Enabling mRNA medicine for brain tumors

mRNA is a new class of drugs that has the potential to revolutionize the treatment of brain tumors. Thanks to the COVID-19 mRNA vaccines and numerous therapy-based clinical trials, it is now clear that lipid nanoparticles (LNPs) are a clinically viable means to deliver RNA therapeutics. However, LNP-mediated mRNA delivery to brain tumors remains elusive. Over the past decade, numerous studies have shown that tumor cells communicate with each other via small extracellular vesicles, which are around 100 nm in diameter and consist of lipid bilayer membrane similar to synthetic lipidbased nanocarriers. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain tumors without undue toxicity. We synthesized LNPs using four components similar to the formulation used in the mRNA COVID19 vaccines (Moderna and Pfizer): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. For the in vitro screen, we tested ten classes of helper lipids based on their abundance in extracellular vesicle membranes, commercial availability, and large-scale production feasibility while keeping rest of the LNP components unchanged. The transfection kinetics of GFP mRNA encapsulated in LNPs and doped with 16 mol% of helper lipids was tested using GL261, U87 and SIM-A9 cell lines. Several LNP formations resulted in stable transfection (upto 5 days) of GFP mRNA in all the cell lines tested in vitro. The successful LNP candidates (enabling >80% transfection efficacy) were then tested in vivo to deliver luciferase mRNA to brain tumors via intrathecal administration in a syngeneic glioblastoma (GBM) mouse model, which confirmed luciferase expression in brain tumors in the cortex. LNPs were then tested to deliver Cre recombinase mRNA in syngeneic GBM mouse model genetically modified to express tdTomato under LoxP marker cassette that enabled identification of LNP targeted cells. mRNA was successfully delivered to tumor cells (70-80% transfected) and a range of different cells in the tumor microenvironment, including tumor-associated macrophages (80-90% transfected), neurons (31- 40% transfected), neural stem cells (39-62% transfected), oligodendrocytes (70-80% transfected) and astrocytes (44-76% transfected). Then, LNP formulations were assessed for delivering Cas9 mRNA and CD81 sgRNA (model protein) in murine syngeneic GBM model to enable gene editing in brain tumor cells. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~94% of brain tumor cells in vivo. In conclusion, we have developed a library of safe LNPs that can transfect GBM cells in vivo with high efficacy. This technology can potentially be used to develop novel mRNA therapies for GBM by delivering single or multiple mRNAs and holds great potential as a tool to study brain tumor biology.

Mid-density Gene Expression profiling of SARSCOV-2 infected samples using Applied BiosystemsTM TaqManTM Flexible Array Panels

Background/Objectives: The broad spectrum of clinical manifestations from SARS-COV-2 infection and observed risk factors for severe disease highlight the importance of understanding molecular mechanisms underlying SARS-CoV-2 associated disease pathogenesis. Research studies have identified a large number of host proteins that play roles in viral entry, innate immune response, or immune signalling during infection. The ability to interrogate subsets of these genes simultaneously within SARSCOV-2 infected samples is critical to understanding how their expression contribute to phenotypic variability of the disease caused by the pathogen. Method(s): 30 Nasopharyngeal swab were obtained and included SARS-CoV-2 infected and control samples. RNA was extracted, reverse transcribed and loaded onto flexible TaqMan array panels designed specifically for targeting the most cited genes related to SARS-COV-2 entry and restriction factors as well as cytokines, chemokines, and growth factors involved in the pathogenesis. Result(s): Our data indicated that not only were the levels of several of these host factors differentially modulated between the two study groups, but also that SARS-CoV-2 infected subjects presented with greater frequency of several important inflammatory cytokines and chemokines such as CCL2, CCL3, IFNG, entry receptors such as ACE2, TMRPS11A, and host restriction factors including LY6E and ZBP1. Conclusion(s): TaqMan array plates provide a fast, midthroughput solution to determine the levels of several virus and host-associated factors in various cell types and add to our understanding of how the pathogenesis may vary depending on gender, age, infection site etc.

A Detection System for Comfortable Locations Based on Facial Expression Analysis While Riding Bicycles

In recent years, the use of bicycle as a healthy and economical means of transportation has been promoted worldwide. In addition, with the increase in bicycle commuting due to the COVID-19, the use of bicycles are attracting attention as a last-mile means of transportation in Mobility as a Service(MaaS). To help ensure a safe and comfortable ride using a smartphone mounted on a bicycle, this study focuses on analyzing facial expressions while riding to determine potential comfort along the route with the surrounding environment and to provide a map that users can explicitly feedback(FB) after riding. Combining the emotions of facial expressions while riding and FB, we annotate comfort to different locations. Afterwards, we verify the relationship between locations with high level of comfort based on the acquired data and the surrounding environment of those locations using Google Street View(GSV). © 2023 Owner/Author.

