Immune checkpoint inhibitors-induced myocarditis presents unique clinical challenges. Here, we assessed post-marketing safety of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), programmed cell death-1 (PD-1), and programmed death-ligand 1 (PD-L1) inhibitors by mining the real-world data reported in two international pharmacovigilance databases.We analyzed immune checkpoint inhibitors (ICIs)-associated fatal adverse drug events (ADEs) reports from the US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) collected from July 1, 2014 to December 31, 2019 and data from EudraVigilance (EV) database accessed on February 29, 2020. Three different data mining approaches were used to detect the signal of fatal myocarditis caused by ICIs.Based on 7613 ICIs-related ADEs reported to the EV database and 5786 ICIs-associated ADEs submitted to the FAERS database, the most frequently reported ADE was ipilimumab-related colitis. For myocarditis, nivolumab-associated myocarditis was the most common. Among the five fatal toxic effects associated with ICIs, the lethality rate of myocarditis was the highest. Therefore, we further analyzed ICI-associated myocarditis and found that elderly patients and male patients were more likely to develop ICIs-related myocarditis. The results of signal detection showed that the risk signal of avelumab-related myocarditis detected by reporting odds ratio (ROR) method and proportional reporting ratios (PRR) method was the highest, whereas the signal strength of ipilimumab-related myocarditis detected by Bayesian confidence propagation neural networks (BCPNN) method was the strongest.The findings of this study indicated the potential safety issues of developing myocarditis when using ICIs, which were consistent with the results of previous clinical trials and could provide a reference for clinical workers when using ICIs.The online version contains supplementary material available at 10.1186/s12885-020-07741-0. Immune checkpoint inhibitors (ICIs), such as cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), programmed cell death-1 (PD-1), and programmed death-ligand 1 (PD-L1) inhibitors that can inhibit the co-inhibitory immune checkpoint pathways have completely changed the treatment landscape of various malignant tumors –4. TheseThe US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) is one of the international spontaneous reporting systems, that is designed to support the FDA’s post-marketing safety surveillance program for drugs approved by the FDA. This database includes all information on adverse drug events (ADEs) and medication errors collected by the FDA. EudraVigilance (EV) is another international spontaneous reporting pharmacovigilance database for ADEs maintained by the European Medicines Agency. Data from these databases can be acquired by the public and utilized to provide evidence for the safe use of the drugs, especially for newly marketed drugs and uncommon ADRs. The objective of this study was to analyze the ICIs-associated myocarditis and to determine the signal of ICIs-associated myocarditis by mining the data reported in the spontaneous reporting systems.https://fis.fda.gov/extensions/FPD-QDE-FAERS/FPD-QDE-FAERS.html), and EV (http://www.adrreports.eu/en/search.html) was accessed and queried on February 29, 2020. In this study, the processing of data downloaded from FAERS database followed the customized strategy described previously   and the risk of ADRs , 13. ThiDespite the limitations, the findings of this study indicated the potential safety issues of developing myocarditis when using ICIs, which was consistent with the results of previous clinical trials and could provide a reference for clinical workers when using ICIs.The results of this study showed that the application of ICIs was associated with an increase in fatal toxic effects, especially myocarditis, which was consistent with previous studies. It was suggested that clinicians should pay attention to these fatal ICIs-associated ADRs and take preventive measures when treating tumor patients with immunotherapy based on ICIs. The findings of this study also provided objective evidence for post-marketing safety of ICIs, thereby ensuring the safe use of these drugs and improving the prognosis of patients with cancer.Additional file 1: Table S1. A 2 × 2 Contingency Table for Disproportionality Analysis. Table S2. Signal Values of ICIs-Associated ADRs.


Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compromises the ability of military forces to fulfill missions. At the beginning of May 2020, 22 out of 70 Belgian soldiers deployed to a military education and training center in Maradi, Niger, developed mild COVID-19 compatible symptoms. Immediately upon their return to Belgium, and two weeks later, all seventy soldiers were tested for SARS-CoV-2 RNA (RT-qPCR) and antibodies (two immunoassays). Nine soldiers had at least one positive COVID-19 diagnostic test result. Five of them exhibited COVID-19 symptoms , while four were asymptomatic. In four soldiers, SARS-CoV-2 viral load was detected and the genomes were sequenced. Conventional and genomic epidemiological data suggest that these genomes have an African most recent common ancestor and that the Belgian military service men were infected through contact with locals. The medical military command implemented testing of all Belgian soldiers for SARS-CoV-2 viral load and antibodies, two to three days before their departure on a mission abroad or on the high seas, and for specific missions immediately upon their return in Belgium. Some military operational settings  were also equipped with mobile infectious disease (COVID-19) testing capacity. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single stranded-stranded RNA beta-coronavirus with a 29,903 nucleotides-long genome . Ph-app/  . Th. Th10]. A military camp is a semi-enclosed environment with a high population density and contained spaces  in which many soldiers  and a number of civilians interact. Communal living and training environments are renowned for person-to-person transmission of disease agents, a frequent problem for militaries worldwide . During https://www.ecdc.europa.eu/en/geographical-distribution-2019-ncov-cases).Other non-Nigerien contacts occurred at least two weeks before the date on which the first COVID-19 cases were presumed to have appeared in Maradi (2 April) and more than six weeks before the onset of COVID-19-like symptoms in the Belgian military service men (30 April–16 May) . ConsideOverall, the data produced by this combined conventional  and genomic  epidemiological analysis, suggest that the Belgian soldiers were infected via direct contacts with locals and subsequently among themselves.The SARS-CoV-2 outbreak in a Belgian military education and training center in Maradi, Niger, was characterized by mild symptoms in five soldiers and asymptomatic infection in two soldiers (one trainer), both having a viral load, as diagnosed upon their timely return to Belgium. The symptoms most strongly associated with COVID-19 infection were anosmia and ageusia. Trainers were disproportionately infected. Conventional and genomic epidemiological data suggest that the Belgian military servicemen were infected by a locally transmitted virus with a recent African common ancestor.Based, among others, on this epidemiological study, the medical military command decided to continue testing Belgian soldiers for SARS-CoV-2 viral load and antibodies, and this two to three days before a mission abroad or on the high seas, and for specific missions immediately upon their return. Soldiers in whom SARS-CoV-2 viral load is detected are isolated. Some military operational settings  were also equipped with mobile infectious disease (COVID-19) testing capacity. More specifically, the training center in Maradi is now using a GeneXpert machine for the rapid detection of SARS-CoV-2 viral load in nasopharyngeal swabs.


The early onset and rapid progression of cutaneous squamous cell carcinoma (cSCC) leads to high mortality rates in individuals with recessive dystrophic epidermolysis bullosa (RDEB). Currently, the molecular mechanisms underlying cSCC development in RDEB are not well understood and there are limited therapeutic options. RDEB-cSCC arises through the accumulation of genetic mutations; however, previous work analyzing gene expression profiles have not been able to explain its aggressive nature. Therefore, we generated a model to study RDEB-cSCC development using cellular reprograming and re-differentiation technology. We compared RDEB-cSCC to cSCC that were first reprogrammed into induced pluripotent stem cells (RDEB-cSCC-iPSC) and then differentiated back to keratinocytes (RDEB-cSCC-iKC). The RDEB-cSCC-iKC cell population had reduced proliferative capacities in vitro and in vivo, suggesting that reprogramming and re-differentiation leads to functional changes. Finally, we performed RNA-seq analysis for RDEB-cSCC, RDEB-cSCC-iPSC, and RDEB-cSCC-iKC and identified different gene expression signatures between these cell populations. Taken together, this cell culture model offers a valuable tool to study cSCC and provides a novel way to identify potential therapeutic targets for RDEB-cSCC. COL7A1 gene, which impair the function of type VII collagen (C7), resulting in blister formation, altered wound-healing processes, and the development of highly aggressive cutaneous squamous cell carcinoma (cSCC) that may lead to metastasis. cSCC, a malignant growth of the cutaneous epithelium, is the second most common skin cancer. In particular, patients with severe recessive DEB (RDEB) have a high risk of early death due to metastasis from their cSCC. This risk reaches up to 87% by the age of 45 years  and an electron coupling reagent . MTS is bioreduced by cells into a formazan product that is soluble in tissue culture medium. The absorbance of the formazan at 490 nm can be measured directly from 96-well assay plates without additional processing. The conversion of MTS into aqueous, soluble formazan is accomplished by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture.The RDEB-cSCC and RDEB-cSCC-iKC were seeded in triplicates and the absorbance was read at 490 nm using the CellTiter 963D human skin equivalents (HSEs) were generated as previously described, using primary fibroblasts and iKCs . After mAll experimental animal protocols were approved by the Institutional Animal Care and Use Committee at Columbia University, and the grafting was performed as previously reported . BrieflyThe In Vivo Imaging System (IVIS) Optical Imaging system was used to detect the bioluminescence signal in the skin grafts made from RDEB-cSCC expressing the luciferase gene. Mice were anesthetized with isoflurane before imaging. Luciferin was administered to the mouse to induce bioluminescence. Once the imaging was complete, mice were kept homeothermic via a warming pad and monitored for recuperation from the isoflurane anesthesia until fully awake and oriented . FollowiFor immunostaining analyses, cells were fixed in chamber slides with 4% paraformaldehyde. Samples were incubated with primary antibodies overnight at 4 °C. The following primary antibodies were used for IF experiments: SOX2 , NANOG , TRA-1-60 , TRA-1-81 . After washing with phosphate-buffered saline, samples were incubated with fluorophore-conjugated secondary antibodies  for 2 h at room temperature. Slides