoma cruzi in contaminated platelet (PLT) components. STUDY DESIGN AND METHODS: Fifteen pools of buffy-coat PLTs (BC-PLTs) were inoculated with approximately 5 x 10(3) to 5 x 10(5) per mL of viable T. cruzi of the G, Tulahuen (T), or Y strains. Samples from BC-PLTs were assayed for infectivity before and after PCT with 150 micromol per L amotosalen and 3 J per cm(2) UVA light. Infectivity was determined with three different methods: 1) in vitro culture to detect viable epimastigotes, 2) [(3)H]thymidine incorporation in culture, and 3) in vivo inoculation into interferon-gamma receptor (IFN-gammaR)-deficient mice. RESULTS: The in vitro assay yielded viable parasite titers of 3.9 x 10(5), 2.8 x 10(4), and 5.6 x 10(3) per mL (corresponding to 5.6, 4.4, and 3.8 logs/mL) for the Y, T, and G strains, respectively. PCT was able to inactivate all three strains of T. cruzi to below the limit of detection (10 parasites/mL) in the sensitive in vivo assay. Because 10-mL samples, each concentrated into a 1-mL sample for inoculation, were tested in the in vivo assay, log reductions achieved were greater than 5.6, greater than 4.4, and greater than 3.8 for the Y, T, and G strains of T. cruzi, respectively. CONCLUSIONS: The pathogen reduction system with amotosalen HCl and UVA demonstrated robust efficacy for inactivation of high doses of three different strains of T. cruzi and offers the potential to make the PLT supply safer.

Hematomas in multicomponent apheresis: searching for related factors.

BACKGROUND: The objective of this study was to identify donation variables that could be related to the development of hematoma during multicomponent apheresis collections (MACs). STUDY DESIGN AND METHODS: This is an observational retrospective study where 1375 donors donated 5177 MACs during a 2-year period with two different machines (Amicus Crescendo [AC], Baxter Healthcare Corp.; and Trima Accel [TA], Gambro BCT). Variable data were recorded prospectively. In the multiple logistic regression analysis, generalized estimating equations were used with an exchangeable correlation matrix to take into account the nonindependence of several measurements from the same donor. RESULTS: During the study period, 170 procedures failed due to hematoma (3.3%). Several variables were related to hematoma development in the adjusted model: operator experience (less than 500 procedures supervised vs. more; odds ratio [OR], 1.66; 95% confidence interval [CI], 1.19-2.31), previous apheresis donations (first time vs. more than 16 donations; OR, 2.87; 95% CI, 1.52-5.45), vein canalized (basilic vs. intermediate antebrachial or cephalic; OR, 1.42; 95% CI, 1.04-1.94), diastolic blood pressure (units divided by 10 mmHg; OR, 0.79; 95% CI, 0.66-0.94), and type of machine used (TA high return limit configuration [RLC] setting configuration vs. AC; OR, 1.94; 95% CI, 1.27-2.96; TA low RLC setting configuration vs. AC; OR, 1.24; 95% CI, 0.83-1.84). CONCLUSIONS: Our study suggests that hematoma in MAC is not a random event. Appropriate machine configuration in the TA could reduce the hematoma rate to a level comparable with that of the AC. Operator training and donor blood pressure are also interesting variables for study because these could be modified to reduce the hematoma rate.

A randomized crossover trial comparing three plateletpheresis machines.

BACKGROUND: The aim of the study was to compare three different apheresis machines with the same donors regarding the processing time required to obtain a 3.5 x 10(11) platelet (PLT) dose and acceptance by donors. STUDY DESIGN AND METHODS: A randomized crossover trial was performed to evaluate the differences between the Amicus Crescendo (Baxter Biotech Corp.), the MCS Plus (Haemonetics Corp.), and the Trima Accel (Gambro BCT). Donations from 51 donors were compared for time adjusted to obtain a standard 3.5 x 10(11) PLT dose (TSD3.5), efficiency, adverse reactions, yield, leukodepletion, machine accuracy, and donor preferences. Processing times were measured by chronometer. The same vein access was used during all three processes in each donor. In the statistical analysis, to take into account the nonindependence of several measurements from the same donor, generalized estimating equations were used with an autoregressive correlation matrix. RESULTS: The Accel produced a TSD3.5 (mean +/- SEM) of 47.9 +/- 1.0 min; the Amicus, 60.3 +/- 1.0 min; and the MCS Plus, 66.7 +/- 1.0 (p < 0.0001). The Amicus presented the greatest efficiency (87.5%; p < 0.0028). The MCS Plus demonstrated the highest capacity for leukodepletion (p < 0.0002) despite one process presenting more than 1 x 10(6) white blood cells per unit. The MCS Plus also measured the processing time with the greatest accuracy. No severe adverse effects were observed. The donors preferred the Accel (61%) followed by the Amicus (35%) and the MCS Plus (4%; p < 0.0001) and the processing speed was the most highly valued measure (55%). CONCLUSIONS: The Accel is the fastest and, because of this advantage, the machine preferred by donors. The Amicus was the most efficient and the MCS Plus was the only one not to underestimate the processing time.

A comparison of PLT collections from two apheresis devices.

BACKGROUND: The aim of this study was to compare two apheresis devices (COBE Trima and COBE Spectra, Gambro BCT). STUDY DESIGN AND METHODS: The study compares 103 Trima procedures with 61 Spectra procedures. The comparison of single-donor PLTs (SDPs) separation parameters and anticoagulant infusion to the donors were the primary targets. Yield and residual amounts of WBCs of SDPs were secondary targets. Residual amounts of WBCs were measured with the Nageotte method. RESULTS: Trima and Spectra groups were comparable before apheresis. Two procedures were terminated before completion in the Trima group owing to vein damage. Trima infused more anticoagulant--352 +/- 104 mL versus 297 +/- 75 mL (p < 0.01)--and was quicker than Spectra. The time to obtain 3.5 x 10(11) was (median) 55.8 minutes for the Trima machine and 80.3 minutes for Spectra machine (p < 0.001). Regarding leukoreduction, all the SDPs had fewer than 1 x 10(6) WBCs per unit except for one in product obtained by the Trima machine. CONCLUSIONS: The Trima machine is faster and more efficient than the Spectra machine, and both machines allow standard leukoreduced SDPs to be obtained. Although donors receive a higher anticoagulant infusion with the Trima machine, their tolerance is acceptable.