Labeling and exocytosis of secretory compartments in RBL mastocytes by polystyrene and mesoporous silica nanoparticles

Maneerat Ekkapongpisit1,*, Antonino Giovia1,*, Giuseppina Nicotra1, Matteo Ozzano1, Giuseppe Caputo2,3, Ciro Isidoro1 1Laboratory of Molecular Pathology and Nanobioimaging, Department of Health Sciences, Università del Piemonte Orientale "A. Avogadro", Novara, Italy; 2Department of Chemistry, University of Turin, Turin, 3Cyanine Technology SpA, Torino, Italy *These authors contributed equally to this workBackground: For a safe ‘in vivo’ biomedical utilization of nanoparticles, it is essential to assess not only biocompatibility, but also the potential to trigger unwanted side effects at both cellular and tissue levels. Mastocytes (cells having secretory granules containing cytokines, vasoactive amine, and proteases) play a pivotal role in the immune and inflammatory responses against exogenous toxins. Mastocytes are also recruited in the tumor stroma and are involved in tumor vascularization and growth.Aim and methods: In this work, mastocyte-like rat basophilic leukemia (RBL) cells were used to investigate whether carboxyl-modified 30 nm polystyrene (PS) nanoparticles (NPs) and naked mesoporous silica (MPS) 10 nm NPs are able to label the secretory inflammatory granules, and possibly induce exocytosis of these granules. Uptake, cellular retention and localization of fluorescent NPs were analyzed by cytofluorometry and microscope imaging.Results: Our findings were that: (1) secretory granules of mastocytes are accessible by NPs via endocytosis; (2) PS and MPS silica NPs label two distinct subpopulations of inflammatory granules in RBL mastocytes; and (3) PS NPs induce calcium-dependent exocytosis of inflammatory granules.Conclusion: These findings highlight the value of NPs for live imaging of inflammatory processes, and also have important implications for the clinical use of PS-based NPs, due to their potential to trigger the unwanted activation of mastocytes.Keywords: secretory lysosomes, inflammation, nanoparticles, vesicular traffi

Pharmacometrics of high dose ivermectin in early COVID-19: an open label, randomized, controlled adaptive platform trial (PLATCOV)

Background: There is no generally accepted methodology for in vivo assessment of antiviral activity in SARS-CoV-2 infections. Ivermectin has been recommended widely as a treatment of COVID-19, but whether it has clinically significant antiviral activity in vivo is uncertain. Methods: In a multicentre open label, randomized, controlled adaptive platform trial, adult patients with early symptomatic COVID-19 were randomized to one of six treatment arms including high-dose oral ivermectin (600 µg/kg daily for 7 days), the monoclonal antibodies casirivimab and imdevimab (600 mg/600 mg), and no study drug. The primary outcome was the comparison of viral clearance rates in the modified intention-to-treat population. This was derived from daily log10 viral densities in standardized duplicate oropharyngeal swab eluates. This ongoing trial is registered at https://clinicaltrials.gov/ (NCT05041907). Results: Randomization to the ivermectin arm was stopped after enrolling 205 patients into all arms, as the prespecified futility threshold was reached. Following ivermectin, the mean estimated rate of SARS-CoV-2 viral clearance was 9.1% slower (95% confidence interval [CI] –27.2% to +11.8%; n=45) than in the no drug arm (n=41), whereas in a preliminary analysis of the casirivimab/imdevimab arm it was 52.3% faster (95% CI +7.0% to +115.1%; n=10 (Delta variant) vs. n=41). Conclusions: High-dose ivermectin did not have measurable antiviral activity in early symptomatic COVID-19. Pharmacometric evaluation of viral clearance rate from frequent serial oropharyngeal qPCR viral density estimates is a highly efficient and well-tolerated method of assessing SARS-CoV-2 antiviral therapeutics in vitro

Clinical antiviral efficacy of remdesivir in coronavirus disease 2019: an open-label, randomized controlled adaptive platform trial (PLATCOV)