were covered with coverslips using VECTASHIELD mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI)  and examined using a Zeiss LSM 5 Exciter confocal laser scanning microscope.p-value of < 0.05 was used to indicate statistical significance.The total RNA was isolated from RDEB-cSCC, RDEB-cSCC-iPSCs and RDEB-cSCC-iKCs according to the manufacturer’s protocol  and RNA-seq was performed at Genewiz, NJ, USA. RNA-Sequencing libraries were prepared by TruSeq Stranded total RNA Library Prep  according to the manufacturer’s standard protocol. Libraries were sequenced on the HiSeq instrument  following the manufacturer’s protocol. FASTQ-formatted sequences were analyzed for quality control by the FASTQC open-source software. Trimmomatic tool was used to trim raw sequences. Sequences with quality score below 24 were excluded. Then HISAT2 software was used to align reads to the reference genome sequence (hg38). Transcript expression was comprehensively quantified with Cufflinks software, which performed: transcript assembly, quantification of expression as FPKM (fragments per kilobase of transcript per million mapped reads) values, and normalization. Normalized FPKM values were used for downstream analysis steps. Exploratory data analysis was used to cluster the gene expression data. The rank candidate genes were typed from each cluster based on functional similarity to training gene list using machine learning algorithm (ToppGene tool). This list was obtained from publication Cho et al. 2018 (ToppGene tool) [p values < 0.05 with a fold change greater than 2. Data were compared between groups using two-tailed unpaired t tests. Statistical significance was set at * p < 0.05.Differential expression analyses were carried out using the non-parametric Wilcoxon rank-sum test comparing the samples and normal human control cells. Significant genes were detected based on the stringent criteria of


The specificity of antibodies for antigens overexpressed or uniquely expressed in tumor cells makes them ideal candidates in the development of bioconjugates for tumor imaging. Molecular imaging can aid clinicians in the diagnosis of gastric tumors and in selecting patients for therapies targeting receptors with a heterogeneous intratumoral or intertumoral expression. Antibodies labeled with an imaging radiometal can be used to detect primary tumors and metastases using whole-body positron emission tomography (PET) or single photon emission computed tomography (SPECT), both during diagnosis and monitoring disease response. Conjugated with fluorescent dyes, antibodies can image tumors by targeted optical imaging. This review provides an overview of the most recent advances in the use of antibodies labeled with radiometals or conjugated with fluorescent dyes for gastric cancer imaging. Gastric (stomach) cancer is a global health concern: in 2018, more than 1,000,000 new cases were diagnosed and 783,000 deaths were registered worldwide  (PD-1 is a T-cell co-receptor (with two ligands: PD-L1 and PD-L2) used in cancer immunotherapy . PD-1 sise model . JS001,  9mTcO4] . While t111In-labeled D2101 in tumors starting 24 h after the radioconjugate was administered and low uptake in surrounding organs. Images with high tumor uptake and low background level were obtained at 96 h after antibody administration. In ELISA assays, D2101 showed high specificity to CDH17-expressing gastric cancer cells. D2101 shown binding to human CDH17 but not murine CDH17, as observed by surface plasmon resonance analysis. Biodistribution and SPECT/CT images demonstrated that in addition to accumulating in the tumor, the radiolabeled D2101 binds other CDH17-positive organs such as the intestine, which may hinder its ability to detect metastases in lymph nodes around the stomach.CDH17 is a transmembrane protein that functions as a peptide transporter and mediates cell-to-cell adhesion . This prHER2 is a transmembrane tyrosine kinase receptor that is part of the epidermal growth factor receptor (EGFR) family. The EGFR family consists of EGFR, HER2, HER3, and HER4 . High le89Zr-labeled trastuzumab has shown success in delineating HER2-positive gastric cancer [89Zr-labeled trastuzumab as an imaging agent in patients with HER2-expressing GC was reported in 2018 by O’Donoghue et al. [89Zr-trastuzumab biodistribution, pharmacokinetics, and dosimetry were evaluated in 10 patients, confirming HER2 status through immunohistochemistry or FISH [89Zr-trastuzumab PET to detect HER2 status in GC is safe and effective. Other preclinical studies demonstrated that 89Zr-trastuzumab is more specific than 18F-FDG in imaging HER2-expressing gastric tumors [HER2-directed PET imaging using radiolabeled trastuzumab has great potential to non-invasively visualize HER2-expressing cancer cells and monitor therapeutic target engagement in patients over a prolonged period ,6,22. 89c cancer ,6 and moe et al. . 89Zr-tr or FISH . PET ima89Zr-labeled anti-HER2 antibodies trastuzumab [Another important biological characteristic of the HER2 antigen relates to its internalization from the cell membrane to the intracellular compartment through an endocytic process. HER2 internalization reduces HER2 availability at the cell membrane and, consequently, decreases anti-HER2 antibody-tumor binding. Pereira et al. used cholesterol-depleting drugs in an attempt to temporarily stabilize HER2 at the cell membrane and enhance the ability of the stuzumab  and pertstuzumab  to bind 64Cu-NOTA-trastuzumab demonstrated higher tumor uptake than the negative control 64Cu-NOTA-hIgG1. Additionally, radiolabeled trastuzumab accumulation was higher in HER2-expressing PDXs than in tumors containing low levels of the protein. Clinical studies further validated the ability of 64Cu-NOTA-trastuzumab to image GC [In addition to zirconium-89, trastuzumab was radiolabeled with the shorter-lived radioisotope copper-64 [111In-labeled 61 IgG showed the highest tumor uptake in microSPECT imaging and biodistribution studies. Additionally, the radiolabeled 61 IgG demonstrated a high internalization rate when compared with H32 IgG, 75 IgG, or trastuzumab. Although the 61 IgG antibody showed positive results, there has been no follow-up preclinical research using the radioconjugate.Others have used molecular imaging during development of new anti-HER2 antibodies . A study89Zr-trastuzumab to image HER2-positive GC before therapy with afatinib, i.e., an inhibitor of EGFR [89Zr-trastuzumab tumor uptake, which is a direct result of the decrease in HER2 expression in the tumors  to image HER3 expression. In vitro studies showed that HER3 was internalized due to lapatinib, which also caused internalization of 89Zr-labeled mAb3481. While there was an observable increase in HER3 expression in vitro, lapatinib did not change HER3 expression in tumor xenografts. As a result, ex vivo results showed no change in HER3 expression with lapatinib treatment and, therefore, 89Zr-labeled mAb348 tumor uptake was similar in control versus lapatinib-treated tumors.Similar to afatinib, lapatinib is an irreversible tyrosine kinase inhibitor ,33. LapaHGF is a heterodimer protein that is present in healthy mesenchymal cells . HGF is 89Zr-DFO-AMG102 can noninvasively visualize HGF overexpression in murine xenografts [89Zr-DFO-AMG102 demonstrated high accumulation in HGF-expressing gastric xenografts and PDXs when compared with low-expressing GC.HGF is inhibited with the HGF monoclonal antibody rilotumumab (AMG102) . Preclinnografts . 89Zr-DF76Br-labeled antibody, 89Zr-labeled onartuzumab demonstrated high tumor accumulation and tumor-to-muscle ratios [Another study demonstrated that onartuzumab, an anti-MET humanized 1-armed monoclonal antibody from the family of IgG1 monoclonal antibodies , radiolae ratios .Endoscopic procedures are used to screen high risk symptomatic patients by detecting morphological changes in gastrointestinal mucosa. Even as a gold standard technique, endoscopic procedures do not allow effective differentiation of neoplastic gastric lesions from healthy or stressed tissue due to their indiscernible microstructural differences . Nam et Optical imaging technologies such as bioluminescence and fluorescence have been increasingly explored over the last decade as an alternative, able to overcome the visual limitations of conventional white-light endoscopy. These cost-effective and minimally invasive optical imaging modalities rely on the light-emitting properties of individual dyes. Antibodies conjugated with such dyes have proven to be excellent tools for targeted optical imaging. Additionally, compounds classified as photosensitizers, upon light activation, serve both diagnosis and therapeutic purposes in photodynamic therapy ,48,49. TFluorescent Endoscopy.While fluorescence imaging has shown positive results, the diagnostic accuracy of this technique is limited by the reduced selectivity to tumor cells of its fluorescent dyes—fluorescein, methylene blue, and indocyanine green (ICG). These are clinically available vascular contrast agents that allow detection of superficial alterations in microvasculature and structural morphology  and require additional confirmation through histopathological analysis of a biopsy sample . However“Red-flagging” tumor-associated antigens with fluorescently labeled antibodies may provide improved specificity and sensitivity compared to currently available diagnostic techniques. In this context, confocal laser endomicroscopy (CLE) uses in vivo microscopy during an ongoing endoscopy to magnify the microstructural aberrations for enhanced visualization . The appEpidermal growth factor receptor (EGFR) is overexpressed in GC; high EGFR protein levels are associated with advanced GC and poor prognosis . For assAs described in the previous section, MG7 is highly expressed in poorly differentiated gastric carcinoma . Li et aThe VEGF signaling pathway plays a pivotal role in promoting angiogenesis in endothelial cells . High VETreatments for all cancers are specific to the primary tumor lesion as well as the specific antigens that may be present in the unique tumor. Most common cancer therapies are in the form of chemotherapy, radiation therapy, or surgery. GC therapies include surgery, chemotherapy, targeted therapies, immunotherapy, or radiation therapy. The type of treatment depends on the stage of the disease and the biomolecular features of the tumors. The results obtained from IHC and ISH can be misleading when selecting patients for targeted therapies. In addition to the infeasibility of collecting all the neoplastic lesions that might be present in a patient at the time of diagnosis, information obtained from biopsies is not an accurate representation of the entire tumor. Gastric cancer is a heterogeneous disease—not only when comparing a primary lesion with metastases, but also intratumorally.Antibody-based imaging strategies specific for certain antigen-overexpressing GC allow the visualization of primary tumors and metastases with high contrast. In this context, antibody-PET and antibody-SPECT have the potential advantage of noninvasively determining changes in antigen  expression and target engagement in both the primary tumor and metastases. A novel integration of fluorescence-tagged antibodies and confocal laser endoscopy for prompt visualization of the dynamic molecular footprint also represents a promising pathway towards individualized therapy.