Angry, Sad, or Scared? Within-valence Mapping of Emotion Words to Facial and Body Cues in 2 to 4-Year-Old Children

The acquisition of emotion words is critical to children's socio-emotional development. Previous studies report that children acquire emotion words gradually during ages 3-5 and beyond. The majority of this work, however, has used demanding tasks for young children (e.g., asking children to label emotion-related facial configurations) and has predominantly relied on facial configurations. Here we designed a child-friendly, word-comprehension task incorporating both facial configurations and body language. In two preregistered online experiments, we asked two to four-year-olds (N = 96) to connect emotion words-happy, sad, angry, and scared-to either facial configurations (Experiment 1) or combined facial and body cues (Experiment 2). We found relatively early competence in understanding emotion words, especially those of the same-valence. All age groups, including 2-year-olds, successfully linked emotion words to corresponding facial configurations (Experiment 1). Experiment 2 replicated this pattern and further showed that children performed equally well (though not substantially better) when given additional body cues. Parental reports of children's exposure to and use of masks during the COVID-19 pandemic did not correlate with children's performance in either experiment. Even before children can produce emotion words in an adult-like manner, they possess at least a partial understanding of those words and can map them to emotion cues within valence domains. © 2023 University of California Press. All rights reserved.

Biodegradable Polyester Nanoparticle Vaccines Deliver Self-Amplifying mRNA in Mice at Low Doses

Delivery of self-amplifying mRNA (SAM) has high potential for infectious disease vaccination due to its self-adjuvanting and dose-sparing properties. Yet a challenge is the susceptibility of SAM to degradation and the need for SAM to reach the cytosol fully intact to enable self-amplification. Lipid nanoparticles are successfully deployed at incredible speed for mRNA vaccination, but aspects such as cold storage, manufacturing, efficiency of delivery, and the therapeutic window can benefit from further improvement. To investigate alternatives to lipid nanoparticles, a class of >200 biodegradable end-capped lipophilic poly(beta-amino ester)s (PBAEs) that enable efficient delivery of SAM in vitro and in vivo as assessed by measuring expression of SAM encoding reporter proteins is developed. The ability of these polymers to deliver SAM intramuscularly in mice is evaluated, and a polymer-based formulation that yields up to 37-fold higher intramuscular (IM) expression of SAM compared to injected naked SAM is identified. Using the same nanoparticle formulation to deliver a SAM encoding rabies virus glycoprotein, the vaccine elicits superior immunogenicity compared to naked SAM delivery, leading to seroconversion in mice at low RNA injection doses. These biodegradable nanomaterials may be useful in the development of next-generation RNA vaccines for infectious diseases.Copyright © 2023 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH.

The preventive effect and mechanism of Dayuanyin in hypoxic pulmonary hypertension through NF-kappaB signaling pathway.

Dayuanyin (DYY) has been shown to reduce lung inflammation in both coronavirus disease 2019 (COVID-19) and lung injury. This experiment was designed to investigate the efficacy and mechanism of action of DYY against hypoxic pulmonary hypertension (HPH) and to evaluate the effect of DYY on the protection of lung function. Animal welfare and experimental procedures are approved and in accordance with the provision of the Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Science. Male C57/BL6J mice were randomly divided into 4 groups: control group, model group, DYY group (800 mg.kg-1), and positive control sildenafil group (100 mg.kg-1). The animals were given control solvents or drugs by gavage three days in advance. On day 4, the animals in the model group, DYY group and sildenafil group were kept in a hypoxic chamber containing 10% +/- 0.5% oxygen, and the animals in the control group were kept in a normal environment, and the control solvent or drugs continued to be given continuously for 14 days. The right ventricular systolic pressure, right ventricular hypertrophy index, organ indices and other metrics were measured in the experimental endpoints. Meantime, the expression levels of the inflammatory factors in mice lung tissues were measured. The potential therapeutic targets of DYY on pulmonary hypertension were predicted using network pharmacology, the expression of nuclear factor kappa B (NF- kappaB) signaling pathway-related proteins were measured by Western blot assay. It was found that DYY significantly reduced the right ventricular systolic pressure, attenuated lung injury and decreased the expression of inflammatory factors in mice. It can also inhibit hypoxia-induced activation of NF- kappaB signaling pathway. DYY has a protective effect on lung function, as demonstrated by DYY has good efficacy in HPH, and preventive administration can slow down the disease progression, and its mechanism may be related to inhibit the activation of NF-kappaB and signal transducer and activator of transcription 3 (STAT3) by DYY.Copyright © 2023, Chinese Pharmaceutical Association. All rights reserved.

