Although young children generally experience mild symptoms following infection with SARS-CoV-2, severe acute and long-term complications can occur. SARS-CoV-2 mRNA vaccines elicit robust immunoglobulin profiles in children ages 5 years and older, and in adults, corresponding with substantial protection against hospitalizations and severe disease. Whether similar immune responses and humoral protection can be observed in vaccinated infants and young children, who have a developing and vulnerable immune system, remains poorly understood. To study the impact of mRNA vaccination on the humoral immunity of infant, we used a system serology approach to comprehensively profile antibody responses in a cohort of children ages 6 months to 5 years who were vaccinated with the mRNA-1273 COVID-19 vaccine (25 μg). Responses were compared with vaccinated adults (100 μg), in addition to naturally infected toddlers and young children. Despite their lower vaccine dose, vaccinated toddlers elicited a stronger functional antibody response than adults, including against variant of concerns (VOCs), without report of side effects. Moreover, mRNA vaccination was associated with a higher IgG3-dependent humoral profile against SARS-CoV-2 compared to natural infection, supporting that mRNA vaccination is effective at eliciting a robust antibody response in toddlers and young children. While most children experience mild symptoms with acute SARS-CoV-2 infection, severe complications can ensue, even in the youngest children, and myocarditis, cardiomyopathy, renal failure, as well as coagulation and hemorrhagic disorders occur at increased rates with COVID-192. Concerningly, COVID-19 deaths in children far exceed deaths from influenza3 and COVID-19 has now become one of the leading infectious cause of death in the Unites States4. SARS-CoV-2-targeting mRNA vaccines have become available for individuals six months of age and older11. These vaccines have provided substantial protection against hospitalizations and severe disease in children ages 5–17 years12. Moreover, detailed humoral profiling of children and adolescents reveals that mRNA COVID-19 vaccines elicit robust, highly functional humoral immune responses in children in a dose-dependent manner13, with strong cross-reactivity against variants of concerns (VOC)14. In children under 5 years of age, mRNA vaccination results in neutralizing immunoglobulin titers comparable to vaccinated adults and vaccination protects against symptomatic infection11. Howerver, detailed humoral profiling in this age group has not yet been investigated. As an individual’s humoral immune response evolves with age15, age-related differences in mRNA vaccine responses must be fully characterized to fully understand the impact of mRNA vaccination in infants and toddlers.Despite the early misconception that children were spared from COVID-19, children continue to account for approximately twenty percent of all documented cases of COVID-19 infection in the United States, with infants and children under 5 years of age disproportionately affected by high rates of hospitalizationIn order to characterize the activation of humoral immunity in young children after SARS-CoV-2-specific mRNA vaccination, we used an unbiased system serology approach to analyze antibody levels and Fc-mediated functions in individuals ages 6 months through 5 years. We comprehensively profiled their antibody response following vaccination with the mRNA-1273 COVID-19 vaccine (25 μg) and compared it with antibody profiles of vaccinated adults (100 μg), as well as children infected with SARS-CoV-2. Our results reveal a strong activation of humoral immunity post-vaccination in these young children, with a highly functional and cross-reactive humoral immunity in comparison to adults and naturally infected infants.Fig. S1).Our first objective was to profile vaccine-induced humoral immunity in infants and toddlers ages 6 months through 5 years (n = 18) after completion of the two doses of the pediatric mRNA-1273 vaccination series (vaccine dose: 25mcg mRNA-1273) and compare these serologic responses to those generated by fully vaccinated adults . Demographics of participants are included in Fig. S2). Interestingly, in contrast to IgG, this young population displayed lower levels of vaccine-induced anti-Spike and anti-RBD IgM and IgA1, which shows the distinct isotype selection between adults and children .Our results demonstrate that despite their young age and receipt of only one quarter of the adult dose, total anti-Spike and anti-RBD IgG levels and IgG subclass in young children were similar to adults , Fig. S2children . We thenchildren , Fig. S1To determine cross-reactivity of the vaccine-induced humoral response against variants of concerns (VOCs), we quantified antibody levels and FcR binding against Spike and RBD for six different SARS-CoV-2 VOCs including wild type (WT), Alpha, Beta, Gamma, Delta, and Omicron. While IgM, IgA1, and the FcR for IgA1 (FcaR) were higher in adults across the different SARS-CoV-2 variants, IgG response was essentially indistinguishable between young children and adults. In fact, the only exceptions were total IgG against RBD Omicron and IgG4 against Spike Gamma, RBD Alpha, RBD Delta, and RBD Omicron, which were significantly increased in young children .To further characterize the capacity of the pediatric population to generate a broad SARS-CoV-2-specific humoral response following mRNA-1273 vaccination, we calculated a Spike and RBD protein breadth score. The breadth score highlights that children less than 5 years old are able to induce a humoral response as robust as adults, with a strong recognition of different VOCs while IgM- and IgA-specific immunity is higher in adults . Taken tFig. S3A). The LASSO-selected features that were used to build the PLS-DA model revealed an enrichment of antibody levels and FcγR binding against HKU1, OC43, and HA in adults . Co-correlates analysis showed strong connections between isotypes and FcγR features against non-SARS-CoV-2 antigens , all of which being enriched in older individuals. These antibody profiles in adults reflect prior exposure to these respiratory viruses over their lifetime, while these young children may remain naïve. Alternatively, the lower antibodies could reflect lower total antigen-specific humoral responses to prior infection or the non-mRNA influenza vaccine, or more rapidly waning immunity in these young children.When looking more broadly at antibody responses against common respiratory infections, including non-SARS-CoV-2 human coronavirus (HCoV) HKU1 Spike (HKU1), HCoV-OC43 Spike (OC43), and Influenza haemagglutinin (HA), we see a strong age-related difference. In contrast to the robust SARS-CoV-2 vaccine-induced humoral immunity across the age spectrum, young children have significantly lower antibodies titers against HKU1, OC43, and HA. Multivariate analysis highlights a clear separation between the two age categories distributions, as attested by the Partial least squares discriminant analysis (PLS-DA) , one month after the first dose (V1), one month after the second dose (V2), six months after vaccination (V6), in addition to one month after boosting (Post-boost) , 3. Afte14. To determine the breadth of antibody responses over time, breadth scores were calculated for each IgG subclass and each FcγR over the time as described above , at Massachusetts General Hospital (MGH) were approached for enrollment in the MGH Pediatric COVID-19 Biorepository (IRB: 2020P000955). Families with children who were infected with SARS-CoV-2 in the past 2–8 weeks and were presenting to MGH for a well-child visit or hospital visit were also approached and offered enrollment in the MGH Pediatric COVID-19 Biorepository. Parents/guardians provided informed consent prior to participation.Families with children 6 months to 5 years of age who were participating in the KidCOVE study, a phase two/three clinical trial to assess the safety and efficacy of the mRNA-1273 two-dose vaccine in healthy children (Blood was collected prior to vaccination (Pre-vaccine), one month following the first vaccination (V1), one month following the second vaccination, (V2), and six months following the second vaccination (V6). If a booster dose was received, blood was collected prior to receipt of the booster dose (if greater than six months from first vaccination), and one month following the booster (post-boost). Participants could opt out of providing blood at any of the time points. Blood was collected by venipuncture or by capillary microneedle device, processed for plasma, and stored at −80°C. All procedures were approved by the MGB IRB.Banked samples from adults who had received the Moderna mRNA1273 vaccine were used for comparison. Detailed information regarding enrollment and specimen collection for this IRB-approved study were previously published [21].SARS-CoV-2 D614G or variants of concern Spike and RBD proteins, in addition to NTD, S1, nucleocapsid, HCoV-HKU1 Spike (HKU1), and HCoV-OC43 Spike (OC43) antigens, were expressed in mammalian HEK293 cells and purchased from Sino Biological. Influenza haemagglutinin (HA) was obtained from Sino Biological. NHS-Sulfo-LC-LC kit was used for antigen biotinylating, according to the manufacturer’s instruction (Thermo Fisher Scientific).33. Plasma samples were diluted between 1:50 and 1:500, depending on the secondary reagent. For functional analyses, antibody-dependent complement deposition (ADCD), cellular phagocytosis (ADCP) and neutrophil phagocytosis (ADNP) were performed as described previously36 with 1:50 diluted plasma samples. MFI values were analyzed by flow cytometry on an iQue analyzer (Intellicyt).Antibody isotype and subclass levels , as well as Fc-receptor (FcR) binding profiles  were measured using a custom multiplex Luminex assay as described previouslySpike and RBD protein breadth score were calculated by categorizing each antigen response as positive or negative and calculating the percentage of Spike and RBD variant antigen responses for each secondary (isotype or FcR) at each timepoint. We defined a positive response as six standard deviations above the mean of the SARS-CoV-2-unexposed controls for the same antigen and isotype or Fc receptor.GraphPad Prism (v.9.2.0) and RStudio (v.1.3 and R v.4.0) were used to perform data analyses. We calculated breadth score by categorizing each antigen response as positive or negative and calculating the percentage of positive Spike and RBD variant antigen responses for each antibody feature at each timepoint. We defined a positive response as six standard deviations above the mean of the COVID-unexposed controls for the same antigen and isotype or Fc receptor.37. Data were normalized using z-scoring, then a least absolute shrinkage and selection operator (LASSO) approach was used for feature selection. For classification and visualization, partial least square discriminant analysis (PLS-DA) models were performed using LASSO-selected features, followed by a ten-fold cross-validation to assess model accuracy. Co-correlates of LASSO selected features were represented in a network format and identified using Spearman method followed by Benjamini-Hochberg correction.Multivariate analyses to compare vaccinated adults and children were built as described previously