Passive immunotherapy applies agents that enhance existing anti-tumour responses, such as antibodies against immune checkpoints. Active immunotherapy uses agents that direct the immune system to attack tumour cells by targeting tumour antigens. Active cellular-based therapies are on the rise, most notably chimeric antigen receptor T cell therapy, which redirects patient-derived T cells against tumour antigens. Approved treatments are available for a variety of solid malignancies including melanoma, lung cancer and haematologic diseases. These novel immune-related therapeutic approaches can be accompanied by new patterns of response and progression and immune-related side-effects that challenge established imaging-based response assessment criteria, such as Response Evaluation Criteria in Solid tumours (RECIST) 1.1. Hence, new criteria have been developed. Beyond morphological information of computed tomography (CT) and magnetic resonance imaging, positron emission tomography (PET) emerges as a comprehensive imaging modality by assessing (patho-)physiological processes such as glucose metabolism, which enables more comprehensive response assessment in oncological patients. We review the current concepts of response assessment to immunotherapy with particular emphasis on hybrid imaging with  Novel response criteria are incorporating positron emission tomography (PET) imaging to assess immunotherapy efficacy.PET-based response criteria refine the assessment of response to immunotherapy.PET can assist in detecting immune-related side effects.Novel PET-ligands targeting molecules in immune-related pathways are under development.e.g., vaccines such as Bacillus Calmette–Guérin in bladder cancer). Active cellular-based therapies are on the rise, most notably chimeric antigen receptor (CAR) T cell therapy, which redirects patient-derived T cells against tumour antigens [Recent immunotherapeutic approaches have emerged as powerful treatment options with high anti-tumour responses. These effects can be achieved by redirecting, stimulating, or genetically reprogramming the patient’s own immune system to target cancer cells. Passive immunotherapy is the most frequent form of immunotherapy, involving agents that enhance existing anti-tumour responses, such as antibodies against immune checkpoints such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 PD-1), and programmed death-ligand 1 (PD-L1). Active immunotherapy uses agents that direct the immune system to attack tumour cells by targeting tumour antigens  protein, its corresponding programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) represent immune checkpoints that are targeted in clinical practice. PD-L1 is often overexpressed in tumour cells and interacts with the membrane bound PD-1 on T cells, thus inhibiting T cell responses . SeveralAntibody therapies have advanced over time and recently the concept of bispecific antibodies or “bispecific T cell engagers” came into the spotlight, as the first bispecific T cell engager, Blinatumomab, was approved by the EMA in 2015 for use in relapsed or refractory B-precursor acute lymphoblastic leukaemia. This antibody is composed of two single-chain variable fragments targeting CD19 or CD3, respectively . The bisYescarta (axicabtagene ciloleucel) and Kymriah (tisagenlecleucel) receiving approval by the EMA in August 2018, shortly after approval in the USA. The treatment is based on genetic modification of patient-derived T cells, obtained by leukapheresis, followed by their reinfusion into the patient. The cells are equipped with artificial CARs, which are composed of an antibody-derived single-chain variable fragments, a transmembrane and a signalling domain [CAR-T cells constitute the latest breakthrough in clinical immuno-oncology, with the two CAR-T cell products g domain . The sinThe consequences of CAR-T cell approval for clinical reality are remarkable, as the overall response rates for Kymriah during pivotal studies in patients suffering from relapsed or refractory diffuse large B cell lymphoma reached 52%, with 40% of the patients experiencing a complete response (CR), of whom 79% would remain relapse-free after 12 months of follow-up . Yescartpseudoprogression  may lead to incorrect determination of the response status using RECIST [Standardised assessment of change in tumour burden is essential in the evaluation of therapies in cancer patients. Most clinical trials use tumour shrinkage (objective response) or development of progressive disease (PD) as endpoints and continuation or modification of therapy regimens depend on it. The Response Evaluation Criteria in Solid tumours (RECIST) guidelines were introduced by an international working group in 2000  and revig RECIST .Table 1immune unconfirmed progression disease that requires to be confirmed by an additional, early follow-up scan within six to eight weeks. Immune unconfirmed progression disease should be considered carefully, as an increase in tumour size is still more likely to be true progression rather than pseudoprogression. The frequency of pseudoprogression varies between different tumour entities and is most frequently observed in melanoma patients (up to 13%) [Since tumour growth or newly detectable tumour lesions are generally classified as PD based on RECIST, pseudoprogression is not diagnosed correctly and may result in an erroneous discontinuation of treatment or an unjustified exclusion of patients from clinical trials. To address this, the RECIST working group developed a modified guideline for response assessment to immunotherapy in 2017, called Immune RECIST (iRECIST) . It is b to 13%) , 27. NotAnother atypical response pattern related to immunotherapy is hyperprogression, a term with various definitions, meaning a pronounced acceleration of tumour growth –30. If hpeak is assessed within a spherical volume of interest in the site of the most metabolically active tumour manifestation.In 1999, the European Organization for Research and Treatment of Cancer’ (EORTC) first introduced PET-based, metabolic information in specified criteria for the response assessment of oncological diseases in general . Of notePET/CT Criteria for Early Prediction of Response to Immune Checkpoint Inhibitor Therapy (PECRIT) [PET Response Evaluation Criteria for Immunotherapy (PERCIMT), which moreover take into account the clinical relevance of the absolute amount of new lesions during immunotherapy [In 2017, Cho et al.  prospect(PECRIT) . Additiootherapy , 36. For18F-FDG-avid lymphomas [First International Workshop on PET in Lymphoma in Deauville, France, a newly established 5-point scale  relative to blood-pool and liver activity  was introduced when an increase of tumour burden, new lesions, or an increase of 18F-FDG-avidity is observed, leading to a consequent follow-up imaging study within twelve weeks in order to rule out or confirm PD or pseudo-progression [In the light of immunotherapy, modified Lugano criteria (lymphoma response to immunomodulatory therapy criteria (LYRIC)) were proposed in 2016 to account for features specific for immunotherapy . Here, tgression .Response Evaluation Criteria in Lymphoma (RECIL) were established by an international working group [18F-FDG PET was reduced in favour of a more pronounced impact of CT-based changes, considering the potential alteration of glucose metabolism by immunomodulatory drugs that may obscure the tumour 18F-FDG-avidity [Most recently, in 2017, ng group . RECIL a-avidity . In Tablper se leading to potential misclassifications. Residual metabolic activity on 18F-FDG PET (similarly to the Deauville score assessment) in melanoma patients treated with anti-PD-1 agents was also associated with residual vital tumour masses. Vice versa, a loss of 18F-FDG-avidity despite remaining morphological masses was associated with improved outcome; however, remaining residual 18F-FDG-avidity despite clinical response was also observed, possibly due to immune infiltrates [PET imaging was initially used for immunotherapy monitoring in patients with solid tumours. Given the early and successful implementation of immunotherapy within the clinical workup of melanoma patients, the first PET-based response assessment using EORTC criteria was applied in melanoma patients . Alreadyiltrates .In a cohort of melanoma patients undergoing immunotherapy, several morphological and functional response assessment criteria were applied, but only a limited agreement among the applied criteria in terms of outcome prediction was observed. Hence, the combination of parameters best suitable for prediction was established (PECRIT criteria) . When dei.e., CR, PR, or SD) and those without . Again, agreement of PERCIMT and EORTC was limited; although PERCIMT showed a significantly higher sensitivity for the prediction of clinical benefit than EORTC, both criteria were equally able to predict the absence of clinical benefit [As a consequence, PERCIMT criteria were also used for interim evaluation in melanoma patients undergoing immunotherapy and compared to EORTC criteria by stratifying patients with metabolic benefit  patients undergoing immunotherapy. Here, 18F-FDG changes in terms of PERCIST criteria (compared to RECIST 1.1) were highly predictive for treatment efficacy in NSCLC patients undergoing nivolumab therapy even at an early stage of 1 month after treatment initiation and was shown to be an independent prognostic factor at multivariate analysis [18F-FDG PET (using EORTC criteria) in NSCLC patients undergoing atezolizumab therapy 6 weeks after initiation were predictive for the further morphological disease course on CT. Moreover, even patients with pseudoprogression could be identified by using 18F-FDG PET [18F-FDG PET imaging in patients classified as PD on PERCIST criteria was able to identify patients with pseudoprogression and immune dissociated-response in more than half of patients previously classified as PD. Importantly, improved clinical outcome was observed in these patients [Beyond melanoma, a few studies have addressed the value of analysis . Also, r-FDG PET . In addipatients .When dealing with haematologic malignancies, first reports of anti-PD1-therapy in Hodgkin lymphoma (HL) were published already in 2014 , where a18F-FDG-avidity in tumour and spleen as well as the general 18F-FDG-avid tumour burden 3 months after initiation of anti-PD-1-treatment were associated with improved clinical outcome [Initially, the metabolic changes over time in patients with relapsed or refractory HL undergoing anti-PD-1-treatment were described by Dercle et al. . Subsequ outcome . Consequ outcome . In the  outcome , a resul outcome , 56, 59.18F-FDG PET 4 weeks after CAR-T cell therapy showed a long-term remission over 2 years and patients with remaining activity had an early relapse. Secondly, Wang et al. [18F-FDG-avid tumour burden prior to therapy was associated with more severe CAR-T cell therapy-related side effects. Interestingly, this study also demonstrated that the phenomenon of pseudoprogression and local immune activation can also occur in patients undergoing CAR-T cell therapy .With regard to PET/CT imaging in CAR-T cell therapy, only limited data is available. Firstly, Shah et al.  demonstrg et al.  showed tSo far, there is only limited literature dealing with the very exact clinical value of PET/CT imaging for the identification of pseudo-progression in patients undergoing immunotherapy. One study in melanoma patients suggests that the new appearance of ≥ 4 metabolically active lesions with a functional diameter < 1.0 cm or ≥ 3 lesions > 1.0 cm is associated with real progression rather than the occurrence of pseudoprogression . AnotherDuring the application of immunotherapeutic agents, there is a reactivation of the immune system that not only has anti-tumour effects, but also might affect healthy tissue leading to new toxicity profiles that require a different management than the toxicity of chemotherapies , 65. The18F-FDG-avidity [18F-FDG-avidity can be observed over time [18F-FDG-avidity of irAEs also enables an exact localisation and identification [The occurrence of irAEs can also affect response assessment with PET, as inflammatory reactions accompany these irAEs consequently leading to an elevated -avidity , which mver time , 71. Vicfication , which gfication , 73.sarcoid-like reactions consisting of lymphadenopathy and pulmonary granulomatosis with elevated glucose consumption have to be kept in mind [Recently, the report from the European Association of Nuclear Medicine symposium on immunotherapy stated that incidental findings related to irAEs should be reported. Although irAEs might not necessarily be associated with clinical symptoms, clinicians should be aware of their presence and ensure clinical monitoring, which, however, might lead to a clinical intervention. First signs of elevated immune activity can be seen as spleen enlargement and/or elevated uptake leading to an inversion of the liver-to-spleen uptake ratio. Also, reactive lymph nodes might be observed in the direct drainage of the tumour. However, these findings have to be compared to the respective baseline scan to assess their pathophysiological relevance, but also to safely relate these findings to the immunotherapy . Moreove in mind . This is18F-FDG-PET [In CAR-T cell therapy, there is, however, another set of rather immediate adverse effects, such as cytokine release syndrome, CAR-T cell-related neurologic toxicities, and B cell aplasia, which are not directly detectable using -FDG-PET , 76, 77.