Pandemics and infodemics: How COVID-19 is reshaping content regulation

The debate about tackling online misinformation and disinformation is not unique to the COVID-19 pandemic. However, the health crisis has elevated the danger of misleading information circulated on social media. Labelled as 'infodemic', the problem of COVID-19-related misinformation and disinformation prompted governments and social media platforms around the globe to impose various speech restrictions. The public and private policy frameworks aiming to curb the spread of the infodemic were adopted in a state of emergency and without proper scrutiny. Yet, they are shaping the future of content regulation, possibly affecting freedom of speech and other democratic values for years to come. This chapter looks at the key problematic aspects of actions taken by governments and social media platforms to address COVID-19-related misinformation and disinformation and discusses the possible long-term effects of these measures. © 2023 the authors.

Increases in expression of carbohydrate sulfotransferases CHST11 and CHST15 and decline in N-acetylgalactosamine-4-sulfatase (Arylsulfatase B, ARSB) require phospho-p38-MAPK following exposure to SARS-CoV-2 spike protein receptor binding domain in human a

Objective: Immunohistochemistry of post-mortem lung tissue from Covid-19 patients with diffuse alveolar damage demonstrated marked increases in chondroitin sulfate and CHST15 and decline in N-acetylgalactosamine-4-sulfatase. Studies were undertaken to identify the mechanisms involved in these effects. Method(s): Human primary small airway epithelial cells (PCS 301-010;ATCC) were cultured and exposed to the SARSCoV- 2 spike protein receptor binding domain (SPRBD;AA: Lys310-Leu560;Amsbio). Expression of the spike protein receptor, angiotensin converting enzyme 2 (ACE2), was enhanced by treatment with Interferon-beta. Promoter activation, DNA-binding, RNA silencing, QPCR, Western blots, ELISAs, and specific enzyme inhibitors were used to elucidate the underlying molecular mechanisms. Result(s): Treatment of the cultured cells by the SPRBD led to increased CHST15 and CHST11 expression and decline in ARSB expression. Sulfotransferase activity, total chondroitin sulfate, and sulfated glycosaminoglycan (GAG) content were increased. Phospho-T180/T182-p38-MAPK and phospho- S423/S425-Smad3 were required for the activation of the CHST15 and CHST11 promoters. Inhibition by SB203580, a phospho-p38 MAPK inhibitor, and by SIS3, a Smad3 inhibitor, blocked the CHST15 and CHST11 promoter activation. SB203580 reversed the SPRBD-induced decline in ARSB expression, but SIS3 had no effect on ARSB expression or promoter activation. Phospho-p38 MAPK was shown to reduce retinoblastoma protein (RB) S807/S811 phosphorylation and increase RB S249/T252 phosphorylation. E2F-DNA binding declined following exposure to SPRBD, and SB203580 reversed this effect. This indicates a mechanism by which SPRBD, phospho-p38 MAPK, E2F, and RB can regulate ARSB expression and thereby impact on chondroitin 4-sulfate and dermatan sulfate and molecules that bind to these sulfated GAGs, including Interleukin-8, bone morphogenetic protein-4, galectin-3 and SHP-2 (Src homology region 2-containing protein tyrosine phosphatase 2). Conclusion(s): The enzyme ARSB is required for the degradation of chondroitin 4-sulfate and dermatan sulfate, and accumulation of these sulfated GAGs can contribute to lung pathophysiology, as evident in Covid-19. Some effects of the SPRBD may be attributable to unopposed Angiotensin II, when Ang1-7 counter effects are diminished due to binding of ACE2 with the SARS-CoV-2 spike protein and reduced production of Ang1-7. Aberrant cell signaling and activation of the phospho-p38 MAPK and Smad3 pathways increase CHST15 and CHST11 production, which can contribute to increased chondroitin sulfate in infected cells. Decline in ARSB may occur as a consequence of effects of phospho-p38 MAPK on RB phosphorylation and E2F1 availability. Decline in ARSB and the resulting impaired degradation of sulfated GAGs have profound consequences on cellular metabolic, signaling, and transcriptional events. Funding is VA Merit Award.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

The Impact of the Pandemic on the Greek Constitution

This chapter discusses the main legal problems Greece faced during the various phases of the COVID-19 crisis. The impact of the pandemic on the protection of civil rights seems to be lasting and nefarious. Fundamental rights were subject to unprecedented restrictions for the sake of public health, without any effective judicial protection. Free movement, the right of assembly, religious liberty, freedom of speech, economic liberty, and ultimately personal autonomy limited by compulsory vaccination, were regulated exhaustively by detailed and intrusive administrative rules and prohibitions. Moreover, the pandemic put under great pressure the existing framework for the constitutional protection of fundamental rights where the principle of proportionality is the cornerstone of judicial review. © The Author(s), under exclusive license to Springer Nature Switzerland AG 2022.