Cancer immunotherapies utilizing genetically engineered T cells have emerged as powerful personalized therapeutic agents showing dramatic preclinical and clinical results, particularly in hematological malignancies. Ectopically expressed chimeric antigen receptors (CARs) reprogram immune cells to target and eliminate cancer. However, CAR T cell therapy's success depends on the balance between effective anti-tumor activity and minimizing harmful side effects. To improve CAR T cell therapy outcomes and mitigate associated toxicities, scientists from different fields are cooperating in developing next-generation products using the latest molecular cell biology and synthetic biology tools and technologies. The immunotherapy field is rapidly evolving, with new approaches and strategies being reported at a fast pace. This comprehensive literature review aims to provide an up-to-date overview of the latest developments in controlling CAR T cell activity for improved safety, efficacy, and flexibility. Over the last decade, therapies with genetically engineered immune cells such as chimeric antigen receptors (CAR) T cells and T cell receptor (TCR) modified T cells have revolutionized personalized cancer treatment. With several products already approved for clinical use and hundreds of products in preclinical development and clinical testing, immunotherapies based on genetically engineered autologous CAR T cells specific for tumor-associated antigens are gaining growing attention from scientists and clinicians, the pharma industry, and the public.The main limitations of current autologous CAR T cell products include high cost and manufacturing time (availability), cancer resistance or evasion leading to relapse and therapy failure (efficacy), and off-target side effects and toxicities (safety). As true living drugs, CAR T cells require special considerations for their formulation, administration, biodistribution, persistence, and mitigation of potential side effects. The first safety concern arises shortly after infusion when CAR T cells engage with their target antigens and expand in response by 100- to 10 000-fold . UnfortuIt is important to note that the activity of CAR proteins can differ depending on the overall construct design. The selection of signaling domains, such as immunoreceptor tyrosine-based activation motifs (ITAMs), incorporated into the CAR protein –15, as wDue to all that reasons, adverse effects can be challenging to manage and pose risks during treatment. Therefore, precise control over infused CAR T cell activation, expansion, and persistence is highly desirable from a clinical perspective. To address these challenges, scientists are implementing various ingenious molecular cell biology and synthetic biology methods and technologies, which are systematically discussed in the respective parts of this review.Many excellent reviews have been published on emerging CAR T cell technologies. The reviews cover a large variety of topics, including CAR switches and modular approaches –25, apprThe suicide genes, molecular safety switches, and inhibitory corticosteroids represent the earliest and still most popular rational approaches to managing unpredictable CAR T cell expansion and toxicities in patients. Essentially, a safety switch is an ectopically expressed safeguard gene, which can be activated on demand to remove the infused therapeutic cells from the body rapidly and permanently, in case of an adverse immune reaction , 44–46. The cell elimination process exploits natural cell death mechanisms such as activating intrinsic or extrinsic apoptotic pathways or complement- or antibody-dependent cell-mediated cytotoxicity (ADCC) with subsequent clearance of cellular remains by phagocytic cells –58. A siOne of the first-in-class and best-characterized suicide genes exploits the herpes simplex virus I–derived thymidine kinase gene. The system is based on the ability of HSV-TK to phosphorylate nucleoside analogs, such as acyclovir or ganciclovir (GCV), to nucleoside monophosphate, which is subsequently phosphorylated into the nucleoside triphosphate. When nucleoside triphosphate analogs incorporate into newly synthesized DNA strands, it potently inhibits DNA synthesis and triggers mechanisms leading to cell death Fig. A 50, 5151. The HMoreover, the HSV-TK suicide system also has potential clinical incompatibility leading to unwanted elimination of modified T cells in the case of cytomegalovirus infection treatment with GCV , 68. ConAnother commonly used suicide gene system utilizes the modified human protein Caspase-9, a key ubiquitously expressed mediator of the extrinsic apoptotic pathway , 70. In Several modifications recently improved the flexibility of the iC9 system. For instance, an alternative method of Cas9 activation based on Rimiducid-inducible MyD88 and CD40 signaling elements can offset potential toxicity risks by an orthogonally regulated, rapamycin-induced, caspase-9-based safety switch , 72. In Like Caspase-9, FADD is a crucial component of the cellular apoptotic pathways, and a suicide switch system analogous to iC9 was developed to activate FADD fused to heterodimerization domains and trigger apoptosis Fig. B. This sThe detection and selection of therapeutic cells modified with intracellular suicide switches require co-expression with the CAR or another marker gene, which is inconvenient for most applications. Therefore, alternative strategies utilizing cell surface elimination markers simplify and streamline cell engineering processes. The most common CD20-based safety switch takes advantage of human (therefore non-immunogenic) protein CD20 and chimeric monoclonal CD20 antibodies, such as the FDA-approved antibody Rituximab, to kill target cells in the ADCC process mediated by NK cells  into chemotherapy drug 5-fluorouracil (5-FU). Since the subsequent cellular metabolites of 5-FU inhibit nucleic acid synthesis in proliferating cells and lead to cell death, ectopically expressed CD can serve as a potent suicide gene approach  represents another promising concept in suicide genes. So called SFas consists of the extracellular anti-fluorescein scFv domain, the D2 and modified transmembrane domains of the erythropoietin receptor, and the intracellular apoptotic signal-transducing domain of Fas. The apoptosis of cells modified with SFas is triggered by low concentrations of fluorescein labeled BSA and results in the nearly complete elimination of cells in vitro . The sysMost current safety strategies rely on stable ectopic expression of transgenes. As a novel concept, CRISPR/Cas9 gene-editing technology provided an elegant way of controlling cell growth by disrupting uridine monophosphate synthetase (UMPS), the key cellular enzyme in the pyrimidine synthesis pathway. Without UMPS, engineered cell proliferation becomes entirely dependent on the external uridine supply. In addition, the same genetic modification confers resistance to 5-fluoroorotic acid, which can be used to select UMPS knockout cells. This promising approach was successfully tested in various cell lines, pluripotent stem cells, and primary human T cells .Eliminating CAR T cells by suicide genes and safety switches constitutes a remedy to excessive adverse reactions but results in permanently losing therapeutic cells. In the following sections, many effective alternative strategies to regulate the proliferation and activity of therapeutic cells at CAR expression and function levels effective alternative strategies will be explored. One of the most popular sets of strategies aims to control the total number of CAR molecules present on the T cell surface via modulating the expression of CAR mRNA by chemical or biophysical inducers Fig. .Fig. 3AnSeveral clinically approved small chemical molecules can be successfully repurposed for use in artificial gene expression systems to switch the expression of transgenes ON or OFF.Tet-ON is a widely used technology that allows precise, reversible, and efficient control of gene expression with the antibiotic Tet or doxycycline (Dox). Briefly, in the presence of the drug, the engineered reverse tetracycline-controlled transactivator (rtTA) transcription factor binds to the Tet-responsive element (TRE) promoter and initiates gene transcription in a dose-dependent manner. Several CAR T cell platforms, including CD19 CAR , 94 and The natural compound Res present in red wine, grapes, and berries has gained attention and popularity as a safe dietary supplement and potential drug candidate . Like TeAnother promising strategy for small molecule-inducible control over effector functions of CAR T cells is focused on zinc finger transcription factor. In this system, the administration of drugs endoxifen or tamoxifen to engineered T cells activates the artificial zinc finger-based transcription factor