-FDG-PET , 79. HenDespite growing success, immunotherapy, adoptive cell therapy in particular, still face many challenges. As the scientific community remains in search for answers as to why significant fractions of patients remain nonresponsive to immunotherapy, new targets for cellular treatments are validated in a fast-growing number of clinical trials.In the context of haematologic malignancies, these new approaches include CAR-T cells targeted against B cell maturation antigen in relapsed or refractory multiple myeloma , 81 and Another emerging concept in cellular immunotherapy is universal or adapter CAR-T cells. The single-chain variable fragments of these CARs are designed to recognize antigens which are physiologically not present on the surface of tumour or healthy cells . Applicaversus-host-disease due to their lack of endogenous T cell receptors, allowing human leukocyte antigen mismatched transfusions [Taking the idea of universal immune cells further, recent reports demonstrate the potential of CAR-transduced natural killer cells to combat lymphoma in a combined phase I and II trial . Interessfusions . These e89Zr or 64Cu and are suitable for in vivo imaging PD-1-expressing tumour-infiltrating lymphocytes [Beyond morphological and glucose-based imaging, new molecular radiotracers arise that directly target the key molecules of immune checkpoint pathways and immune responses , 89. Antphocytes .89Zr-nivolumab in patients with advanced NSCLC and showed a significant 89Zr-nivolumab tumour uptake, that was higher in patients with immunohistochemically proven PD-1 positive tumour-infiltrating immune cells as compared with PD-1 negative tumours. Interestingly, PD-(L)1 PET-CT demonstrated highly heterogeneous tumour uptake inter-individually, but also intra-individually with divergent uptake between different tumour lesions [89Zr-atezolizumab (an antibody to PD-L1) PET showed stronger correlation with the clinical outcome than immunohistochemistry- or ribonucleic acid-sequencing-based biomarkers in patients subsequently undergoing PD-L1-targeted therapies.First studies were already performed in humans. Niemeijer et al.  publishe lesions , 92. Mor89Zr-avelumab PET in NSCLC  or 89Zr-durvalumab in head-and-neck squamous cell cancer . Also, dual imaging approaches with 18F-FDG PET are on the way, for example combining 18F-FDG PET and 18F-PD-L1 PET in oral cavity squamous cell cancer Several trials in humans aimed at establishing novel immuno-PET ligands in a broad range of cancer entities such as 89Zr-anti-IFN-γ) that allows imaging of activated lymphocytes inside tumour lesions [68Ga-NOTA-GZP allowed prediction of response to immunotherapy with high accuracy in preclinical models [99mTc-labeled interleukin-2 (99mTc-HYNIC-IL2), which demonstrated feasibility for visualisation and quantification of tumour infiltrating lymphocytes in a small set of melanoma patients undergoing immunotherapy, so providing a potential non-invasive tool for the differentiation between progression and pseudoprogression [Beyond imaging PD-L1 using PET, several interesting biomarkers were introduced to molecular imaging in preclinical settings such as interferon-γ immuno-PET ( lesions . Anotherl models . Beyond gression .integrated diagnostics’, i.e., combining multiparametric diagnostic data from imaging, pathology, molecular genetics, and liquid biopsies, with final aim of therapy guidance.These promising efforts in both preclinical and clinical setting underline the further investigation of immuno-PET and the comprehensive translation into clinical imaging to further improve pretreatment patient selection, response assessment and clinical management. Moreover, artificial intelligence algorithms are increasingly used to evaluate treatment response by evaluating image-derived biomarkers , 97, whi


Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of mammalian germline cells. A large proportion of ERVs lose their open reading frames (ORFs), while others retain them and become exapted by the host species. However, it remains unclear what proportion of ERVs possess ORFs (ERV-ORFs), become transcribed, and serve as candidates for co-opted genes.gag, pro, pol, and env) in 19 mammalian genomes. The fractions of ERVs possessing ORFs were overall small (~ 0.15%) although they varied depending on domain types as well as species. The observed divergence of ERV-ORF from their consensus sequences showed bimodal distributions, suggesting that a large proportion of ERV-ORFs either recently, or anciently, inserted themselves into mammalian genomes. Alternatively, very few ERVs lacking ORFs were found to exhibit similar divergence patterns. To identify candidates for ERV-derived genes, we estimated the ratio of non-synonymous to synonymous substitution rates (dN/dS) for ERV-ORFs in human and non-human mammalian pairs, and found that approximately 42% of the ERV-ORFs showed dN/dS < 1. Further, using functional genomics data including transcriptome sequencing, we determined that approximately 9.7% of these selected ERV-ORFs exhibited transcriptional potential.We investigated characteristics of 176,401 ERV-ORFs containing retroviral-like protein domains (These results suggest that purifying selection operates on a certain portion of ERV-ORFs, some of which may correspond to uncharacterized functional genes hidden within mammalian genomes. Together, our analyses suggest that more ERV-ORFs may be co-opted in a host-species specific manner than we currently know, which are likely to have contributed to mammalian evolution and diversification. ERVW-1) gene, which is derived from an envelope (env) gene in human endogenous retrovirus (HERV) type W (HERV-W), functions in cell fusion during placental development , all of which are commonly found in viral amino acid sequences. In this study, we performed genome-wide comparisons on the number, divergence, genomic distribution, and transcriptional potential for each protein domain in the ERV-ORFs of mammals. Our findings are expected to improve the understanding of the evolution and potential roles of unannotated ERV-derived genes.To understand the characteristics of possible protein-coding ERVs that possess ORFs for retroviral-like protein domains (ERV-ORFs), including their transcriptional potential in mammalian genomes, we comprehensively examined ERV-ORFs in 19 mammalian species. We previously developed a database called gEVE , which means that the amount of ERV-ORFs in each genome reflected that of the total ERVs in the genome.To characterize ERVs that are expressed as proteins in mammalian species, we first compared 176,401 possible protein-coding ERV-ORFs from 19 mammalian species genomes obtained from the gEVE database . ERV-ORF∆F) from the mammalian average for each domain fraction . By contrast, the ∆F values for pro and pol were similar among mammalian species, many of which had ∆F within ±0.25. Exceptions were found in mice in which the pro gene showed a dramatic increase (∆F = 1.8). Platypuses showed quite unique compositions in their gag and pro genes with very small ∆F values .We further compared mammalian ERV-ORFs at the level of gene domains. To reduce the influence of genome qualities on the number of domains detected, we calculated the relative fraction change  corrected, see Methods) in many mammalian species save for dogs, sheep, goats, and opossum, with two modes of approximately 2–7% (younger) and 39–45% (older). It should be noted that in specific species the distribution patterns revealed that bimodality was not statistically supported in dogs, sheep, goats and opossum . We hypothesized that newer elements would be closer to the consensus, and older ones would be further. Thus, we were able to estimate the age of ERV-ORFs by comparing the divergence spectra to their consensus sequences. We first determined the medians of Kimura 2-parameter divergence values  between We next determined how many ERV-ORFs serve as raw materials for the assembly of multi-exon genes. Comparison of our observed ERV-ORFs with those in Ensembl gene and protein annotations revealed that 15 and 22 ERV-ORFs were constituents of ordinary multi-exon genes in humans and mice, respectively Table S. The numenv genes, many do not [F in gag and pro, and lower ∆F in env. These results indicate that many ERV-ORFs in mice may be derived from the young ERVs that possess ORFs by chance. Similar phenomena may have occurred for other species, such as primates, as the frequencies of the divergence with modes of approximately 2–7% were larger than those with 39–45%, which is likely to be boosted by the younger ERVs, such as ERVK  of ERV-ORFs using the CAGE data in the FANTOM5 database . CAGE dadN/dS) of ERV-ORFs. To this end, we first identified syntenic sequences to human ERV-ORFs in non-human mammalian genomes, and extracted the sequences when the length of non-human ERV-ORFs was > 90% of that of humans  expressed in simians . To deteapproach . The paipol and env domains, were found 1821 bp to 7086 bp downstream of the TSS , and the second had no limitation for dN/dS ratio or distance to TSS applied . We extracted 705 ERV-ORFs with transcriptional potential from human-chimpanzee pairs and compared their divergence distributions with those of all ERV-ORFs , demonstrating that the divergence of ERV-ORFs with transcriptional potential was significantly higher than that of all ERV-ORFs. Furthermore, the statistical differences in divergence between ERV-ORFs with transcriptional potential and all ERV-ORFs were also observed between human-gorilla and human-orangutan pairs . This confirmed that ERV-ORFs with relatively small divergence (≤ 7%), which were likely recently inserted into the genomes, may include ERV-ORFs that remain by chance alone. Of note, ERV groups showing a significant decrease in the proportion compared to those expected from all ERV-ORFs in humans were HERV-H and HERV-K groups ; however, this result was not statistically significant .To predict which ERV-ORFs are expressed in a variety of tissues, we performed comparative analyses using a wide range of transcriptome and histone mark data generated by next-generation sequencing. We first compared observed ERV-ORFs from our study with quantified transcriptome data derived from 31 human tissues generated in the GTEx study  from theSince myoblast cells were reported to express syncytins during myogenesis , 45, we pro domain was found exclusively in liver cells. We also analyzed the relationship between TSSs and ERV-ORFs with transcriptional potential, which were detected by comparing RNA-Seq and histone mark data .One of the features associated with transcribed gene body is the chromatin mark of trimethylated histone H3 at lysine 36 (H3K36me3) , 47. We,p -value < 0.05, chi-squared test, FDR corrected). Some enriched ERV groups were more prominent in ERV-ORFs with transcriptional potential compared to all ERV-ORFs detected in humans. For example, when comparing top 10 enriched ERV groups, the proportion of the HERVK3 group significantly increased in all three functional datasets compared to that in all ERV-ORFs in humans , which accounts for nearly 60% of ERV groups in the ERV-ORFs from myoblast RNA-Seq. However, enrichment of the HERV-K group was observed only in ERV-ORFs from myoblast RNA-Seq. Hence, the differences in the ERV-ORF groups may result from differences in sample types used in each project. Indeed, the ERV-ORFs detected via three different functional datasets were quite different , and compared them with those of all ERV-ORFs detected in the human genome. The top 10 enriched ERV groups in ERV-ORFs with transcriptional