Association of SARS-COV-2 viral RNAemia, IL- 6 gene polymorphism, serum IL-6 and peripheral blood lymphocytes and monocytes with disease severity in COVID-19 patients

Coronavirus disease 2019 (COVID-19) is a fatal pandemic viral disease caused by the severe acute respiratory syndrome corona virus type-2 (SARS-CoV-2). The aim of this study is to observe the associations of IL-6, SARS-COV-2 viral load (RNAemia), IL- 6 gene polymorphism and lymphocytes and monocytes in peripheral blood with disease severity in COVID-19 patients. This study was carried out from March 2021 to January 2022. RT-PCR positive 84 COVID-19 patients and 28 healthy subjects were enrolled. Blood was collected to detect SARS-COV-2 viral RNA (RNAemia) by rRT-PCR, serum IL-6 level by chemiluminescence method, SNPs of IL-6 by SSP-PCR, immunophenotyping of lymphocytes and monocyte by flow cytometry. Serum IL-6 level (pg/ml) was considerably high among critical patients (102.02 +/- 149.7) compared to severe (67.20 +/- 129.5) and moderate patients (47.04 +/- 106.5) and healthy controls (3.5 +/- 1.8). Serum SARS-CoV-2 nucleic acid positive cases detected mostly in critical patients (39.28%) and was correlated with extremely high IL-6 level and high mortality (R =.912, P < 0.001). Correlation between IL-6 and monocyte was statistically significant with disease severity (severe group, p < 0.001, and 0.867*** and critical group p < 0.001 and 0.887***). In healthy controls, moderate, severe and critically ill COVID-19 patients, IL-6 174G/C (rs 1800795) GG genotype was 82.14%, 89.20%, 67.85% and 53.57% respectively. CC and GC genotype had strong association with severity of COVID-19 when compared with GG genotype. Significant statistical difference found in genotypes between critical and moderate groups (p < 0.001, OR-10.316, CI-3.22-23.86), where CC genotype was associated with COVID-19 severity and mortality. The absolute count of T cell, B cell, NK cell, CD4+ T cells and CD8+ T cells were significantly decreased in critical group compared to healthy, moderate and severe group (P < 0.001). Exhaustion marker CD94/NKG2A was increased on NK cells and CD8+ cytotoxic T cell among critical and severe group. Absolute count of monocyte was significantly increased in critical group (P < 0.001). Serum IL-6, IL-6 174 G/C gene and SARS-CoV-2 RNAaemia can be used in clinical practice for risk assessment;T cell subsets and monocyte as biomarkers for monitoring COVID-19 severity. Monoclonal antibody targeting IL-6 receptor and NKG2A for therapeutics may prevent disease progression and decrease morbidity and mortality.Copyright © 2023 Elsevier Inc.

Masked Student Dataset of Expressions

Facial expression recognition (FER) algorithms work well in constrained environments with little or no occlusion of the face. However, real-world face occlusion is prevalent, most notably with the need to use a face mask in the current Covid-19 scenario. While there are works on the problem of occlusion in FER, little has been done before on the particular face mask scenario. Moreover, the few works in this area largely use synthetically created masked FER datasets. Motivated by these challenges posed by the pandemic to FER, we present a novel dataset, the Masked Student Dataset of Expressions or MSD-E, consisting of 1,960 real-world non-masked and masked facial expression images collected from 142 individuals. Along with the issue of obfuscated facial features, we illustrate how other subtler issues in masked FER are represented in our dataset. We then provide baseline results using ResNet-18, finding that its performance dips in the non-masked case when trained for FER in the presence of masks. To tackle this, we test two training paradigms: contrastive learning and knowledge distillation, and find that they increase the model's performance in the masked scenario while maintaining its non-masked performance. We further visualise our results using t-SNE plots and Grad-CAM, demonstrating that these paradigms capitalise on the limited features available in the masked scenario. Finally, we benchmark SOTA methods on MSD-E. The dataset is available at https://github.com/SridharSola/MSD-E. © 2022 ACM.

Glucosaminylmuramyl dipeptide efficacy in post-COVID-19 patient rehabilitation treatment.