to induce gene expression. In vitro and in vivo experiments demonstrated precise induction of CD20 CAR expression with tamoxifen in primary T cells, which was accompanied by rapid induction of CAR T cell effector functions .Spatiotemporal confinement and activation of therapeutic cells in the body represent one of the critical outstanding challenges in cell immunotherapy. While small molecule-based inducible expression strategies offer simple, fast, and flexible control, it is difficult to achieve tissue specificity and temporal resolution due to cell permeation and diffusion kinetics. Therefore, an efficient spatiotemporal control approach enabling precise and on-point action of CAR T cells might be groundbreaking, especially for developing improved solid tumor cell therapies. Several strategies based on synthetic biology and biophysical methods were invented to control CAR expression in a specific tissue, thus addressing “on-target, off-tumor” toxicity often associated with CAR T cell products.Avena sativa phototropin 1 [Arabidopsis thaliana cryptochrome 2 (AtCRY2) and its light-dependent interaction partner CIB1 [Various optogenetic protein dimerizer approaches rely on plant-derived light-sensing domains of photoreceptors mediating light-dependent protein–protein interactions – e.g., the light-inducible nuclear localization signal (NLS) technology (LINuS) based on the LOV2 domain of tropin 1  or the Cner CIB1 . The LINner CIB1 . The LINner CIB1 . HoweverReCoM is another emerging modular method to trigger tissue-specific expression of CARs remotely and noninvasively in T cells by using a combination of ultrasound and mechanosensory proteins . The ultphotothermal switch CAR approach, T cells are engineered to trigger the expression of CAR and other genes (such as IL-15) in response to mild (40–42 °C) temperature elevations. The expression cassette under the control of the heat-shock protein (Hsp) promoter can dynamically upregulate gene expression more than 20-fold at 42 °C in primary T cells in vivo [At least two distinct approaches employing thermal control of CAR expression have been reported. One example of a synthetic thermal-specific gene switch approach is utilizing thermal energy from near-infrared light converted by plasmonic gold nanorods to induce expression of the CAR gene . In this in vivo . HoweverFUS-CAR system comprises a Cre-lox gene switch that utilizes Hsp-driven Cre recombinase to activate CD19 CAR gene expression. When expressed, the Cre recombinase removes the lox-flanked ZsGreen-STOP sequence, resulting in sustained production of the CD19 CAR with ZsGreen expression switched on [Local heat generated by focused ultrasound (FUS) represents an elegant acoustogenetics method that allows inducible and reversible CAR expression in T cells at the desired time and location without requiring additional exogenous cofactors . Brieflytched on . The phohypoxia-induced CAR (HiCAR) combines two oxygen-sensing mechanisms. First, the anti-CD19, anti-AXL, or anti-HER2 CAR expression cassettes are driven by hypoxia response elements upstream of EF1α promoter  exhibiting dismal basal expression under normoxia but strong expression under hypoxia. Second, CAR was fused to an oxygen-dependent degradation domain that is actively degraded under normoxia but stabilized under hypoxia. The resulting chHE-HiCAR T cells demonstrated significantly increased cytotoxicity against tumors under hypoxia compared to normoxia in vitro and anti-tumor efficacy comparable to conventional CAR T cells in vivo [HypoxiCAR T cells with inducible expression of pan-anti-ErbB (T4) CAR, which also co-expressed a chimeric cytokine receptor (4αβ) [The solid tumor microenvironment is often characterized by inadequate oxygen supply. Thus, intratumor hypoxia can be exploited to control the expression of CAR, specifically in the tumor tissue, to effectively mitigate on-target/off-tumor toxicities often associated with CAR T cell therapies , 107. Fo in vivo . A very or (4αβ) . HypoxiCSynNotch CAR system illustrates a rather distinct sophisticated approach to spatiotemporal control of CAR expression and reducing on-target/off-tumor toxicities of T cell therapies via better separation of tumor and normal cells. Instead of being triggered by biophysical methods, the SynNotch CAR system is an artificial dual-receptor genetic circuit that requires a combination of two different antigens (AND-gate) to initiate CAR expression and activate T cell cytotoxicity. The first step of the AND-gate signaling circuit requires activating an engineered synNotch receptor recognizing the first antigen A. Antigen A recognition leads to cleavage of synNotch and release of an orthogonal transcription factor that activates the expression of CAR directing T cells toward a second tumor antigen B –113. TheSince only a fully assembled cell surface CAR receptor can activate cytotoxic T cell functions, other rational strategies for controlling CAR T cell activities implement mechanisms for modulating CAR surface presentation, turnover, and stability. In particular, the approaches for rapid and reversible control of CAR protein degradation provide much-desired flexibility to manage therapy-associated toxicities in the case of T cell hyperactivation without CAR T cell removal. These strategies incorporate various small molecule inducible CAR receptor degradation systems to stop CAR-mediated signal transduction Fig. .Fig. 4A As a proof of concept, reversible CAR degradation strategy, the switch-off (SWIFF) CAR technology integrates the target site of HCV-NS3, HCV-NS3, and a degradation moiety (degron) into the intracellular part of the CAR construct (SWIFF-CAR) . Under sA slightly different chemical genetic technology achieves CAR degradation by incorporating IKZF3 zinc finger degron into the CAR construct , 121. UpFinally, yet another strategy for inducible CAR degradation exploits the PROTAC technology. Lee and colleagues modified the anti-CD19-CAR construct with BRD4-derived bromodomains, targeted by PROTAC compounds ARV-771 and ARV-825 to induce reversible protein degradation by VHL and CRBN E3 ubiquitin ligases and a proteasome pathway . InteresAmong many strategies developed to control the function of CAR T cells, the inducible assembly or disassembly of a functional CAR represents one of the earliest approaches Fig. . The CARON-switch split CAR [The inducible formation of a functional CAR receptor was originally described as plit CAR . The autplit CAR .dimerizing agent–regulated immunoreceptor complex (DARRIC) also consists of two physically separated membrane-bound components equipped with FKBP12-FRB* dimerization module, but in this case, in their extracellular parts [A similar split CAR approach denoted as the ar parts . One polar parts .Avidity-controlled CAR (AvidCAR) platform exploits the natural avidity effects of antigen recognition to develop CAR constructs with a drug inducible ON-switch function [Furthermore, the function . In thisfunction .lenalidomide-gated ON switch split CAR [The continuous research into CAR ON-switches ultimately has led to the developing of alternative, highly efficient systems engineered from nonimmunogenic human proteins controlled by non-immunosuppressive FDA-approved drugs. One example of such a novel design is the plit CAR . The sysplit CAR .lipocalin-based conformation-specific ON-switch CAR system [Similarly, the R system  was desiFKBP/FRB multichain CAR (FKBP/FRB-mcCAR) technology was developed by modifying the hinge domain of FcεRI-based CAR scaffold with FKBP and FRB (or FRB*) domains [As an alternative to split ON-switch CAR strategies, the  domains . Interes domains .light-switchable CAR (LiCAR) design [Interestingly, instead of using small molecule drugs, ON-switch CARs can also be engineered using optogenetic protein dimerizer systems to respond to light stimulation, as demonstrated in the ) design . Briefly) design .Chemically Regulated SH2-delivered Inhibitory Tail (CRASH-IT) switch, which exploits natural signaling modalities of immunoreceptors [A slightly different concept to engineering an ON-switch CAR was applied in the eceptors . Rather eceptors . In addiSTOP-CAR, is based on a rational computer-assisted design of a chemically disruptable heterodimer originating from two human protein scaffolds (Bcl-XL and the BH3 domain of Bim) [minocycline TetCAR, exploits the spontaneous heterodimer formation between tetracycline repressor protein B and a small peptide tetracycline mimic, disruptable with the FDA-approved small molecule antibiotic minocycline [As opposed to ON-switch CAR designs, several OFF-switch genetic systems were developed to modulate CAR T cell activity at the level of CAR disassembly. For instance, one approach, denoted  of Bim) . The CARocycline . SuccessCondCAR OFF-switch, implements antibody scaffolds with inducible binding affinities [Finally, a slightly different technology of conditional CAR modulation, called finities . In Condfinities .One area of intensive CAR development focuses on solving the problem of the “on-target, off-tumor” toxicity of CAR T cells to healthy cells and bystander tissues with target antigen expression , 26, 27.iCAR, was constructed from scFv targeting the prostate-specific membrane antigen (PSMA) fused to the transmembrane segment and signaling domains of CTLA-4 or PD-1 [The first proof-of-concept inhibitory CAR design, called  or PD-1 . When co or PD-1 . TherefoThe application potential of iCAR technology was further corroborated by a similar approach to mitigate the on-target off-tumor toxicity of novel CD93-specific CAR T cells in acute myeloid leukemia (AML). The CD93 CAR T cells demonstrated adequate leukemic clearance and minimal hematopoietic toxicity; however, with substantial off-target toxicity to endothelial cells with shared CD93 antigen expression . The feaDouble-Arm CAR is another inhibitory CAR that works as a NOT-logic gate [gic gate . Double-gic gate .iKP (KIR2-PD1) CAR, was engineered using the extracellular domain of KIR2DL2 and the intracellular domain of PD-1 [In addition to markedly