potential are shown in Fig. ent Fig. e. AlthoudN/dS ratios between all ERV-ORFs and ERV-ORFs with transcriptional potential in primates, and found that the dN/dS ratios with transcriptional potential only for baboons, macaques and marmosets were significantly smaller than those of all ERV-ORFs . Moreover, the dN/dS ratios of ERV-ORFs between apes were significantly small when they were shared by at least four non-human primates . In the sets for chimpanzee, gorilla, and orangutan, approximately 81, 74, and 72% of ERV-ORFs were below dN/dS 1, respectively. This showed that ERV-ORFs detected by functional data still contain sequences that are not under purifying selection. Out of 408 ERV-ORFs, which were detected using three different functional datasets, 93 had dN/dS < 1, demonstrating that approximately 22.3% of ERV-ORFs considered to exhibit high transcriptional potential, were under purifying selection.We also compared pol genes . Although nine clusters contained known annotated genes or reported transcripts  of the ERV-ORFs showed dN/dS ratios < 1, which can be considered to be under purifying selection. Further, we confirmed that approximately 9.7%  of these ERV-ORFs with dN/dS < 1 exhibited transcriptional potential. Although the proportion of ERV-ORFs that function as potential candidates for ERV-derived genes was small, there may be unidentified protein-coding genes in these ERV-ORFs.We comprehensively identified ERV-ORFs in 19 mammalian species, with differences in their domain fractions noted between species  containing splice variants. This suggests that, as reported in a previous study [CNBP, GIN1, SUGP2, in chickens and CTSE in humans and mice), these ERV-ORFs may have been co-opted before the evolutionary divergence of birds and mammals.We also noted that entire ERV-ORFs were significantly depleted near the TSS in humans and mice. This may support the low transcriptional potential of ERV-ORFs as a whole. If specific epigenetic changes occur in close proximity to ERV-ORFs with low transcriptional potential, they subsequently begin to be expressed, which may lead to development of diseases as described in previous studies , 51. Thius study , ERV-ORFdN/dS ratios in each ERV-ORF loci varied. This showed that ERV-ORFs in the same ERV group experienced different levels of selection including neutral selection. Indeed, we detected 93 human ERV-ORFs showing dN/dS < 1 in the comparisons with functional data. We believe this level of detailed expression analysis will assist in revealing the function of ERV-ORFs by identifying individual ERV-ORF loci.The ERV-ORF expression results may also suggest that the promoter regions of ERVs were co-opted rather than the ORFs. Previous studies have reported that promoter regions, most notably for ERV LTRs, have been co-opted as tissue-specific, or alternative promoters in mammalian cells (well summarized in ). The LTERVW-1, are expressed in limited tissues [dN/dS < 1 were not significantly different from those of entire ERV-ORFs in humans. Moreover, we observed that the fractions of viral-like protein domains among mammalian linages were quite different, implying that the fractions of retroviral-like domains within ERV-derived genes also differ among mammalian lineages, and are differentially transcribed in different tissues, which may affect lineage-specific characteristics at varying levels.Combinatorial analysis of functional datasets and cluster analysis for ERV-ORFs in humans and mice revealed that many expressed ERV-ORFs were tissue- and lineage-specific. This is consistent with previous studies showing that specific ERV genes, such as  tissues , 54–56 a tissues . In addiTogether, our results suggest that a certain portion of mammalian ERV-ORFs may be co-opted in a lineage-specific manner, many of which were not described before. Such sequences are likely to have contributed to mammalian evolution and diversification. However, functional data is currently very limited. Thus, further ERV-ORF studies employing the functional data from multiple species may provide a deeper understanding of mammalian evolution.dN/dS < 1, which exhibit high transcriptional potential in humans. We also showed that fractions of each protein domain in the ERVs varied among mammalian species, indicating that the function of ERV-derived genes, which have not been identified yet, may be different among mammals. Taken together, our study suggests that more ERVs may have been co-opted as genes in a lineage-specific manner than we know, which are likely to have contributed to mammalian evolution and diversification.We investigated what proportion of ERVs possessing ORFs (ERV-ORFs) are under purifying selection, become transcribed, and serve as candidates for co-opted genes in mammalian genomes using various functional data. We found that approximately 9.7% of the ERV-ORFs showed http://geve.med.u-tokai.ac.jp). Species names and corresponding genome versions used in this study are summarized in Table Shmmer.org) with HMM profiles and/or RetroTector [pol and LINE-ORF2 domains as previously described [All sequences and annotations for potential protein-coding ERV-ORFs from 19 mammalian genomes were used in this study were obtained from the gEVE database  . Using adjusted standardized residuals obtained from the test, we further performed the residual analysis. The final p-values were calculated by pnorm function of R and adjusted by FDR. For Fig. ∆F), we first obtained the total number of each domain  and its fraction within a species. Using the fractions, we calculated mammalian average fractions for four domains. Then, each species ∆F was calculated by dividing the domain fraction of the species by the mammalian average.We first obtained all ERV sequences in the human and mouse genomes using RepeatMasker version 4.03 [p-value using the R package diptest [dN/dS < 1 using Wilcox.test function of the R package. The p-values were adjusted by FDR [We used a utility script “calcDivergenceFromAlign.pl” in RepeatMasker  to calcu diptest . Wilcoxod by FDR  using thwww.ncbi.nlm.nih.gov/sra). For humans, we used 12 RNA-Seq datasets from skeletal muscle cells under the SRA study number SRP033135 [RNA-Seq data for human and mouse myoblast differentiation were downloaded from the NCBI SRA; https:// RP033135 . We obtaRP033135  with theRP033135  with theRP033135  and DESeRP033135  were useRP033135 .To investigate whether ERV-ORFs were incorporated in known trassnscript in mammals, we obtained exon coordinates of protein-coding genes from Ensembl Genes 84 database in BioMart. We compared the coordinates of each exon with those of ERV-ORFs using the intersect function of BEDTools version 2.26.0 . We idenTo investigate the functionality of ERV-ORFs, we obtained RNA-Seq datasets from the GTEx project  in GFF fWe obtained histone mark data for H3K36me3 from the Peak Browser in ChIP-atlas  with thrTo predict which ERV-ORFs become transcribed, we analyzed CAGE data from FANTOM5 . Human adN/dS ratios with PAML version 4.9i  [dN/dS ratios between ERV-ORF groups. The p-values were corrected by FDR in the R package.To predict signals of selections on ERV-ORFs, we generated human-centered pairwise alignments by selecting only non-human sequences with length ≥ 90% of human ERV-ORFs. The nucleotide and amino acid alignments were aligned using MAFFT version 7.394 using the auto option . These ates = 0) . WilcoxoWe obtained ERV-ORF alignments and clustered each domain with the CD-HIT program  for 50–9For all analyses employed in this study, we used in-house programs developed with Perl, Python, AWK, and Shell script as well as R package dplyr  and rehaAdditional file 1.


On September 23, 2020, administrators from a Florida high school were notified of a confirmed COVID-19Among the 14 team members with COVID-19, seven were symptomaticFactors that likely contributed to team transmission included 1) infrequent mask use in the weight room or during practice; 2) inadequate physical distancing and air ventilation on buses transporting players (windows remained closed); 3) infrequent cleaning and disinfection of locker rooms, weight room equipment, and communal areas  before and after practices; and 4) insufficient sanitizing of shared hydration system drinking nozzles between uses.SARS-CoV-2 transmission among team members likely occurred during practice. Football and other contact sports involve frequent, direct contact, as well as physical exertion that can result in heavy respiration and higher rates of emission of virus particles (The findings in this report are subject to at least two limitations. First, testing was voluntary; not all players and classroom close contacts sought testing during their quarantine. Second, some asymptomatic persons with COVID-19 might not have been identified; therefore, the extent of SARS-CoV-2 transmission might have been underestimated.To prevent school transmission of SARS-CoV-2 and lost quarantine-related in-person school days, school sports teams should implement recommended CDC strategies to prevent spread of COVID-19, including maintaining a distance of ≥6 ft between persons, routine mask use during practice, and testing to identify asymptomatic infected players and staff members.


The most successful ACT, anti-CD19 chimeric antigen receptor T-cell therapy directed against B-cell lymphoma, has proved to be efficacious. However, current efforts to utilize this approach for solid tumors, like breast cancer, have shown only modest improvement. Nevertheless, the potential efficacy of CAR-T therapy is promising in an era of immunological advances. By appropriately manipulating CAR T-cells to combat the immunosuppressive forces of the tumor microenvironment, significant eradication of the solid tumor may occur. This review discusses CAR T-cell therapy and its specificity and safety in adoptive cell transfers in breast cancer. We will highlight novel discoveries in CAR T-cell immunotherapy and the formidable barriers including suppression of T-cell function and localization at tumor sites.Chimeric antigen receptor (CAR) T-cell therapy is an innovative form of immunotherapy wherein autologous T-cells are genetically modified to express chimeric receptors encoding an antigen-specific single-chain variable fragment and costimulatory molecules. Moreover, CAR T-cell therapy can only work successfully in patients who have an intact immune system. Therefore, patients receiving cytotoxic chemotherapy will be immunosuppressed making CAR-T therapy less effective. In adoptive CD8+ T-cell therapy (ACT), numerous tumor-specific, engineered T-cells are sourced from patients, expanded  Breast cancer has consistently been characterized as a leading cause of death in women. One-fourth of all women with cancer, or 1.5 million women, are diagnosed with breast cancer each year throughout the world ex vivoIn 2016, the American Joint Committee on Cancer updated breast cancer staging to include T, N, M, tumor grade, and expression of estrogen and progesterone receptors and HER2 to groups patients into risk categories that help define and guide treatment recommendation As described in Figure Current literature highlights CAR T-cell therapy as a highly effective treatment modality in hematological cancers, like Acute Lymphocytic Leukemia (ALL), exhibiting a 92% full recovery Clinical trials often focus on disseminated tumor cells that lay dormant for years but have metastatic potential in breast cancer patients. Many of these clinical trials involve the triple-negative breast cancer (TNBC) type, a very aggressive malignant type of tumor that is frequently resistant to standard breast cancer therapies due to the absence of estrogen receptors (ER), progesterone receptors, and epidermal growth factor receptors (EGFR), making novel therapeutic modalities such as CAR T-cells particularly attractive As mentioned earlier, modified CAR T-cells contain hybrid receptors that include both an extracellular and intracellular antigen recognizing domain with extensive costimulatory regions that together allow for enhanced antigen affinity The various types of antigens recognized by CAR T-cells can be extended to carbohydrates and glycolipids that are often modified in cancer cell Another tumor antigen known as integrin αvβ3 is expressed in several tumor entities including melanoma, glioblastoma, breast, pancreatic, and prostate cancer and promotes tumor cell survival and metastasis One study explored the cell-surface molecule c-Met, which is expressed in ~50% of breast tumors, prompting the construction of a CAR T-cell specific for c-Met in