The COVID-19 pandemic has altered people's lifestyles around the world. Prevention of recurrent episodes of the disease and mitigation of its consequences are especially associated with effective post-COVID-19 rehabilitation in patients. The aim of the study was to evaluate the effects of the drug Likopid (glucosaminylmuramyl dipeptide, GMDP) for post-COVID-19 rehabilitation in patients. Material and methods. Patients who recovered from mild to moderate COVID-19 (n=60, mean age 54+/- 11.7 years) were randomized into the observation group (n=30, 15 men and 15 women) who received 2 courses of Licopid (1 mg twice a day) and the comparison group (n=30, 15 men and 15 women). Analysis of the phenotypic and functional characteristics of the innate immune cellular factors was carried out before the start of immunomodulatory therapy, immediately after the end of the course, and also after 6 months observations. In order to assess the quality of life of all patients, we used the SF-36 Health Status Survey and the Hospital Anxiety and Depression Scale questionnaires. Results. During assessing the effect of immunomodulatory therapy on the parameters of innate immunity of patients at the stage of rehabilitation after COVID-19, an increase in the protective cytolytic activity of CD16+ and CD8+Gr+ cells, as well as a persistent increase in TLR2, TLR4 and TLR9 expression was found, which indicates the antigen recognition recovery and presentation at the level of the monocytic link of the immune system. The use of GMDP as an immunomodulatory agent resulted in an 8-fold reduction in the frequency and severity of respiratory infections due to an increase in the total monocyte count. As a result of assessing patients' quality of life against the background of the therapy, a positive dynamic in role functioning was revealed in patients. In the general assessment of their health status, an increase in physical and mental well-being was noted during 6 months of observation. The comparison group showed no improvement in the psychoemotional state. Discussion. The study demonstrated the effectiveness of GMDP immunomodulatory therapy in correcting immunological parameters for post-COVID-19 rehabilitation in patients. The data obtained are consistent with the previously discovered ability of GMDP to restore impaired functions of phagocytic cells and induce the expression of their surface activation markers, which in turn contributes to an adequate response to pathogens. Conclusion. The study revealed that the correction of immunological parameters with the use of GMDP in COVID-19 convalescents contributed not only to a decrease in the frequency and severity of respiratory infections, but also to an improvement in the psycho-emotional state of patients, and a decrease in anxiety and depression.Copyright © Eco-Vector, 2023. All rights reserved.

Two-year outcome of extracorporeal membrane oxygenation in COVID-19 related severe respiratory failure

Objectives: We sought to evaluate 2-year outcome of V-V ECMO support for COVID-19 related severe respiratory failure in our center. Method(s): Retrospective analysis of 41 consecutive patients (73% male, mean age 51.6+/-14.2 years, mean BMI 35.1+/-12.5 kg/m2) with critical hypoxemic and/or hypercapnic refractory respiratory failure (mean P/F ratio 67.9+/-14.3 mmHg, mean pCO2 77.6.0+/-185.7 mmHg, Murray Score 3.71+/-0.4) on V-V ECMO support from October 2020 to January 2022 Results: With mean support duration of 234.4+/-63.2 hours, 29 patients (70.7%) were successfully weaned off. Finally, 19 of them (46.3%) were discharged home with good neurological outcome (CPC 1,2). During followup, 30-day, 6-, 12-, and 24 -month survival rate was 61.3%, 46.2%, 41.9%, and 41,9% respectively. In survivor group shorter symptoms onset to respiratory failure time (4+/-4.7 vs. 7+/-6.7 days, p=0.04), higher P/F ration (86+/-41.5 vs. 65+/-37.5 mmHg, p=0.04) and norepinephrine support (0.03+/-0.06 vs. 0.09+/-0.12 ug/kg/min, p=0.04), and lower IL-6 level (12.3+/-7.5 vs. 25.9+/-8.8 ng/l, p=0.03) p=0.01) were analysed before cannulation. Mean in-ICU stay and in-hospital stay in survivors;groups reached 32.5+/-27.7 days and 42.6+/-35.8 days, respectively. All long-term survivors (17 patients) complained about slight functional health limitation only with normal 6MWT (542.6+/- 89.2 min), near to normal spirometry parameters (FEV/VC 87+/-7.4%, DLCO 63.1+/-13.7%, KCO 82.,1+/-19.4%) and minimal neurological disability (CPC 1-2) Conclusion(s): 2-year outcome of V-V ECMO support in COVID-19 severe respiratory failure is acceptable even in the scope of low-volume ECMO centre. Reported functional status of long-term survivors was good despite the complicated and prolonged in-hospital stay. (Table Presented).

Clinical and laboratory indicators in predicting the risk of chronic heart failure in COVID-19 survivors.