increasing the specificity of conventional CAR-based therapies, inhibitory CARs might allow precise discrimination and targeting of tumor cells with genetic AND/NOT signal integrators. In particular, the clonal loss of heterozygosity (LOH) in a large majority of cancer cells provides a unique therapeutic window that can be exploited by reprogrammed immune cells. One approach took advantage of downregulated expression of human leukocyte antigen HLA-C1 to discriminate between normal cells (HLA-C1 positive) and tumor cells (HLA-C1 negative). HLA-C1 is naturally recognized in normal cells by the inhibitory receptor KIR2DL2, predominantly expressed in NK cells. Thus, an inhibitory CAR, denoted  of PD-1 . When co of PD-1 . InteresT-cell module 2 (Tmod2) [Neoplasm-targeting Allele-Sensing CAR (NASCAR) [anti-HLA-DR iCAR was co-expressed with anti-CD19 or anti-CD33 activating CARs and mediated highly selective killing of HLA-DR-negative tumor cells in vitro and in a xenograft mouse model [Other promising approaches denoted as  (Tmod2)  and Neop(NASCAR) , applied(NASCAR) . Similar(NASCAR) . Beyond se model .The following section will discuss a summary of the expanding body of literature on various other CAR T cell control strategies and their combinations that do not fall into the previous chapters. Most discussed strategies employ modular two-component switchable antigen receptors, often referred to as “adaptor CARs” or “universal immune receptors” Fig. . The firThe Fc Receptor (FcR)-based CARs belong amongst the first reported two-component CAR platforms. The FcR-based CARs utilize the natural binding of FcγRIIIa (CD16) to Fc parts of IgG antibodies combined with inherent FcR signaling properties to activate T cell cytotoxic functions via ADCC pathway . MoreoveSimilarly, experiments with the second-generation FcR CARs further modified with costimulatory signaling domains of CD28  or 4-1BBMany other approaches rely on combining existing therapeutic mAbs with universal transmembrane signaling receptors to create mAb-coupled CARs. Since the antibodies are usually tagged with small molecules or peptides for binding to CAR, this CAR technology is also termed Anti-TAG CAR. Compared to FcR CARs discussed in the previous section, Anti-TAG CARs provide another layer of specificity due to the more restricted interaction of tagged mAbs with the signaling receptor present only in engineered therapeutic cells.Anti-Fluorescein isothiocyanate (Anti-FITC) CAR is a conventional CAR with specificity for FITC-labelled therapeutic antibodies, which mediate T cell targeting to TAAs [The  to TAAs , 145–148 to TAAs . Interes to TAAs , 146. Fu to TAAs , 148.Another level of Anti-FITC CAR modulation was recently achieved by including ortho-nitrobenzyl ester photocleavable linker into folate-FITC module  or by coBiotin-binding Immunoreceptor (BBIR) CAR and Monomeric Streptavidin 2 (mSA2) CAR platforms based on strong interaction properties of biotin to avidin or its variants [AdCAR system, utilizes a biotinylated antigen-targeting adaptor and CAR receptor derived from mBio3 scFv, which recognizes biotin in the context of a specific linker. This strategy applied in primary T cells and NK-92 cells was highly effective in eliminating lymphoma cells in vitro and in vivo [Other examples of anti-TAG CARs include variants –153. In variants . Another in vivo , 156. In in vivo .Peptide neoepitope (PNE) specific CARs [Convertible CAR (cCAR) technology leverages the robust binding affinity between the inert form of NKG2D receptor ectodomain to its cognate ligand MICA to engineer a bispecific antigen-targeting adapter molecule (MicA-body™), which can also serve as a vehicle for targeted delivery of cytokines in vitro and in vivo [+ reservoir T cells in vitro and ex vivo [In addition to chemical modifications with FITC or biotin, antibodies, antibody fragments, or other tumor-targeting receptors can be engineered with non-immunogenic small peptide tags for Anti-TAG CAR systems such as fic CARs , 158–160 in vivo . The cCA in vivo  and to e ex vivo .Chemically programmed antibody fragment (Cp-Fab) CAR [Interestingly, a proof-of-concept study adapted PNE CAR technology to enable the targeting of the pocketome fraction of cancer cell surface receptors as Fab) CAR . In thisFab) CAR .SpyCatcher CAR is based on the SpyCatcher-SpyTag chemistry, which allows a rapid and spontaneous post-translational covalent attachment of targeting ligands to universal CARs. The SpyCatcher CAR T cells were successfully tested in vivo in combination with Herceptin-SpyTag targeting Her2-positive ovarian cancer cells SKOV3 [SNAP-CAR, achieves covalent bonding of CAR with mAbs using the SNAPtag self-labeling enzyme present in the extracellular part of CAR, which reacts with benzylguanine (BG)-conjugated mAbs [Finally, two technologies were employed to link therapeutic mAbs to universal CAR receptors covalently, and both represent up-and-coming innovations of universal Anti-TAG CARs –167. Thels SKOV3  and antils SKOV3 . The othted mAbs , 167. Thted mAbs , 166. Moted mAbs , 165. ThIn this final section, a summary of the development of CAR technologies, most often referred to as “universal”, including UniCARs, RevCARs, and SUPRA CARs will be discussed. However, these universal CARs can be considered part of the Anti-TAG CAR category as they share similar overall modular CAR logic.UniCAR represents the first reported and most extensively tested universal CAR technology [+ tumor cells [chnology , 21, 168chnology . The solchnology –172. Thechnology . The Unichnology –175, CD1or cells , carcinoor cells , 178, EGor cells , 180, CDor cells , and proor cells , 183. Inor cells .RevCAR, was established using a fully humanized bispecific targeting module (RevTM) and an overall smaller size than UniCARs [A similar switchable, split, and adaptable universal CAR platform, termed  UniCARs . Like ot UniCARs , includi UniCARs  and glio UniCARs .Split, universal, and programmable (SUPRA) CAR technology also integrates several improved comprehensive features to enhance the specificity, safety, and cell-specific programmability of CARs [ of CARs . The SUP of CARs . Later,  of CARs .The field of cellular immunotherapy has seen significant progress in recent years, with the initial clinical success of CAR T cell therapies in hematological malignancies leading to a surge of follow-up research. The focus has been on developing new gene engineering technologies for improved control over therapeutic cells after administration to the patient and overall product safety, efficacy, and flexibility. As is summarized in the respective sections of this review and Table The ability to target multiple antigens with a single CAR T cell population allows for genetic cell programming to perform Boolean logic operations , and some new multicomponent technologies are being developed such as the colocalization-dependent protein CAR system (Co-LOCKR) . In addiMany of these advances were greatly facilitated by splitting the original CAR architecture into the soluble antigen recognition module and universal membrane receptor in various adaptor and universal immune receptor platforms , 20, 22.https://clinicaltrials.gov). In addition to conventional T cells, several other immune cell types, including NK cells and γδ T cells, are being successfully explored for cancer immunotherapy [Another aspect of cellular immunotherapy rapidly developing in parallel with advanced CAR technologies aims to produce superior and more affordable allogeneic “off-the-shelf” therapeutic cells enhanced by gene editing technologies –196. Theotherapy –205.This review article aimed to comprehensively cover novel gene engineering technologies applied to control CAR T cells, and improve virtually all aspects of the original CAR design. Due to space limitations, the discussion could not include all studies and some other technologies, including the use of bispecific antibodies –208 and


Protein kinases (PKs) represent one of the most important targets in the discovery of new drug candidates in oncology based on their crucial roles in the processes of cellular growth and proliferation . Kinase However, most of the currently available kinase inhibitors were recorded to be less effective over time, especially in the treatment of complex and mutagenic tumors due to their design to act on a single target. On the contrary, multitargeted kinase inhibitors were observed to be more effective due to their ability to inhibit multiple pathways in cancer cell proliferation which results in decreasing the possibility of drug resistance development .https://www.frontiersin.org/research-topics/33242/recent-advances-in-the-research-and-development-of-kinase-inhibitory-anticancer-molecules.Herein, four research articles were published on this Research Topic received from China, Sudan, Saudi Arabia, and Germany. Tian et al., inhibitors isolated from Zanthoxylum simulans were reported as inhibitors of multiple janus kinases (JAKs). The authors developed JAKs based affinity ultrafiltration method coupled with LC/Q-TOF-MS to enable them to study the affinity of Z. simulans quaternary alkaloids against JAKs. They discovered that Berberine and Chelerythrine exhibited superior selectivity against JAK1, JAK2, and JAK3 over Tyk2. Chelerythrine demonstrated promising antiproliferative activity against AGS gastric cancer cells. Both Berberine and Chelerythrine showed obvious inhibition against LO2 human hepatocyte cells. Chelerythrine-mediated inhibition and apoptosis against AGS cells happened via the Estrogen Pathway. This is in addition to direct inhibition of JAK1, JAK2, and JAK3 kinases. The authors studied the putative binding interactions of the quaternary alkaloids to reveal the reasons for the selectivity towards the three JAK isozymes.Not only synthetic medicinal chemistry contributes to the discovery and development of kinase inhibitors, many natural compounds also have been reported as kinase inhibitors. In the original research article of Mohamed et al. reported another contribution of natural products to kinase