order to halt tumor growth in immune-incompetent mice with tumor xenografts Several CAR T-cell trials are currently enrolling, including trials targeting CEA, HER2, mesothelin, MUC1, NKG2D ligands, ROR1, CD70, and CD133Another potential target focuses on mesothelin, a cell-surface antigen overexpressed in 67% of TNBC samples in vitro and in vivo in a xenograft mouse model, with minimal off-tumor cytotoxicity. The activation of the interferon γ, granzyme-perforin-PARP and Fas-FADD-caspase signaling pathways in TNBC cells further confirm that CAR-T as an immunotherapy tool may be used to treat TNBC in the clinical setting One study reported on the evaluation of disialoganglioside GD2 expressed in 35.5% of metastatic TNBC as a breast cancer stem cell specific target antigen for immunotherapy TEM8, a marker overexpressed on the vasculature of some solid tumors, has been proposed as a target of controversy in vitro studies to determine the cytotoxicity of K-CAR toward MDA-MB-231, a triple-negative breast cancer cell line, and significantly greater lysis was demonstrated for this cell line using K-CAR from breast cancer patients. In vivo studies with immunodeficient mice revealed that tumor growth, size, and weights were significantly decreased in mice bearing MDA-MB-231 or MDA-MB-435.eB1 transfectant cancer cell lines, which express a 258-fold increase in the cell growth protein c-erbB-2, were treated with K-CAR T-cells compared to control T-cells or no treatment in vivoAn intriguing antigen that is overexpressed in most cancers including breast cancer is the human endogenous retrovirus family K (HERV-K) The future of antitumor therapeutics will most likely encompass cell-based therapy in addition to chemotherapy as it is unlikely that CAR T-cell treatment will successfully eradicate a tumor on its own Additionally, two recent reports have linked the gut microbiota composition to the promising clinical response of CAR T-cells in terms of efficacy and toxicity. Specifically, the immunostimulation by intestinal bacteria includes cross-reactions between microbial and tumor antigen stimulation of pattern-recognition receptors (PRRs) and the production of bacterial metabolites that might exert systemic modulatory effects In infant cases of ALL, CAR T-cells achieve up to 90% eradication of the tumor, while in solid tumors, the clinical efficacy is less rewarding due to toxic side effects and fewer clinical trial implementations 1. For instance, HER2 can undergo proteolysis to cleave the extracellular domain without compromising kinase activity. By using a dual-targeting CAR system, engineered T-cells coexpress two CARs that recognize two distinct antigens so that T-cells can be activated in the presence of either antigen to mitigate antigen-loss escape Another possible concern with cell-based therapies is immunoediting of tumor antigens. As tumor cells are destroyed by CAR T-cells, which are formed from memory cells, they are directed against the tumor antigen they were originally engineered to recognize. In this setting, immunomodulation may prove to be a problem as antigenic shift may cause tumor cells to evolve in a way to create new tumor antigens that may not be recognized by the original CAR T-cells. Thus, this notable antigenic shift, or molecular alteration of an antigen due to recombination, may inadvertently cause CAR T-cells to act against the tumor antigen they were intended to recognize Metastasis to the brain from breast cancer poses a significant clinical challenge that may be treated with CAR-based immunotherapy in situ and on to invasive levels. Further exploration as to the direct role of these cells will be imperative in diagnosing breast cancer in its early stages through the identification of discreet cell populations expressing markers commonly associated with a pro-tumor phenotype While the results of immunotherapy through CAR T-cells have been monumental in cancer therapy, further understanding of the inhibitory tumor microenvironment is needed. Combinatory use of multiple agents in a patient's treatment protocol is found to be advantageous in malignancies like breast cancer that express high immunogenicity and a heterogenous antigen profile. Collectively, studies comparing normal with benign breast tissue indicated early immune infiltration of B cells, T cells, and macrophages as the tumor progresses to ductal carcinoma The emergence of immuno-oncology has provided a unique opportunity for researchers and clinicians to concentrate on cancers like TNBC that lack targeted therapy universally and are recognized as the most complex and challenging breast cancer subtype to treat, with chemotherapy remaining the standard of care. Therefore, significant effort has been placed on the discovery of novel treatment strategies for subtype TNBC with the hope to broaden this therapeutic approach to other breast cancer subtypes in the future. One strategy in particular targets the use of immune checkpoints for immune escape, specifically the tumor's ability to suppress PD-L1 expression and decrease the activity of cytotoxic T-cells Healthy donor allogeneic CAR T-cells can be derived from an HLA-matched hematopoietic stem cell transplant donor. However, for the non-HLA-matched patient, current efforts to pursue gene-editing approaches using CRISPR/Cas9 have shown to be advantageous. Nevertheless, unwanted side effects of gene editing include off-target cleavage of genes and undesired translocations


Coronavirus 2 (CoV) Severe Acute Respiratory Syndrome (SARS-CoV2) is causing a highly infectious pandemic pneumonia. Coronaviruses are positive sense single-stranded RNA viruses that infect several animal species, causing symptoms that range from those similar to the common cold to severe respiratory syndrome. The Angiotensin Converting Enzyme 2 (ACE2) is the SARS-CoV2 functional receptor. Measures are currently undertaken worldwide to control the infection to avoid disruption of the social and economic equilibrium, especially in countries with poor healthcare resources. In a guarded optimistic view, we hope that the undertaken preventive and treatment measures will at least contribute to contain viral diffusion, attenuate activity, or even eliminate SARS-CoV2. In this review, we discuss emerging perspectives for prevention/treatment of COVID-19 infection. In addition to vaccines under development, passive immunization is an open opportunity since patients develop neutralizing antibodies. A full spectrum of potential drugs for COVID-19 infections could in turn affect virus binding or enzymatic activities involved in viral replication and transcription. Furthermore, clinical trials are currently evaluating the safety and efficacy of anti-inflammatory drugs, such as tocilizumab. Bioinformatics may allow characterization of specific CD8+ and CD4+ T cell responses; thus, CoV2 T cells’ frequency can be correlated with the disease severity and outcome. Combinatorial antibody phage display may be empowered to identify the immune repertoire of CoV2-specific neutralizing antibodies. The 2019 novel coronavirus disease (COVID-19) is a highly infectious pneumonia recently recognized as “pandemic” by the WHO [COVID-19 has been also named SARS (Severe Acute Respiratory Syndrome)-Coronavirus (CoV)2 by the Coronavirus Study Group (CSG) of the International Committee on Taxonomy of Viruses (ICTV). The novel virus is indeed part of the taxonomic Coronaviridae family (subfamily Coronavirinae) . CoronavFurthermore, the viral spike glycoproteins include a transmembrane anchor and a short intracellular tail at C-terminal . ACE2 isACE2 is widely expressed from fish to amphibians, birds, and reptiles to mammals; all these animal species can be natural hosts of SARS-CoV2. Since ACE2 is highly expressed in the lung, intestine, testis, and kidney, in addition to salivary droplets, other routes of transmission of the viral infection are possible . Indeed,Wang et al. (2020)  first deConsidering the impact of COVID-19 ongoing global public health emergency, measures to reduce person-to-person transmission of the virus, thus containing its spread, are currently undertaken all over the world ,17.At present, vaccines are under development, and there are no specific antiviral treatments to defeat COVID-19 infection in humans and other animal species ,19,20,21In this review, we discuss emerging perspectives for the appropriate preventive and treatment strategies to tackle COVID-19 infection. These include vaccine development based on definition of immunodominant viral epitopes, passive immunization, several anti-viral therapeutics, and anti-inflammatories. We also highlight tools that may elucidate mechanisms governing viral infection and clearance at the different stages of disease and advance effective immunotherapies based on monoclonal antibodies discovery.9 copies in less than an hour at 65 °C [In the past, MERS and SARS-CoV have caused large-scale epidemics; however, from recent epidemiological data, SARS-CoV2 presents a higher rate of transmission; on average, 2 or 3 people become infected from one already infected person . Moleculat 65 °C .Serological immunoassays are currently used to estimate seroconversion in seroprevalence surveys in people suspected of current or previous SARS CoV2 infection through anti-viral IgG and IgM immunoglobulins testing. Accuracy of antibodies detection is assessed by enzyme-linked immunosorbent, chemiluminescence, and flow laminar immunoassays. IgGs persist for the longest time and may reflect a longer-time immunity, although it is the last to rise after infection since positivity occurs between 13–21 days. Reported time to IgM positivity ranges from 5 to 10 days following disease onset but declines soon after an infection is cleared . CombiniOther abnormalities were detected in COVID-19 patients, such as lymphopenia, increased levels of C reactive protein (CRP), lactate dehydrogenase (LDH), erythrocyte sedimentation rate (ESR), and D-dimer . In addiIt is known that SARS patients develop strong neutralizing antibody responses against the virus . Indeed,A potential alternative challenge is to directly administrate neutralizing monoclonal antibodies (mAbs) to subjects encountering the infection. Generated mAbs can interrupt any stage of the viral lifecycle or the host cell receptor proteins, thereby affecting virus binding, attachment on the cell surface, and entry into the host cell . SimilarAnother possible route of investigation is the safe passive immunization of exposed individuals and patients throughout the use of human single chain antibodies (Hu-scFvs) or humanized nanobodies (single-domain antibodies (sdAb)). These molecules or transbodies directed to CoV intracellular pathways/non-structural proteins can go across the membrane of virally infected cells thus leading to block viral replication and transcription. Transbodies have already being experimented for the treatment of other infections, including those caused by influenza, hepatitis C, Ebola, and Dengue viruses .The future use of mAbs could overcome many drawbacks that are associated with the administration of plasma and intravenous immunoglobulin preparations, as above outlined, especially in terms of specificity, safety, purity, and low risk of transmission from blood-borne pathogens . We needManagement of COVID-19 infection aims first to ameliorate clinical manifestations and to provide supportive care . NeverthTo date, there is a full spectrum of potential drugs for COVID-19 infections that affects virus binding or enzymatic activities involved in viral replication and transcription. Essential viral proteins may be target of small molecule inhibitors i.e., helicase, proteases, inhibitors of host cell proteases, and endocytosis and fusion core blockers. Furthermore, anti-sense RNA (siRNAs), ribozyme, neutralizing antibodies, mAbs binding host receptor or that affect S1 RBD interaction, antiviral peptides that act on S2, and other natural products can be exploited ,50.Experimentation of drugs for COVID-19 management should certainly start to screen for efficacy and safety of clinically assessed MERS- and/or SARS-CoVs inhibitors, although some precautions derive from the still unexplored properties of the novel COVID-19 virus . Indeed,Clinical trials are required in particular for confirming safety and efficacy for COVID-19 RNA synthesis inhibitors , remdesivir, neuraminidase inhibitors, peptide (EK1), anti-inflammatory drugs, and arbidol . In vitrOf note, binding of the S1 COVID-19 protein to ACE2 has an effect on the renin–angiotensin system (RAS), leading