Coronavirus disease has many systemic disease symptoms and has severe consequences for the cardiovascular system. Objective. To assess the role of clinical and laboratory indicators in determining the risk of chronic heart failure (CHF) in COV-ID-19 survivors. Material and methods. In total, 151 patients treated in a monoinfectious hospital from 03.11.20 to 10.02.21 with a confirmed diagnosis of COVID-19 were retrospectively selected. Medical history and laboratory data were collected by reviewing electronic medical records. The data included age, gender, body mass index, smoking status, and comorbidities. The laboratory data included the results of hematology and blood chemistry, coagulation, and the levels of acute-phase proteins. The CHF occurrence was used as the study endpoint. Results and discussion. The study patients were divided into two groups depending on the presence of CHF: group 1 included 46 patients with CHF, and group 2 included 105 patients without CHF. The median age was 66.2 (50-92) years;91 (60.3%) were females. Laboratory tests, such as levels of the hs-C-reactive protein, lactate dehydrogenase, procalcitonin, creatinine, and bilirubin, were statistically significantly different in patients of the study groups, and the median values were higher in patients with CHF. Neutrophil-lymphocyte ratio (NLR) showed statistically significant differences between groups: in patients with CHF, the median was 4.97% compared to 3.62% (p=0.011) in those without CHF. The most significant predictors of an increased risk of CHF were age >=66 years (OR=8.038, p<0.001), procalcitonin level >=0.09 ng/mL (increased the CHF risk by 3.8 times, p<0.001), thrombocy-topenia <=220x109/L (p=0.010), an NLR ratio >=4.11% (p=0.010), and a history of chronic kidney disease (p=0.018). Conclusion. A model has been developed to determine the factors closely associated with the risk of chronic heart failure in CO-VID-19 survivors.Copyright © 2023, Media Sphera Publishing Group. All rights reserved.

Angiotensin-converting enzyme 2 (ACE2) in COVID-19 and other diseases.

The coronavirus SARS-CoV-2 was detected in isolates of pneumonia patients in January 2020. The virus cannot multiply extracellularly but requires access to the cells of a host organism. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as a receptor, to which it docks with its spikes. ACE2 belongs to the renin angiotensin system (RAS), whose inhibitors have been used for years against high blood pressure. Renin is an endopeptidase that is predominantly formed in the juxtaglomerular apparatus of the kidney and cleaves the decapeptide angiotensin I (Ang I) from angiotensinogen. Through the angiotensin-converting enzyme (ACE), another 2 C-terminal amino acids are removed from Ang I, so that finally the active octapeptide angiotensin II (Ang II) is formed. The biological effect of Ang II via the angiotensin II receptor subtype 1 (AT1-R) consists of vasoconstriction, fibrosis, proliferation, inflammation, and thrombosis formation. ACE2 is a peptidase that is a homolog of ACE. ACE2 is predominantly expressed by pulmonary alveolar epithelial cells in humans and has been detected in arterial and venous endothelial cells. In contrast to the dicarboxy-peptidase ACE, ACE2 is a monocarboxypeptidase that cleaves only one amino acid from the C-terminal end of the peptides. ACE2 can hydrolyze the nonapeptide Ang-(1-9) from the decapeptide Ang I and the heptapeptide Ang-(1-7) from the octapeptide Ang II. Ang-(1-7) acts predominantly antagonistically (vasodilatory, anti-fibrotic, anti-proliferative, anti-inflammatory, anti-thrombogenetically) via the G protein-coupled Mas receptor to the AT1-R-mediated effects of Ang II. In the pathogenesis of COVID-19 infection, it is therefore assumed that there is an imbalance due to overstimulation of the AT1 receptor in conjunction with a weakening of the biological effects of the Mas receptor.Copyright © 2022 Dustri-Verlag Dr. K. Feistle.

Mastocytosis and COVID19 infection and vaccination: A pediatric series

Introduction: Mastocytosis encompasses a heterogeneous group of diseases characterized by an accumulation of clonal mast cells (MC) in the skin and/or internal organs, and symptoms of MC activation. This MC activation can be elucidated by several factors, including infections or vaccination. Objective(s): We present our experience with COVID infection and vaccination in a series of 133 patients with pediatric mastocytosis. Method(s): Between January 1998 and December 2022, 133 pediatric patients have been referred to our hospital owing to clinically suspected MC disorder, mainly with mastocytosis in the skin. The final diagnoses of mastocytosis were established by the presence of typical skin lesions together with an increase of MC numbers in a biopsy from lesional skin or activating KIT mutations in lesional skin tissue. Serum baseline tryptase and total immunoglobulin E levels were measured, and patients underwent a comprehensive allergy workup to confirm atopic status and history of anaphylaxis. Regarding vaccination, REMA's (Spanish Network on Mastocytosis) protocol was followed. Result(s): 13 patients with COVID infection were identified, of which 25 (56,8%) were female and 0% had symptoms of MC activation. All of them had an asymptomatic or mild course of COVID infection. None of the patients experimented MC activation symptoms during viral illness. Regarding COVID vaccination, all patients received premedication with antihistamine 60 minutes prior vaccination. No one experimented immediate reactions and only one patient (0,75%) referred worsening of MC activation symptoms (baseline pruritus, urtication and brain fog) only after the first doses, recovering without changes in his treatment (oral cromoglycate and antihistamine) in two months. Discussion(s): Although MC have been implicated in the pathogenesis of cytokine storm in COVID19, there is no clinical evidence of SARSCoV- 2-induced MC activation, perhaps related to the fact that bone marrow MC lack angiotensin-converting enzyme 2 receptors.