inhibitors discovery. In silico studies including molecular docking, molecular dynamic simulation, and pharmacophore modeling led to discovery of dual MNK/PIM as potential antiproliferative candidates for treatment of acute myeloid leukemia. The authors started their study with pharmacophore modeling of ligands bound to MNK-2 and PIM-2 crystal structures, and the obtained pharmacophoric features were screened against 270,540 natural compounds from ZINC database. The matched natural compounds were docked into the binding sites of MNK-2 and PIM-2 kinases.The other article authored by Fu et al. reported the design and synthesis of a series of flavone-based cyclin-dependent kinase 1 (CDK1) inhibitors. The most potent CDK1 inhibitor among this series exhibited higher inhibitory activity against RAW 264.7 than MCF-7 cells. This hit compound can be useful for further optimization towards antiproliferative and anti-inflammatory candidates development. The authors studied the putative binding interactions of the most active inhibitor with CDK1 crystal structure.The original research article by Heine et al. , where the authors investigated the effect of alectinib and crizotinib on human monocyte-derived DCs (moDC) as the most powerful antigen-presenting cells. Crizotinib-treated DCs were observed to reduce the activation markers such as CD83, chemokine-guided migration, antigen uptake, and pro-inflammatory cytokines, especially Interleukin-12. These effects were superior to those of alectinib and therefore crizotinib could dampen the anti-tumor immunity more effectively. Briefly, crizotinib alone was confirmed to have immunosuppressive effects on DCs phenotype and reduced DC function, accordingly potentially impairing anti-tumor immunity.The fourth published article entitled


Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation-induced DNA double-strand breaks (DSBs) in human cells and is essential for the generation of mature T and B cells in the adaptive immune system via the process of V(D)J recombination. Here, we review how recently determined structures shed light on how NHEJ complexes function at DNA DSBs, emphasizing how multiple structures containing the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) may function in NHEJ. Together, these studies provide an explanation for how NHEJ proteins assemble to detect and protect DSB ends, then proceed, through DNA-PKcs-dependent autophosphorylation, to a ligation-competent complex. Non-Homologous end-joining (NHEJ) is the major pathway for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in both quiescent and dividing human cells . NHEJ isFor mechanistic purposes, NHEJ can be considered to occur in three stages, (i) detection of the DSB, (ii) processing of IR-induced damaged or modified DNA ends to make them suitable for ligation, and (iii) ligation of the broken DNA ends. The main players in NHEJ have been known for many years, but structural insights into how these proteins interact to form large, multiprotein complexes have only recently begun to emerge . In the Detection: DSB ends are detected by the Ku70/80 heterodimer which forms a basket-shaped structure with a preformed ring that encircles a molecule of double stranded (ds)DNA . In addiProcessing: IR leads to multiple forms of DNA damage including DNA ends with non-ligatable groups. Thus, before DNA ends can be re-ligated, this damage must be repaired and ligatable end groups restored. DSB end processing is thought to involve polynucleotide kinase phosphatase (PNKP), which has 5′-DNA kinase and 3′-DNA phosphatase activity  and inteLigation: DSB end ligation in NHEJ requires DNA-Ligase IV which exists in complex with a homodimer of XRCC4. XRCC4 is composed of a globular head domain, a coiled–coil stalk and a disordered C-terminal tail , and intHuntingtin, Elongation Factor 3, A subunit of protein phosphatase 2A, Target of Rapamycin/TOR) domain (residues 1–891), a central HEAT region, often termed the circular cradle (residues 892–2799) and a C-terminal kinase domain (residues 3580–4100) that is flanked by conserved FAT  and FATC  domains . In. In64]. Recently, elegant mutational studies by Meek and colleagues suggest that the LR complex and the domain-swap dimer support opposite end processing activities, with the LR complex promoting fill-in end processing, while the domain-swap dimer promotes DNA end resection . They prTogether, these new structural studies suggest that DNA-PKcs can participate in multiple complexes, adopting many different orientations and conformations. Moreover, higher-order structures composed of multiple DNA-PK-containing complexes have also been observed  suggestiIn summary, the past few years have yielded a plethora of exciting and informative NHEJ structures that provide the first glimpses into how NHEJ proteins assemble at a DSB, tether the DSB ends and subsequently make them accessible for ligation. Nevertheless, many questions remain. For example, when and how are the processing enzymes recruited to the NHEJ complex? For example, the FHA domains of PNKP and APLF interact with XRCC4  while APFinally, as a protein kinase required for repair of IR-induced DSB through NHEJ, DNA-PKcs is an attractive target for therapeutic intervention using small molecule inhibitors . MoreoveNon-Homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation-induced DNA double-strand breaks (DSBs) in human cells. A better understanding of NHEJ has major implications for improving treatment of cancer patients with radiation therapy and chemotherapy as well as better understanding other repair and non-repair processes.Although individual components have been identified and well-studied, how NHEJ proteins combine and interact at sites of damage to repair DSBs has been unclear. Here, we discuss recent structural advances that show how NHEJ proteins assemble at DSBs, and reveal how DNA-dependent protein kinase catalytic subunit (DNA-PKcs) autophosphorylation regulates the transition from protected to ligatable DNA ends.These new studies reveal how DSB ends are detected, protected and ligated during NHEJ. How processing enzymes that repair ionizing radiation (IR)-damaged DNA ends are recruited to and function at these newly described NHEJ machines remains to be determined.


The intestinal epithelial Caco-2 cell monolayer is a well-established in vitro model useful for predicting intestinal drug absorption in humans. Coculture models of Caco-2 and goblet-cell-like HT29-MTX cells have been developed to overcome the lack of a mucus layer; however, those models are much leakier compared to the intestinal epithelium. Here, we developed a partially laminated culture model where HT29-MTX cells were superimposed onto a Caco-2 monolayer to overcome this issue. A morphological study showed that the piled HT29-MTX cells were voluntarily incorporated into the Caco-2 monolayer, and mucus production was confirmed via periodic acid-Schiff and mucin protein 2 staining. Permeability was evaluated in terms of transepithelial electrical resistance (TEER) and the apparent permeability of paracellular markers with different molecular sizes. The partially laminated model maintained the high barrier function of the Caco-2 monolayer, whose permeability appeared adjustable according to the HT29-MTX/Caco-2 cell ratio. In contrast, the coculture models showed abnormally high permeability of those markers, correlated with low TEER. Thus, the partially laminated model enabled in vitro recapitulation of effective mucosal barrier function. Consequently, this novel model may be useful as an in vitro high-throughput evaluation system for enteral mucosal permeability and mucus-penetrating efficiency of drugs and nanocarriers. Culture monolayers of Caco-2 intestinal epithelial cells derived from human colorectal adenocarcinoma have been used for decades as the golden standard model of the small intestinal epithelium to predict oral drug absorption in humans ,2,3,4. CThe mucus layer covering the gut epithelial surface functions as a barrier to certain drugs and delivery systems, as well as microorganisms and toxins . Goblet ® plate to reduce the required culture time to three days and simplify the Caco-2 cell culture -L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester during the first six days of semi-wet interface with mechanical stimulation increased the mucus layer thickness to 25–30 μm for the 28 days of incubation. This report indicates that the incubation time and stimulation in our study were inadequate for the formation of a mucus layer. We examined mucus production in the partially laminated model using western blotting; however, glycosylated MUC2 enhancement was difficult to detect in the whole-cell lysate and supernatant, although we could observe the mucus layer via PAS staining and MUC2 immunostaining. Consequently, further studies are required to improve the partially piled model in terms of mucus layer thickness.With respect to mucus production, PAS staining and MUC2 immunostaining revealed the mucus layer in the partially laminated model. The thickness of the mucus layer can affect intestinal absorption ,16,30,31Papp with Caco-2 monolayers [In a recent study, Sahoo et al. reported successful fabrication of canine-driven three-dimensional (3D) organoid monolayers and compared them using nolayers . Human onolayers . This coWe developed a simple intestinal epithelial model, where HT29-MTX cells are partially laminated on a Caco-2 monolayer, resulting in a physiological structure with higher practical permeability than the conventional coculture models. It is suggested that the partially laminated model could be used to evaluate intestinal mucosal permeability of biologics and formulations, such as mucus-permeable nano-delivery systems, in a high-throughput screening process. In addition, the partially laminated technique may provide a new strategy for constructing a hybrid cell layer with different cell types in 3D models, although further intensive research is required to validate the in vitro assessment model of intestinal drug permeability.