Background Uncertainty over the therapeutic benefit of parenteral remdesivir in coronavirus disease 2019 (COVID-19) has resulted in varying treatment guidelines. Methods In a multicenter open-label, controlled, adaptive, pharmacometric platform trial, low-risk adult patients with early symptomatic COVID-19 were randomized to 1 of 8 treatment arms including intravenous remdesivir (200 mg followed by 100 mg daily for 5 days) or no study drug. The primary outcome was the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance (estimated under a linear model fit to the daily log10 viral densities, days 0–7) in standardized duplicate oropharyngeal swab eluates, in a modified intention-to-treat population. This ongoing adaptive trial is registered at ClinicalTrials.gov (NCT05041907). Results The 2 study arms enrolled 131 patients (remdesivir n = 67, no study drug n = 64) and estimated viral clearance rates from a median of 18 swab samples per patient (a total of 2356 quantitative polymerase chain reactions). Under the linear model, compared with the contemporaneous control arm (no study drug), remdesivir accelerated mean estimated viral clearance by 42% (95% credible interval, 18%–73%). Conclusions Parenteral remdesivir accelerates viral clearance in early symptomatic COVID-19. Pharmacometric assessment of therapeutics using the method described can determine in vivo clinical antiviral efficacy rapidly and efficiently

Evaluation of hydroxychloroquine or chloroquine for the prevention of COVID-19 (COPCOV): A double-blind, randomised, placebo-controlled trial

Background: Hydroxychloroquine (HCQ) has proved ineffective in treating patients hospitalised with Coronavirus Disease 2019 (COVID-19), but uncertainty remains over its safety and efficacy in chemoprevention. Previous chemoprevention randomised controlled trials (RCTs) did not individually show benefit of HCQ against COVID-19 and, although meta-analysis did suggest clinical benefit, guidelines recommend against its use. Methods and findings: Healthy adult participants from the healthcare setting, and later from the community, were enrolled in 26 centres in 11 countries to a double-blind, placebo-controlled, randomised trial of COVID-19 chemoprevention. HCQ was evaluated in Europe and Africa, and chloroquine (CQ) was evaluated in Asia, (both base equivalent of 155 mg once daily). The primary endpoint was symptomatic COVID-19, confirmed by PCR or seroconversion during the 3-month follow-up period. The secondary and tertiary endpoints were: asymptomatic laboratory-confirmed Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection; severity of COVID-19 symptoms; all-cause PCR-confirmed symptomatic acute respiratory illness (including SARS-CoV-2 infection); participant reported number of workdays lost; genetic and baseline biochemical markers associated with symptomatic COVID-19, respiratory illness and disease severity (not reported here); and health economic analyses of HCQ and CQ prophylaxis on costs and quality of life measures (not reported here). The primary and safety analyses were conducted in the intention-to-treat (ITT) population. Recruitment of 40,000 (20,000 HCQ arm, 20,000 CQ arm) participants was planned but was not possible because of protracted delays resulting from controversies over efficacy and adverse events with HCQ use, vaccine rollout in some countries, and other factors. Between 29 April 2020 and 10 March 2022, 4,652 participants (46% females) were enrolled (HCQ/CQ n = 2,320; placebo n = 2,332). The median (IQR) age was 29 (23 to 39) years. SARS-CoV-2 infections (symptomatic and asymptomatic) occurred in 1,071 (23%) participants. For the primary endpoint the incidence of symptomatic COVID-19 was 240/2,320 in the HCQ/CQ versus 284/2,332 in the placebo arms (risk ratio (RR) 0.85 [95% confidence interval, 0.72 to 1.00; p = 0.05]). For the secondary and tertiary outcomes asymptomatic SARS-CoV-2 infections occurred in 11.5% of HCQ/CQ recipients and 12.0% of placebo recipients: RR: 0.96 (95% CI, 0.82 to 1.12; p = 0.6). There were no differences in the severity of symptoms between the groups and no severe illnesses. HCQ/CQ chemoprevention was associated with fewer PCR-confirmed all-cause respiratory infections (predominantly SARS-CoV-2): RR 0.61 (95% CI, 0.42 to 0.88; p = 0.009) and fewer days lost to work because of illness: 104 days per 1,000 participants over 90 days (95% CI, 12 to 199 days; p < 0.001). The prespecified meta-analysis of all published pre-exposure RCTs indicates that HCQ/CQ prophylaxis provided a moderate protective benefit against symptomatic COVID-19: RR 0.80 (95% CI, 0.71 to 0.91). Both drugs were well tolerated with no drug-related serious adverse events (SAEs). Study limitations include the smaller than planned study size, the relatively low number of PCR-confirmed infections, and the lower comparative accuracy of serology endpoints (in particular, the adapted dried blood spot method) compared to the PCR endpoint. The COPCOV trial was registered with ClinicalTrials.gov; number NCT04303507. Interpretation: In this large placebo-controlled, double-blind randomised trial, HCQ and CQ were safe and well tolerated in COVID-19 chemoprevention, and there was evidence of moderate protective benefit in a meta-analysis including this trial and similar RCTs. Trial registration: ClinicalTrials.gov NCT04303507; ISRCTN Registry ISRCTN10207947