GENE ANALYSIS REVEALED THE USEFULNESS OF TRIPLE THERAPY WITH BARICITINIB, RITUXIMAB AND TACROLIMS FOR ANTI-MDA5 ANTIBODY POSITIVE DERMATOMYOSITIS (MDA5-DM)

BackgroundAnti-MDA5 antibody positive dermatomyositis (MDA5-DM) is characterized by high mortality due to rapid progressive ILD. MDA5 is a cytosolic protein and a family of RIG-I like receptor, which functions as a virus RNA sensor and induces the production of such as type-1 IFN. Although little is known about the pathogenesis of MDA5-DM, it is notable that the similarities were reported between COVID-19 infection and MDA5-DM. It may suggest that there is a common underlying autoinflammatory mechanism. We reported that in MDA5-DM, (1) RIG-I-like receptor signaling is enhanced and (2) antiviral responses such as type 1 IFN signaling are also enhanced as compare with anti-ARS-antibody positive DM, and (3) the key for survival is suppression of RIG-I-like and IFN signaling (EULAR2022, POS0390). We also found that a significant role for uncontrolled macrophage in the pathogenesis of ILD by our autopsy case. Recently, it has been reported that tacrolimus (TAC) and cyclophosphamide (CY) combination therapy (TC-Tx) has improved the prognosis of cases with early onset of the disease, but there are cases that cannot be saved. Therefore, we devised BRT therapy (BRT-Tx). The Tx combines baricitinib (BAR), which inhibits GM-CSF and IFN-mediated signaling and effectively suppresses uncontrolled macrophages, with rituximab (RTX) and TAC, which rapidly inhibits B and T cell interaction and ultimately prevents anti-MDA5 antibody production.ObjectivesTo determine the differences in gene expression between BRT and TC-Tx for MDA5-DM in peripheral blood.MethodsTotal of 6 MDA5-DM (TC: 3, BRT: 3) were included and all of them had multiple poor prognostic factors. Peripheral whole blood was collected at just before and 2-3 months after the treatment. RNA was extracted, and quantified using a next-generation sequencer. Differentially Expressed Genes (DEGs) were identified by pre vs. post treatment. Gene Ontology (GO), clustering and Gene Set Variation Analysis (GSVA) were performed to DEGs. As one BRT case was added since our last year's report, we also reanalyzed the surviving vs. fatal cases. The IFN signature was scored separately for Types 1, 2, and 3, and the changes between pre- and post-treatment were investigated.ResultsTwo of three cases with TC died during treatment, while all three cases on BRT recovered. The cluster analysis of the DEGs separated deaths from survivors, not by type of treatment. Comparing surviving and dead cases, GO analysis revealed that the immune system via immunoglobulins and B cells was significantly suppressed in surviving cases. GO analysis of DEGs in each therapeutic group showed that expression of B cell-related genes such as lymphocyte proliferation and B cell receptor signaling pathway were significantly suppressed in BRT-Tx. On the other hand, TC-Tx significantly suppressed such pathways as cell proliferation and cell surface receptor signaling, and was less specific for the target cells than BRT-Tx. The changes in IFN signature score after treatment showed an increase in type 2 and 3 IFN scores in all fatal cases and an increase in type 1 IFN score in one fatal case.ConclusionBRT-Tx significantly suppressed gene expression associated with B cells, while TC-Tx was characterized by low specificity of therapeutic targets and suppression of total cell proliferation. Comparison of surviving and dead cases revealed that the combination of RTX contributed to the success of treatment, as suppression of the immune system mediated by immunoglobulins and B cells is the key for survival. Analysis of the IFN signature revealed an increase in IFN score after treatment in fatal cases, indicating that the combination of BAR is beneficial. The superiority of BRT-Tx seems clear from the fact that all patients survived with BRT-Tx while only one/three patients survived with TC-Tx.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsMoe Sakamoto: None declared, Yu Nakai: None declared, Yoshiharu Sato: None declared, Yoshinobu Koyama Speakers bureau: Abbvie, Asahikasei, Ayumi, BMS, Esai, Eli-Lilly, Mitsubishi Tanabe, Grant/research support from: Abbvie, GSK.