SEL). Proteomics demonstrated a highly significant representation of cardiovascular development functions and angiogenesis in CPC exosomes, and RNAseq analysis yielded about 350 different miRNAs; among the exo-miRSEL population, miR-935 was confirmed as the miRNA most significantly up-regulated; interestingly, miR-935 was also found to be preferentially expressed in mouse primary cardiac Bmi1+high CPC, a population highly enriched in progenitors. Furthermore, it was found that transfection of an miR-935 antagomiR combined with oxidative stress treatment provoked a significant increment both in apoptotic and necrotic populations, whereas transfection of a miR-935 mimic did not modify the response. Conclusion. miR-935 is a highly differentially expressed miRNA in exo-miRSEL, and its expression reduction promotes oxidative stress-associated apoptosis. MiR-935, together with other exosomal miRNA members, could counteract oxidative stress-related apoptosis, at least in CPC surroundings.Oxidative stress-induced myocardial apoptosis and necrosis are critically involved in ischemic infarction, and several sources of extracellular vesicles appear to be enriched in therapeutic activities. The central objective was to identify and validate the differential exosome miRNA repertoire in human cardiac progenitor cells (CPC). CPC exosomes were first analyzed by LC-MS/MS and compared by RNAseq with exomes of human mesenchymal stromal cells and human fibroblasts to define their differential exosome miRNA repertoire (exo-miR Stem cell therapy is being developed to promote repair and eventually regenerate the damaged heart, but currently its putative therapeutic effect is hampered by poor engraftment/survival of transplanted cells ,2. CardiIt is accepted that a substantial part of the CDC/CPC repair effects are mediated by secreted paracrine factors . These fEach exosome source demonstrates a different composition, but probably the most relevant source of regulatory signaling is associated with their RNA content, including mRNA, microRNA (miRNA), and long non-coding RNA . In partCPC- and CDC-derived exosomes are enriched in some cardioprotective miRNAs, such as miR-146a-3p (reviewed in ). A largHere, we have thoroughly analyzed the preferential proteins (LC-MS/MS) and miRNA exosomal repertoire of human CPC. We have identified miR-935 as a highly differentially expressed miRNA in CPC compared with other reference cell lineages; in agreement, miR-935 is also preferentially represented in murine CPC. Moreover, its functional evaluation demonstrated the induction of oxidative stress-associated apoptosis after its inhibition. Hence, miR-935, combined with other exosomal miRNAs, could act together to counteract oxidative stress-related apoptosis in different contexts.2/5% CO2 atmosphere, which mimics closer physiologic conditions and reduces the senescence evolution of the cultures  software .It was carried out as previously described . In brieIMM or CPC-derived exosomes  were co-cultured with target cells  on subconfluent cultures (seeded at 4 × 104 cells/cm2 on 0.1% gelatin-treated plates) and compared with appropriate controls. B-CPCIMM-conditioned medium was obtained as previously described . In. InIMM).partment C. AimingMM cells . We confd medium D. Finalld medium D. ResultGene Ontology (GO) analysis of the functional categories of target genes associated with the mouse E and hum2O2 treatment (20–30 mM); these treatments did not provoke a reduction in miR-935 levels but a moderate increase (1.5–2 fold) in both compartments, although not statistically significant  F,G. Resunificant A; these nificant B.2O2 during 48 h). Results clearly indicated that whereas miR-935 mimic overexpression did not modify the response to oxidative stress damage, compared with controls (+ PI−) and necrotic (Annx+ PI+) populations (+ PI−) but again, transfection of the miR-935 mimic did not provoke any significant effect on survival upon exposure to H2O2 . Next, using a protein array designed for detection of proteins involved in apoptosis, we compared HL-1 cells subjected to oxidative stress, pre-transfected with INH, and transfected with the scramble construct. Results found that 70% of proteins included in the array were upregulated in HL-1 cells pre-transfected with INH; only BCL-W was found to be moderately downregulated  transfection, compared with controls (scramble), cells were exposed to oxidative stress , cIAP2 (6.5-fold), CASP8 (6.5-fold), and DR6 (5-fold) D. Most oThese results, therefore, demonstrated that under moderate oxidative damage in several cell lines, miR-935 levels were not significantly modulated, but reduction of miR-935 levels was associated with the induction of oxidative-stress-associated apoptosis. This suggests that miR-935 could be participating in the pathway(s) involved in antioxidant stress defense.Several miR-935 targets, evaluated in other cellular models or predicted in silico, were defined . We seleARNT2, NOTCH1 and CASP10 did not show a significant reduction, and SOX7 expression seemed not to be affected (ARNT2 and SOX7); CASP10 showed a similar response, but it was not statistically significant. On the contrary, NOTCH1 did not show modulation by miR-935 mimic overexpression. In addition, miR-935 INH transfection in HDF cells only promoted a moderate trend to up-regulation for the four target genes (1.5–3.5 fold) but was not statistically significant  to assist in the results’ interpretation. Results demonstrated that all of them were significantly up-regulated upon oxidative stress induction  but was non-statistically significant. These results could be related to the low level of up-regulation of NOTCH1 and ARNT2 provoked by oxidative damage in HDF  could participate in the prevention of apoptosis/necrosis observed in HL-1 and human cardiac fibroblasts upon inhibition of miR-935 in the context of oxidative damage B,C.Based on the previous data obtained with the human model of CPC, the confirmed high levels of expression in murine cardiac progenitors (B-CPC cells), and the clear reduction in miR-935 levels after AMI, especially in CM B, we aimWe hypothesized that partially counteracting the severe reduction of miR-935 levels as a consequence of AMI could positively impact cardiac recovery. The vector AAV-TnT-miR935 (AAV-935) and a control vector (AAV-control) that expresses a scrambled miRNA sequence were used D. In theA significant functional improvement was not found after miR-935 treatment, despite the fact that a slight positive trend in cardiac EF was observed after 2 months of miR-935-AAV treatment in comparison with the control group F. All thHuman adult cardiac progenitors (CPC/CDC) promote functional improvements upon transplantation in large infarcted animals ,5,6. MosSEL has the potential to regulate a relevant number of transcription factors  were found, and bioinformatic analysis defined more than 2200 genes as potential targets of exo-miR, including Actin Alpha Cardiac Muscle 1 (ACTC1) and Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C), regulated with high probability by 5 and 7 exo-miR, respectively (unpublished data). In addition, exo-miR factors . Finally cancers .SEL elements displaying antagonistic roles, most probably depending on the specific futures of each cell type and even conditioned by external signals .Taking together both proteomic and mRNA signatures, CPC-exosomes showed a highly significant angiogenic profile and plausible, direct or indirect, involvement in cardiovascular physiology regulation and repair, contributing to heart repair after damage. A first conclusion noted a high variable regulation potential but with several miRNAWithin the exo-miR population, 58 were preferentially represented in the CPC exosomal compartment. MiR-2110 and miR-1911-3p were those that showed the highest exo/cell ratio, being considered almost specific for the exosomal compartment . Both haSEL in exosomes with the whole cell levels (exo/cells ratio) indicated that miR-1268 is almost exclusively found in the exosomal compartment, but no specific function has yet been ascribed to miR-1268, although some weak association has been reported in prostate cancer [SEL but with a low-to-medium absolute level of expression. Concerning miR-625-3p, serum levels have only been linked with cholesterol levels in the metabolic syndrome and the cardiovascular field [Comparison of the levels of expression of exo-miRe cancer . Interese cancer . In our ar field . In addiar field ; more rear field . Finallyar field .SEL population showed that the above-mentioned code fit well with the miRNA with the highest differential representation in CPC exosomes. The analysis of miRNAs that are highly overrepresented in CPC relative to MSC/HDF, independently of their preferential sorting to exosomes  by which miRNAs get sorted into exosomes or retained in cells are not fully understood. Recently, it has been demonstrated that miRNAs possess sorting sequences (EXOmotifs) that facilitate their secretion loading in exosomes in a cell-dependent way. The global conclusions stated that miRNAs that display a higher CG content have a higher probability of being enriched in exosomes . The anaexosomes , confirmon miRNA .SEL, we focused our study on miR-935, which showed the highest differential expression ratio between CPC and MSC/HDF, both represented in the whole cell and the exosomal compartment (exo-miRSEL). Altered levels have been again associated with several human cancer types, mostly relating miR-935 overexpression with malignant stages [Then, as a first attempt to characterize the functional role of some exo-miRt stages . In ostet stages . Therefot stages . This adt stages .In our cell system, however, we showed that manipulation of miR-935 levels did not significantly affect proliferation or survival in different cell lineages. Gene Ontology bioinformatic