to possible exacerbation of severe pneumonia. Currently, ACE1 and angiotensin type-1 receptor (AT1R) inhibitors are believed to reduce pulmonary inflammation, thus reducing the mortality rate ,54.Remarkably, antiviral drugs already experimented for CoV infections can potentially treat COVID-19. The use of HIV protease inhibitor lopinavir in association with ritonavir in recent Chinese trials on SARS-CoV2 patients requires further evaluation and investigation ,56. ThesOf note, cytokines play important roles in the immunopathology of viral infections since innate immunity is the first line defense against viruses. Dysregulated proinflammatory immune responses cause the ‘cytokine storm’ in the pathogenesis of CoV2 infection. This is closely related to the development and progression of the Acute-Respiratory Disease Syndrome (ARDS) or extrapulmonary multiorgan failure, leading to infection exacerbation or death. Therefore, immunomodulators and cytokine antagonists represent a potential effective strategy. In this regard, evidences were produced on the efficacy of anti-inflammatory drug tocilizumab to ensure the possibility to timely control the cytokine storm at the early stage to improve success rate of treatment and reduce mortality rate. Tocilizumab is a recombinant humanized monoclonal antibody raised to both soluble IL-6 receptor (sIL-6R) and bound to the membrane (mIL-6R) . This drRegarding the antiviral activity of Type 1 interferons (IFN-1), their beneficial effects are at an early stage of infection. At a later stage, their administration may worsen the cytokine storm and exacerbate inflammation . Type 1 Further, Chinese physicians have used steroids to control cytokine storm related to the infection and occurrence of lung fibrosis. However, steroids have a narrow therapeutic window since these should be used only when SARS-CoV2 has been already eliminated by the human immune system. Otherwise, virus replication and shedding and related symptoms of the infection may exacerbate .The utility of combined administration of IVIgG (intravenous Immunoglobulin G) and low molecular weight heparin (LMWH) anticoagulant therapy was suggested since infection, inflammation, and other factors may lead to disseminated intravascular coagulation .Chloroquine (CQ) and hydroxychloroquine (HCQ) have been used for prevention and treatment of malaria . They haTeicoplanin is another drug under investigation for COVID-19 infection. This is a glycopeptide antibiotic, currently used in the treatment of Gram-positive bacterial infection, especially in Staphylococcal infections. Already, this antibiotic showed antiviral activity in vitro against viruses, including Ebola, influenza, flavivirus, hepatitis C, HIV, MERS-CoV, and SARS-CoV ,73,74. TThe emergency to combat the COVID-19 pandemic has propelled the search for vaccine development in the scientific community. The urgent need for a vaccine raises from several concerns, which are not only control of the infection in its spreading from its real geographical origin in Wuhan and the whole country of China, but also worldwide with the consequent disruption of the social and economic equilibrium, especially in countries with poor healthcare resources . FurtherIn the past 10 years, approaches were already attempted to combat CoV viruses ,77. SurfAs for previous attempts in vaccine preclinical experimentation to SARS-CoV, virus studies are needed to ascertain the most suitable animal models, especially electing small animals that closely resemble the clinical disease caused by SARS-CoV2 infection in humans and its etiopathogenetic mechanisms . These aRegarding the present pandemic, immunoinformatics databases can support the identification of strategic target epitopes, promising candidates for immune recognition to be used in COVID-19 vaccine developments. Indeed, effective binding of identified epitopes for cytotoxic T lymphocytes (CTL) responses and the peptide binding grooves on MHC class I molecules was proven, indicating their potential to generate immune responses ,81.https://www.darwinresearch.com/astrazeneca-ramps-up-covid-19-vaccine-manufacturing-distribution-capacity-with-two-more-deals, accessed on June 2020) [It is estimated that more than 100 vaccines are currently being produced (ne 2020) . These pne 2020) .The immunity generated by CoV2 vaccines is currently assessed in preclinical studies and started trials through humoral antibody induction and/or T cell effector responses . Of notehttps://www.bristol.ac.uk/news/2020/april/codid-19-vaccine-platform.html, accessed on June 2020). In the UK, the Jenner Institute and the Oxford vaccine group  has produced the ChAdOx1 nCOV-19 vaccine that employs the replication-deficient chimpanzee adenoviral vector isolate Y25. This vectored vaccine encodes a codon-optimized full-length S-protein with a human tPA (tissue plasminogen activator) leader sequence. Preclinical studies have already demonstrated that it is immunogenic in mice by eliciting a strong humoral and not Th2 dominated cell-mediated response. A single vaccination was effective in rhesus macaques. A reduced viral load was observed in bronchoalveolar lavage and respiratory tract of vaccinated animals respect to controls [10 viral particles . Vaccination with a lipid nanoparticle-encapsulated mRNA-1273 encoding SARS-CoV2 S-protein developed by Moderna (NCT042283461) [https://investors.modernatx.com/news-releases/news-release-details/moderna-announces-positive-interim-phase-1-data-its-mrna-vaccine/, accessed on June 2020). Adenovirus type 5 vector expressing S-protein (Ad5-nCoV) from CanSino Biological (NCT04313127) is in Phase I. Participants with pre-existing Ad5 neutralizing antibodies exhibited lower humoral and cellular responses. BNT162  employed a modified RNA delivered in a lipid nanoparticle [Bifidobacterium longum to deliver CoV2 S-protein into intestinal cells (NCT 04334980) [Several nucleic acid vaccines are currently under development for COVID-19 . Recombicontrols . This vaa NCT0422461 [83,82283461)  prevente4334980) . Preclin4334980) . ShenzheSARS-CoV2 has recently emerged as a novel unknown virus. Today, we are facing the health and economic consequences of its rapid diffusion worldwide. In a guarded optimistic view, we hope that the undertaken preventive and treatment measures will at least contribute to contain diffusion, attenuate activity or even eliminate the virus. Although today there are no specific treatments for COVID-19, early diagnosis and improved management of severe patients may reduce mortality. As above underlined an effective measure is to treat a cytokine storm by administering anti-inflammatory drugs, such as tocilizumab, at early stage of the infection for timely control of the disease and to avoid the risk of mortality. Modern technologies may subside in a near future to validate vaccination strategies in eradicating viral infection from humans. In this regard, reverse vaccinology based on computational identification of immunodominant epitopes may contribute to novel preventive strategies for COVID-19. In providing examples, indeed, epitope-based immune-derived vaccines (IDV) are generally considered to be safe when compared to other vectored or attenuated live vaccines. These may also provide essential T-cell help for antibody-directed vaccines . Several


Extracellular vesicles (EVs), which are cell released double layered membrane particles, have been found in every circulating body fluid, and provide a tool for conveying diverse information between cells, influencing both physiological and pathological conditions. Viruses can hijack the EVs secretory pathway to exit infected cells and use EVs endocytic routes to enter uninfected cells, suggesting that EVs and viruses can share common cell entry and biogenesis mechanisms. SARS-CoV-2 is responsible of the coronavirus disease 2019 (Covid-19), which may be accompanied by severe multi-organ manifestations. EVs may contribute to virus spreading via transfer of virus docking receptors such as CD9 and ACE2. Covid-19 is known to affect the renin angiotensin system (RAS), and could promote secretion of harmful EVs. In this scenario EVs might be linked to cardiovascular manifestations of the Covid-19 disease through unbalance in RAS. In contrast EVs derived from mesenchymal stem cells or cardiosphere derived cells, may promote cardiovascular function due to their beneficial effect on angiogenesis, fibrosis, contractility and immuno-modulation. In this article we assessed the potential impact of EVs in cardiovascular manifestations of Covid-19 and highlight potential strategies to control the extracellular signaling for future therapies. Cells are continuously secreting extracellular vesicles (EVs) that include large apoptotic bodies (1–5 μm), microvesicles  or small exosomes (30–100 nm), which vary in their abundance, size, and composition. Enclosed by a lipid bilayer, EVs are thus very stable and convey cytoplasmic molecules including proteins, RNAs, lipids, and metabolites .Exosomes originate from processing of early endosomes by endosomal sorting complex to form intraluminal vesicles within larger multivesicular bodies (MVB) that traffic in the cytoplasm . The fusMicrovesicles have size overlap and share some surface markers, such as CD9 and CD63  making ein vitro data show that alveolar cells infected with SARS-CoV-1 present whole virions in the secretory vesicles near the plasma membrane of infected cells  carried by EVs. Whether EVs released from infected cells have harmful or protective roles is controversial: vesicles containing only viral fragments might stimulate the immune system for their antigen priming activity, while the whole virion hidden in bigger vesicles could potentially elicit viral dissemination by escaping the immune recognition and further spread the virus within the body.In addition to CD9, it has been shown that exosomes can transfer the angiotensin converting enzyme 2 (ACE2) receptor , which SIt has been reported that during SARS-CoV-1 and MERS-CoV infections there were increased levels of circulating exosomes enriched in lung-associated self-antigens as well as viral antigens . Thus, i1R and AT2R) through a feedback process that promotes myocardial hypertrophy  activation promotes cardiac remodeling . Currentertrophy . Moreovemodeling . Post-momodeling . SARS-Comodeling , and it modeling .Both SARS-CoV-1 and SARS-CoV-2 uses ACE2 as docking station to infect target cells and the internalization of virus/enzyme complex leads to loss of enzymatically active ACE2 at the cell surface .As RAS-inhibitors can upregulate expression of ACE2, there were initial concerns that use of RAS-antagonists may increase risk or severity of Covid-19 as more docking stations would theoretically be present. However, the use of RAS inhibitor therapy was not associated with increased infectivity or severity of disease .2R agonist is used as therapy to counterbalance the potentially harmful RAS effects during Covid-19   .Although EVs release as results of RAS disturbance during SARS-CoV-2 infection has not yet been demonstrated, we here provide a provocative hypothesis of a link between EVs, Covid-19 and RAS based on two demonstrated evidences: (i) SARS-CoV-2 infection causes disturbance of RAS; (ii) RAS-dependent release of exosomes in e.g., hypertension are known to aggravate cardiovascular diseases and myocardial remodeling.Severe Covid-19 display a systemic pro-inflammatory state often referred to as a cytokine storm with critical illness and manifestations such as acute respiratory distress syndrome (ARDS), pulmonary edemea, thromboembolism, and acute cardiac injury, which come with high mortality .Mesenchymal stem cells (MSCs) have powerful immuno-modulatory effects and are considered potentially effective therapeutic assets to combat a wide spectrum of inflammatory-driven diseases but also clinically useful in preventing inflammatory activation from acute graft vs. host disease following allogenic hematopoietic stem-cell transplantation . Thus, MInterferon gamma (INF-γ) is a major proinflammatory cytokine secreted by activated T cells and natural killer cells, which promotes macrophage activation and mediate host defense against pathogen infection. Decreased plasma levels of INF-γ in the early stage of the disease (before the onset of cytokine storm) were associated with increased risk of pulmonary fibrosis in Covid-19 patients . Indeed Therapeutic approaches for Covid-19 based on MSCs-derived EVs aimed to enhancing the healing process following lung injury, have been taken in consideration . HoweverThe timing of an intervention seems to be a critical aspect in Covid-19 disease. The acute phase of Covid-19 illness is characterized by three pathological phases: (i) the early infection phase when SARS-CoV-2 infiltrates and replicate; (ii) the pulmonary phase characterized by respiratory dysfunction; (iii) finally the hyperinflammation phase driven by the host immunity with an exaggerate inflammatory response . The speAlthough EVs containing viral components might elicit the adaptive immune response by presenting the antigen in a process similar to a vaccination, EVs secreted from infected/injured cells could activate macrophage secreting cytokines and contribute to the cytokine storm formation during Covid-19 . Indeed,Thus, a therapeutic application of EVs for Covid-19 must include donor cells with anti-inflammatory activity such as mesenchymal stem cells or cardiosphere derived cells. The anti-inflammatory activity of EVs indicate that this platform can work well in xenogenic applications , which cWe have discussed the possible function of EVs in Covid-19 and highlight potential therapeutic application for EVs in modulating the virus infection, the pro-inflammatory state and cardiovascular disease in Covid-19. EVs, such as exosomes could be involved in aggravated cardiovascular manifestations of Covid-19 either by promoting viral docking in cardiac cells or shifting RAS to promote inflammation, coagulation, fibrosis and endothelial dysfunction. Furthermore, EVs derived from engineered MSCs or CDC used as drug delivery system to modulate the cytokine storm of virus spreading represent a promising strategy to combat Covid-19. In summary, understanding the molecular mechanisms behind the entry, replication, and spreading of SARS-CoV-2, in conjunction to EVs trafficking, may provide tools to limit the manifestation of Covid-19.GP substantially contributed to the conception and design of the perspective article, literature search, drafting the article and revising the article critically for important intellectual content. DA and LL contributed in literature search, drafting the article and revising the article critically for important intellectual content. All the authors approved the final version of the article to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a global health crisis and will likely continue to impact public health for years. As the effectiveness of the innate immune response is crucial to patient outcome, huge efforts have been made to understand how dysregulated immune responses may contribute to disease progression. Here we have reviewed current knowledge of cellular innate immune responses to SARS-CoV-2 infection, highlighting areas for further investigation and suggesting potential strategies for intervention. We conclude that in severe COVID-19 initial innate responses, primarily type I interferon, are suppressed or sabotaged which results in an early interleukin (IL)-6, IL-10 and IL-1β-enhanced hyperinflammation. This inflammatory environment is driven by aberrant function of innate immune cells: monocytes, macrophages and natural killer cells dispersing viral pathogen-associated molecular patterns and damage-associated molecular patterns into tissues. This results in primarily neutrophil-driven pathology including fibrosis that causes acute respiratory distress syndrome. Activated leukocytes and neutrophil extracellular traps also promote immunothrombotic clots that embed into the lungs and kidneys of severe COVID-19 patients, are worsened by immobility in the intensive care unit and are perhaps responsible for the high mortality. Therefore, treatments that target inflammation and coagulation are promising strategies for reducing mortality in COVID-19.  NK cells are thought to undergo mass migration to the lungs, depleting circulating populations and may contribute to tissue damageComplement activation is linked to virus removal, but is implicated in neutrophil activation and thrombosisA robust early macrophage IFN-response is associated with mild disease, while high levels of the cytokines IL-6, IL-10 and IL-1β in the early stages of COVID-19 are linked to poor outcomeNeutrophil activation causes tissue damage and an increased risk of potentially fatal immunothrombotic events in the lungs and kidneys of COVID-19 patientsMonocyte HLA-DR expression, neutrophil activation and the ratio of neutrophils to lymphocytes might be a potential identifier of high-risk patientsThe first responders to viral infection in coronavirus disease 2019 (COVID-19) are the cells of the innate immune system, including natural killer (NK) cells, monocytes, macrophages and neutrophils. After migrating to the site of infection and recognizing the virus from conserved features, innate cells initiate responses to eliminate the virus and coordinate the adaptive immune system. However, in some cases, innate cells have been shown to contribute to viral dissemination and tissue damage which is associated with severe COVID-19 disease and fatal outcomes. Therefore, it is critical to evaluate innate immunity in the context of COVID-19 to predict outcome and facilitate development of therapeutic interventions.A hallmark of life-threatening COVID-19 is a pathological hyperactive immune cell response brought about by the inability to control virus. NK cells that are lost in the circulation traffic to the lungs where they may contribute to inflammatory damage. Alveolar macrophages and monocytes are implicated in cytokine propagation. Macrophage virus infection and activation may inhibit their ability to cloak inflammation. Non-classical monocytes and neutrophils are increased in the blood of severe COVID-19 patients suggesting high bone marrow turnover. A cytokine storm has also been reported in COVID-19. Though high cytokine levels such as interleukin (IL)-6, IL-10 and IL1-β are linked to disease severity, these cytokine elevations are thought to only occur early in the disease followed by reported immunosuppression. Complement, damage and cytokines are implicated in excessive neutrophil activation, triggering fibrosis and damaging thrombotic events, thought to be the key drivers of mortality in COVID-19. As such, a high ratio of neutrophils to lymphocytes (including NK cells and T cells) and high D-dimer levels can predict COVID-19 disease outcome.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus causing coronavirus disease 2019 (COVID-19). COVID-19 has had devastating effects on human health and social and economic aspects worldwide. SARS-CoV-2 is highly infectious and while a proportion of patients present with asymptomatic disease, clinical manifestations can include fever, cough, loss of smell/taste, fatigue, difficulty breathing and organ dysfunction [acute respiratory distress syndrome (ARDS)], which for a small percentage of patients will be fatal. The risk of developing severe disease is associated with old age, gender, ethnicity and underlying health conditions including diabetes, obesity and heart disease. Severe outcomes likely involve a defective viral sensing and a dysregulated immune response. Part 1 of this review discussed the mechanisms of SARS-CoV-2 host cell entry and immune evasion. Part 2 assesses the current understanding of how innate immune responders  contribute to pathogenesis.The innate immune system provides the first line of defence against infection, rapidly respond to pathogens through recognition of conserved features, but are also important for adaptive immune responses. However, such lack of specificity can contribute to tissue damage and undermine adaptive immunity, thus a fine balance is required to protect the host.The complement system comprises several plasma proteins that opsonize or lyse pathogens and orchestrate host defence . Dysreguet al. [In addition to complement, pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) can also induce cytokine release. Cytokines play an important role in promoting immunopathology and immune dysfunction: ‘Cytokine storms’ reported in severe SARS-CoV-2 infected patients  exhibited higher levels of cytotoxic mediators [In severe disease, CD56ediators , suggestMacrophages, monocytes, and dendritic cells (DCs) are sentinels of the immune system that express a broad repertoire of PAMP, DAMP and complement receptors. They also present antigen to trigger adaptive immune responses and are equipped with antibody Fc receptors to be effector cells. Under physiological conditions, macrophages are tissue accessory cells that dampen inflammation via scavenging of dead cells and DAMPs such as ATP . Tissue-As macrophages express ACE-2, direct infection with SARS-CoV-2 could prevent IFN I production  impairing viral control and causing hyperinflammation . Mild SA+CD16−) pass through an intermediate state (CD14+CD16+) to eventually develop into non-classical monocytes (CD14−CD16+). Increased demand for monocytes in inflammation is associated with elevated intermediate and non-classical monocytes in the blood [Circulating monocytes are recruited to the lungs of COVID-19 patients where monocyte attracting chemokines including CCL2 and CCL7 are increased . Monocythe blood  and obsehe blood . Intermehe blood . These cIn mild COVID-19, circulating monocytes express high levels of Human Leukocyte Antigen (HLA)-DR, CD11c and CD14 and are characterized by an IFN-driven transcriptional programme. In sharp contrast, patients with severe COVID-19 monocytes showed low expression of HLA-DR while expressing anti-inflammatory associated genes , 74. Lowin vitro [SARS-CoV-2 spike protein can induce IL-8, IL-6 and TNF-α secretion in monocyte-derived macrophages (MDMs) and specifically primes COVID-19 patient-derived MDMs for IL-1β production in vitro . IL-6 isin vitro . IL-6 isin vitro . Macrophin vitro , while Iin vitro . CytokinMuch less data have been collected on the role of DCs in COVID-19, though studies demonstrate a decrease of plasmacytoid DCs (pDCs) in the lung and blood of patients , 82, whiNeutrophils serve as the first wave of immune cells recruited to infection and are prominent in viral defence and response to cellular damage . A rat cIn COVID-19, neutrophil activation/degranulation is the largest gene signature in blood cells  predictiet al. [Along with activation, markers of systemic NETosis have been identified in COVID-19 patients’ blood and are linked to disease severity  (recentlet al. . Extensiet al. , the reset al.  that canet al. . NETs aret al. , 105–107et al. , as are et al. , 110, whet al. . Thromboet al. . Such aset al. , 116.Chronic kidney disease is another common outcome in COVID-19 : as NETsIn Part 1 of this review, we concluded that SARS-CoV-2 deliberately sabotages early innate immunity, which along with inborn errors of innate sensing and IFN signalling results in increased viral load in severe COVID-19. The heavy viral burden causes cellular damage and triggers a hyperinflammatory response involving several innate immune cell types, summarized in NK cells are principal anti-viral innate lymphocytes and display reduced circulating numbers perhaps attributed to lung recruitment in COVID-19. In severe cases, NK cells display ‘exhausted’ phenotypes and an enrichment of specialized subsets with a bias towards cytotoxicity. Without healthy phagocyte clearance of NK-targeted apoptotic host cells, such activity may result in further inflammation, however, further research is needed to evaluate the role of NK cells in COVID-19. NK cells also likely join macrophages and monocytes in contribution to the cytokine environment identified in COVID-19. There is no consensus for a cytokine storm in COVID-19. Though certain cytokines, such as high early IL-6 levels can predict the worse outcomes. In mild cases, macrophages have an early robust IFN I response, associated with viral clearance, i.e. lacking in severe patients. Additionally, low monocyte HLA-DR expression, neutrophil activation, and the ratio of neutrophils to lymphocytes have been suggested as potential identifiers of high-risk patients.Neutrophils express an IFN I signature and produce NETs which have been implicated in immunopathology, particularly the formation of potentially fatal thrombotic events in COVID-19 patients. It is likely that activated leukocytes and NETs provide a nucleation point for the complement and coagulation pathways, and these resulting thrombi can become clogged in the small capillaries of inflamed lungs and kidneys. The heightened systemic inflammation and poor circulation in ICUs increase the likelihood of such clotting events, and thus treatments  which ta