The utility of an AMR dictionary as an educational tool to improve public understanding of antimicrobial resistance

Background: Communicating about antimicrobial resistance (AMR) to the public is challenging.   Methods: We developed a dictionary of terms commonly used to communicate about AMR. For each term, we developed learning points to explain AMR and related concepts in plain language.  We conducted a pilot evaluation in 374 high school students in Ubon Ratchathani, Thailand. In three 50-minute sessions, students were asked to answer five true/false questions using a paper-based questionnaire. The first session assessed their understanding of AMR at baseline, the second after searching the internet, and the third after the provision of the printed AMR dictionary and its web address.    Results: We developed the AMR dictionary as a web-based application (www.amrdictionary.net). The Thai version of the AMR dictionary included 35 terms and associated learning points, seven figures displaying posters promoting AMR awareness in Thailand, and 66 recommended online videos. In the pretest, the proportion of correct responses to each question ranged from 10% to 57%; 10% of the students correctly answered that antibiotics cannot kill viruses and 57% correctly answered that unnecessary use of antibiotics makes them ineffective. After the internet searches, the proportions of correct answers increased, ranging from 62% to 89% (all p&lt;0.001). After providing the AMR dictionary, the proportions of correct answers increased further, ranging from 79% to 89% for three questions (p&lt;0.001), and did not change for one question (p=0.15). Correct responses as to whether taking antibiotics often has side-effects such as diarrhoea reduced from 85% to 74% (p&lt;0.001). The dictionary was revised based on the findings and comments received. Conclusions: Understanding of AMR among Thai high school students is limited. The AMR dictionary can be a useful supportive tool to increase awareness and improve understanding of AMR. Our findings support the need to evaluate the effectiveness of communication tools in the real-world setting.</ns3:p

Clinical antiviral efficacy of favipiravir in early COVID-19 (PLATCOV): an open-label, randomised, controlled, adaptive platform trial

Brief summary: In early symptomatic COVID-19 treatment, high dose oral favipiravir did not accelerate viral clearance. Background: Favipiravir, an anti-influenza drug, has in vitro antiviral activity against SARS-CoV-2. Clinical trial evidence to date is inconclusive. Favipiravir has been recommended for the treatment of COVID-19 in some countries. Methods: In a multicentre open-label, randomised, controlled, adaptive platform trial, low-risk adult patients with early symptomatic COVID-19 were randomised to one of ten treatment arms including high dose oral favipiravir (3.6g on day 0 followed by 1.6g daily to complete 7 days treatment) or no study drug. The primary outcome was the rate of viral clearance (derived under a linear mixed-effects model from the daily log10 viral densities in standardised duplicate oropharyngeal swab eluates taken daily over 8 days [18 swabs per patient]), assessed in a modified intention-to-treat population (mITT). The safety population included all patients who received at least one dose of the allocated intervention. This ongoing adaptive platform trial was registered at ClinicalTrials.gov (NCT05041907) on 13/09/2021. Results: In the final analysis, the mITT population contained data from 114 patients randomised to favipiravir and 126 patients randomised concurrently to no study drug. Under the linear mixed-effects model fitted to all oropharyngeal viral density estimates in the first 8 days from randomisation (4,318 swabs), there was no difference in the rate of viral clearance between patients given favipiravir and patients receiving no study drug; a -1% (95% credible interval: -14 to 14%) difference. High dose favipiravir was well-tolerated. Interpretation: Favipiravir does not accelerate viral clearance in early symptomatic COVID-19. The viral clearance rate estimated from quantitative measurements of oropharyngeal eluate viral densities assesses the antiviral efficacy of drugs in vivo with comparatively few studied patients