analysis also suggested a potential implication in regulating cell death in response to oxidative stress. Therefore, we explored the potential role of miR-935 under oxidative stress conditions, finding a clear implication in the responses of several cell lineages. These results suggested that miR-935 could be a relevant actor for an anti-apoptotic response associated with moderate oxidative stress. Protein array analysis on HL-1 cells confirmed that transfection of the miR-935 inhibitor (INH) coupled to moderate oxidative stress provoked a significant upregulation of an important number of apoptosis mediators. It is worth considering that miR-935 levels in CPC are high and are not reduced by moderate oxidative stress, neither in whole cells nor exosomes; this could be an interesting attribute for participating in such an anti-oxidative stress pathway. A few additional examples of exosomal miRNA and circular RNAs, both cardiac or from heterologous cells, have been demonstrated to participate in regulating oxidative stress-related apoptosis (reviewed in ). ConcerNOTCH1 showed a trend to be upregulated upon miR-935 inhibitor (INH) transfection. Transfection with miRNA-935 mimic (25 nM) significantly reduced the expression of NOTCH1 and ARNT2, with a clear similar trend for SOX7  were evaluated in HDF under oxidative stress conditions C. Only Nfor SOX7 C. The evfor SOX7  and undefor SOX7 C, demonsNotch1 has a critical function during development, but in adult hearts, Notch1 signaling seems to be only activated in response to several forms of damage, favoring angiogenesis and demonstrating cardioprotective effects [In mouse myocardium,  effects . Therefo effects , so the  effects .Aryl Hydrocarbon Receptor Nuclear Translocator 2 , regulates the translocation of the ligand-bound aryl hydrocarbon receptor (AhR) complex to the nucleus, modulating sensitivity to apoptosis ,71,72. WInterestingly, a link between NOTCH1 and ARNT2 has been proposed that depends on AhR heterodimerization with ARNT or ARNT2 for signaling (through IL22), both in homeostasis or in acute inflammation and regeneration. NOTCH1 favors expression of ligands (ARNT/ARNT2) for the aryl hydrocarbon receptor (AhR) (reviewed in ) TherefoCASP10/Casp8 can be directly associated with the phenotype described. Upon oxidative stress induction, CASP10 expression is highly augmented in HDF (more than 2 × 104 fold), so the level of inhibition provoked by mimic overexpression, although not statistically significant, could be considered a relevant response with regard to the modulation of apoptosis/necrosis coupled to oxidative stress.Due to the profiles obtained in HDF (pre-transfected with mimic or INH), in homeostasis, and after the induction of oxidative stress, only the expression profile of Aiming to confirm the proposed role for miR-935 in B-CPC  we foundInterestingly, Zhang et al. reported, in a rat osteoporosis model, a significant decline in miR-935 levels associated with ovariotomy; reconstitution of miR-935 levels reduced the severity of osteoporosis . Based oOur study, therefore, possesses some limitations, as we could not confirm the expected in vivo cardioprotective role of miR-935 when injecting the AAV-935 vectors into the ischemic hearts. Probably, the combination of miR-935 with other described cardioprotective miRNAs such as miR145-5p ,57, and/SEL) has been defined in human CPC. MiR-935 has been identified as one of the most overrepresented miRNAs in exo-miRSEL, defined by comparison with reference cell lineages; this profile has also been confirmed in murine cardiac progenitors (B-CPC). In vitro functional evaluation strongly suggests that miR-935 plays a relevant role in the oxidative stress defense response, and its inhibition, combined with oxidative stress regimens, promotes apoptosis/necrosis in several cell lineages, although with significant differences among them.A preferential exosomal miRNA repertoire (exo-miR


Obsessive-compulsive disorder (OCD) is a frequent and debilitating mental illness. Although efficacious treatment options are available, treatment resistance rates are high. Emerging evidence suggests that biological components, especially autoimmune processes, may be associated with some cases of OCD and treatment resistance. Therefore, this systematic literature review summarizing all case reports/case series as well as uncontrolled and controlled cross-sectional studies investigating autoantibodies in patients with OCD and obsessive-compulsive symptoms (OCS) was performed. The following search strategy was used to search PubMed: “(OCD OR obsessive-compulsive OR obsessive OR compulsive) AND (antib* OR autoantib* OR auto-antib* OR immunoglob* OR IgG OR IgM OR IgA)”. Nine case reports with autoantibody-associated OCD/OCS were identified: five patients with anti-neuronal autoantibodies  and four with autoantibodies associated with systemic autoimmune diseases . Six patients (67%) benefited from immunotherapy. In addition, eleven cross-sectional studies  were identified with inconsistent results, but in six studies an association between autoantibodies and OCD was suggested. In summary, the available case reports suggest an association between OCD and autoantibodies in rare cases, which has been supported by initial cross-sectional studies. However, scientific data is still very limited. Thus, further studies on autoantibodies investigated in patients with OCD compared with healthy controls are needed. Approximately 2% of the population worldwide suffer from obsessive-compulsive disorder , freThere is increasing evidence that biological components have substantial influence on the development of this disorder. A moderate genetic component with heritability estimates ranging from 27 to 65% has been reported , 14. BesAn autoimmune hypothesis in some cases was initially postulated based on an association between OCS in children and their exacerbation after infections with beta-hemolytic streptococci . This suOriginal research findings comprising case reports, case series, uncontrolled and controlled cross-sectional studies of patients with OCD or OCS reporting on autoantibodies detected in blood/and or cerebrospinal fluid (CSF) were included. Patients of all ages were analyzed, and no restrictions were placed on the methodology of autoantibody testing.Preliminary results as well as case reports/series and cross-sectional studies reporting findings of patients with OCD and comorbid Tourette syndrome were excluded. Studies conducted exclusively in animals or with pathogen-associated antibodies  were ruled out. Furthermore, all articles that were published in languages other than English or German were excluded.The aim was to provide a descriptive presentation of autoantibody-associated case reports of patients with OCD, along with the findings from all cross-sectional studies.The literature search was performed in PubMed in line with the PRISMA guidelines using the following search terms: “(OCD OR obsessive-compulsive OR obsessive OR compulsive) AND (antib* OR autoantib* OR auto-antib* OR immunoglob* OR IgG OR IgM OR IgA)”. All articles available until 17 February 2021 were searched. Titles and abstracts of all articles were screened independently by two expert raters (DD and KR). Subsequently, a full text analysis was conducted for papers that met the eligibility criteria. Disagreements were resolved by a third reviewer or consensus-based discussion. Additionally, references of literature reviews or meta-analyses specific to the immunological topic of the current literature search were screened for additional eligible references. In total, 13 reviews , 33–43, The literature search resulted in a total of 473 reports. After screening, nine case reports –52 and eIn the identified nine case reports, three patients with well-characterized anti-neuronal autoantibodies , two patients with non-well-characterized anti-neuronal autoantibodies , and four with autoantibodies associated with systemic autoimmune diseases  were described. The detailed findings are summarized in Table Seven patients in a case series with anti-NMDA-R encephalitis developed initial cognitive deficits and persisting impairment of neuropsychological function (mean follow-up = 35 months). One out of these seven patients developed persistent OCS .OCS where the patient arranges and frequently rearranges objects in distinct lines, were the first symptoms diagnosed in a 69-year-old woman. Four weeks later, she showed initial neurological symptoms  that steadily deteriorated until her death. Immunological treatment was unsuccessful. Brain autopsy revealed global cerebral amyloid with extensive neuronal loss and perivascular/parenchymal infiltration of T-lymphocytes limited to the striatum. Anti-CV2 autoantibodies were positive in the serum after her death. A tumor was not found, however, the autopsy was limited to the brain only .OCS consisting of checking central heating, light, doors, and so forth were the first signs of paraneoplasia in a 39-year-old male who later on started to develop severe neurological symptoms . Orchiectomy revealed a differentiated teratoma and seminoma in situ. Thoracic computer tomography (CT) confirmed metastasis of the teratoma. Magnetic resonance imaging (MRI) of the brain showed a T2 hyperintense signal alteration in the left hippocampus. EEG indicated left temporal sharp slow-wave activity, and laboratory analysis (CSF and serum) identified anti-Ma2 autoantibodies. Neither surgical removal of the pulmonary metastasis nor chemotherapy (bleomycin/cisplatin/etoposide) yielded clinical improvements in contrast to the antiepileptic and immunological treatment .A 64-year-old man without comorbidities presented with sudden OCS related to his garden . He additionally developed faciobrachial dystonic seizures. Anti-VGKC autoantibodies were significantly elevated in the CSF, whereas