Assessment of the Impact of the Egyvir on Rats Experimental Animals, a Preclinical Study for Sars-Cov-2 Treatment

Background: The SARS-COV-2 is a worldwide pandemic problem. We developed a herbal extract with potent in-vitro virucidal, anti-inflammatory and immunomodulatory effects called EGIVIR. Our aim is to assess the bioavailability and cytotoxicity of EGYVIR on different organs and biological systems in Sprague Dawley rats as a model of experimental animals.Methods: 128 rats were divided into 16 groups (8 rats each), where Egyvir was assessed in oral doses of 20, 30, and 40 mg/kg body weight, and by inhalation in 0.2, 0.3, and 0.4 mg/kg body weight, four times/day, compared to the control groups.Results: The Egyvir had no significant effect on the blood pressure, pulse, motor activity, histological, hematological, and coagulation profiles. Also, the blood levels of triglycerides, cholesterol, blood glucose, lactate dehydrogenase (LDH), and creatine phosphor kinase (CPK) were not significantly affected. Egyvir had no harmful effect on the kidney and liver functions, blood electrolytes levels and urinary levels of sodium, potassium, and chloride. There was no significant effect on the serum levels of interleukin-113 (IL -113), IL-2, IL-4, IL-6, IL-10, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). Additionally, there was no significant change in the levels of Superoxide dismutase (SOD), catalase, reduced glutathione (GSH), and malonaldehyde (MDA) in comparison to the control groups (P<0.05).Conclusion: Egyvir is considered a safe antiviral natural drug. It could be used for the treatment of SARS-COV-2 without any adverse effects when used with the recommended doses. However, these data are a preliminary step for validation in a clinical setting.

Multi-objective Evolutionary Discretization of Gene Expression Profiles: Application to COVID-19 Severity Prediction

Machine learning models can use information from gene expressions in patients to efficiently predict the severity of symptoms for several diseases. Medical experts, however, still need to understand the reasoning behind the predictions before trusting them. In their day-to-day practice, physicians prefer using gene expression profiles, consisting of a discretized subset of all data from gene expressions: in these profiles, genes are typically reported as either over-expressed or under-expressed, using discretization thresholds computed on data from a healthy control group. A discretized profile allows medical experts to quickly categorize patients at a glance. Building on previous works related to the automatic discretization of patient profiles, we present a novel approach that frames the problem as a multi-objective optimization task: on the one hand, after discretization, the medical expert would prefer to have as few different profiles as possible, to be able to classify patients in an intuitive way;on the other hand, the loss of information has to be minimized. Loss of information can be estimated using the performance of a classifier trained on the discretized gene expression levels. We apply one common state-of-the-art evolutionary multi-objective algorithm, NSGA-II, to the discretization of a dataset of COVID-19 patients that developed either mild or severe symptoms. The results show not only that the solutions found by the approach dominate traditional discretization based on statistical analysis and are more generally valid than those obtained through single-objective optimization, but that the candidate Pareto-optimal solutions preserve the sense-making that practitioners find necessary to trust the results. © 2023, The Author(s), under exclusive license to Springer Nature Switzerland AG.

Establishment of an infrastructure for rapid pandemic response at the Frederick National Laboratory

The sudden onset of the 2019 SARS-CoV-2 pandemic required agile development of standards and efficient validation of assays to assess prevalence of infection as well as immune responses to infection and vaccination. Leveraging their experience in HPV serology and standards, the Vaccine, Immunity and Cancer Directorate (VICD) at the Frederick National Laboratory for Cancer Research (FNCLR) pivoted to address this unmet need in SARS-Co-V2 serology clinical testing and research. This standardization effort required the collection and processing of large volumes of blood from SARS-Co-V2 infected and uninfected individuals into serum and peripheral blood mononuclear cells (PBMCs). Collaborations with specimen collection sites across the United States were established. Following qualification for anti-SARS-CoV-2 IgG and IgM levels in independent laboratories, VICD assembled reference evaluation panels, which were used to assist the FDA's performance evaluation of commercial assays submitted for EUA approval. To date, 185 different shipments of the standard or validation panel have been sent to both domestic and international labs. These materials are also available to the SARS-CoV-2 serology community for assay calibration and performance evaluation which greatly facilitates assay data harmonization. In addition, the NCI Serological Sciences Network (SeroNet) was born from this initiative and expertise, resulting in the establishment of Capacity Building Centers (CBCs) for sample collection from different healthy, cancer and immunocompromised cohorts at Mount Sinai, Arizona State University, the University of Minnesota, and Northwell Feinstein. The NCI and FNLCR simultaneously collaborated to develop a network of investigators focused on advancing research on the immune response to SARS-CoV-2 infection and vaccination among diverse and vulnerable populations, including cancer patients. Their research has resulted in over 326 peer-reviewed publications. The CBC's have enrolled patients in longitudinal studies, resulting in a centralized collection of annotated, well characterized serum, PBMCs and clinical data. Numerous cancer cohorts, but predominantly Multiple Myeloma, are included. Furthermore, technology development was supported at the CBC's. Based upon this success, the VICD in collaboration with NCI is pursuing an even more innovative effort in pandemic preparedness to establish a Center for Serology and Data Emergency Preparedness (CESDEP);a global network able to activate and pivot to address pandemic-level threats, while continuing to expand the development of immunological assays that can inform clinical decisions for cancer and other immunocompromised patients.