the other parameters were normal. Metabolic brain imaging with 18F-Fluorodeoxyglucose positron-emission tomography (FDG-PET) revealed increased symmetrical uptake in the caudate and lentiform nuclei while no association between seizures and EEG alterations were detected. After treatment with cytostatics and prednisolone, complete remission of OCS and seizure symptoms was observed, along with normalization of anti-VGKC autoantibody levels and reduced FDG uptake in the basal ganglia .Zhu and colleagues  describeOCS with psychotic tendencies were observed in a 22-year-old male patient who had ANAs with anti-nucleosome specificity detected in his blood and CSF accompanied by abnormalities in the MRI  and EEG (slowing); improvement was reported by methylprednisolone, methotrexate, and hydroxychloroquine .A 40-year-old female patient with xerostomia and xerophthalmia presented with OCS that included washing, checking behavior, and fear of contamination. The patient was diagnosed with OCD, and treatment with CBT and psychotropic drugs (fluoxetine/risperidone/aripiprazole) was initiated. Furthermore, the diagnosis of Sjögren syndrome was ascertained based on increased titers of ANAs with anti-Ro/SS-A and -La/SS-B specificity, a positive Schirmer test, and positive lisamin green staining. Ultrasound and scintigraphy showed dysfunction of the salivary glands. Both her rheumatological symptoms and OCS improved significantly after treatment with hydroxychloroquine and nutrition supplements .Ong and colleagues (2017) described the case of a 17-year-old woman who initially presented with symptoms of OCD that included contamination fears, hoarding, symmetry, washing, cleaning and checking, and comorbid depression, which was managed with a low dosage of psychotropic drugs  and psychotherapy. Subsequently, she developed severe neuropsychiatric symptoms  and was additionally diagnosed with Sjögren syndrome based on the positive titers of ANAs with anti-Ro/SS-A and anti-La/SS-B specificity. Magnetic resonance spectroscopy revealed a reduction in N-acetylaspartate in both hippocampi, while brain MRI presented insignificant findings. The CSF displayed pleocytosis, elevated protein, increased neopterin, immunoglobulin G (IgG) elevation, and a significant increase of CSF IgG to albumin ratio and oligoclonal bands, while anti-neuronal autoantibodies were negative. OCS completely disappeared with immunotherapy using plasmapheresis and intravenous methylprednisolone .A 5-year-old girl developed rapid-onset OCD after an ear infection without evidence of streptococcal infection, but the patient had elevated anti-phosphatidylethanol-amine autoantibodies and the antibody levels correlated with OCS. Immunotherapy was not administered since she improved with low dose sertraline .The findings are summarized in Table N = 48) comprising seven patients with OCD and 52 healthy controls, two out of seven patients with OCD were positive for autoantibodies against Purkinje cells, for which all healthy volunteers were negative. Overall, Purkinje cell antibodies were identified in 11 of 48 patients (22.9%) with mental disorders but in 0% of controls / thyroglobulin [Tg]/ and streptolysin O [ASO]) were screened in a study of 40 OCD patients and compared to 14 patients with other mental disorders including mood, adjustment, psychotic, or anxiety disorders. Furthermore, OCD patients were divided into two subgroups based on disease onset (early- vs. late-onset OCD). No significant differences in autoantibody prevalence emerged between OCD subgroups and controls. However, early-onset OCD was associated with higher anti-streptolysin O levels .This systematic literature review examined the association between OCD/OCS and different autoantibodies. It includes all articles on this topic published until February 2021 to provide a comprehensive and broad overview. Patients with PANDAS and PANS patients were not content of the paper. Nine case reports of autoantibody-associated OCD have been published, of which six patients (67%) benefited from immunotherapy. Six of eleven cross-sectional studies have also provided evidence for an association between autoantibodies and OCD.A total of five patients with OCS described as case reports had anti-CNS autoantibodies , 50, 52;Previous cross-sectional studies have mostly focused on autoantibodies associated with dysfunction along the cortico-striato-thalamo-cortical circuits and serotonin or dopamine receptor pathways, based on established OCD pathophysiology. In this context, several studies investigated ABGA , 60, 62,Four case reports with autoimmune forms of OCD in the context of systemic autoantibodies were identified , 49, 51.Only PubMed (and the references of included papers and important reviews) were searched. Specific limitations related to the data from patients with OCD in the included studies were as follows: Several studies were lacking data for age and sex , 58, 62,Case reports suggest a possible association of OCS with autoantibodies. Some cross-sectional studies also showed correlations between OCD and different autoantibodies in rare cases, but were mostly conducted with small sample size or were not replicated. Therefore, larger studies analyzing different autoantibodies are needed. The concept of a rare subtype of “autoimmune OCD” should be further investigated as it may open up novel avenues for targeted therapeutic approaches in the future.


COVID-19 infections are known to cause numerous systemic complications including cardiovascular disorders. In this regard, clinicians recently noticed that patients recovering from COVID-19 infections presented with diverse set of cardiovascular disorders in addition to those admitted to ICU (intensive care unit). COVID-19 heart has multifaceted presentation ranging from dysrhythmias, myocarditis, stroke, coronary artery disease, thromboembolism to heart failure. Atrial fibrillation is the most common cardiac arrhythmia among COVID-19 patients. In the background section, we briefly discussed epidemiology and spectrum of cardiac arrhythmias in COVID-19 patients.In this state-of-the-art review we present here, we present the information regarding COVID-19-induced A-fib in sections, namely mechanism of action, clinical presentation, diagnosis and treatment. Unfortunately, its occurrence significantly increases the mortality and morbidity with a potential risk of complications such as cardiac arrest and sudden death. We included separate sections on complications including thromboembolism and ventricular arrhythmias. Since its mechanism is currently a gray area, we included a separate section on basic science research studies that are warranted in the future to comprehend its underlying pathogenic mechanisms.Taken together, this review builds upon the current literature of COVID-19-induced A-fib, including pathophysiology, clinical presentation, treatment and complications. Furthermore, it provides recommendations for future research moving forward that can open avenues for developing novel remedies that can prevent as well as hasten clinical recovery of atrial fibrillation in COVID-19 patients.The online version contains supplementary material available at 10.1186/s43044-023-00359-0. There has been an uptick in the occurrence of atrial fibrillation (A-fib) cases in COVID-19 patients during the recently concluded pandemic. Out of 9574 patients, COVID-19 patients admitted to ICU in Northwell Health System in New York, A-fib developed in 1687 (17.6%) of them and these patients experienced significantly higher in-hospital mortality compared to those with sinus rhythm (55.2% vs 46.8%) . CliniciThe prevalence of arrhythmias in COVID-19 patients admitted to the hospital is around 18% and 15% during admission and discharge, respectively . In thisAtrial fibrillation is the most common cardiac arrhythmia in COVID-19 patients. The incidence of A-fib in COVID-19 patients without and with cardiovascular abnormalities is approximately 19–21% and 36%, respectively . The inc2DS2-VASc score [Demographically, the incidence of A-fib is more common in Europeans 15%) followed by Americans (11%), Asians (6%) and Africans (2%) in COVID-19 patients . RetrospSc score . Multice% followeSc score .Although atrial fibrillation is one of the most common arrhythmias in COVID-19 patients, its exact mechanism is not elucidated definitely yet. Nevertheless, researchers hypothesized some speculative mechanisms for its occurrence which can be summarized as downregulation of angiotensin-converting enzyme 2 (ACE2), CD-147, sialic acid-spike protein interaction, cytokine storm, endothelial damage, electrolyte disturbances, hypoxemia and hyperactive sympathetic nervous system . Loss ofICaL, INa) currents and the same time antagonism of outward repolarizing currents . Fourth, direct invasion of cardiomyocytes can also lead to myocardial inflammation due to combined effect of inflammatory infiltrate, immune response and cytokines [Upon entering into the atrial myocytes, it undergoes replication and provokes multifarious pathological derangements that underlie the susceptibility to cardiac arrhythmias. First, it can impair the intercellular communication between adjacent cardiomyocytes by downregulating the expression of connexins (Cx40 and Cx43), thus leading to derailed impulse conduction and protracted cardiac cycle duration . Second,ytokines  Fig. 1)ICaL, INaytokines . These pIcal  [Due to disturbance of the delicate balance between Th1 and Th2 lymphocytes, cytokine storm is unleashed with production of pro-inflammatory cytokines takes place . Systemicurrent)  current) , 25, 26.Electrolyte disturbances  are considered as one of the most important risk factors for development of tachyarrhythmias including atrial fibrillation  Fig. 1)1). The o+ channels, thereby resulting in lengthening of ventricular depolarization [QT prolongation which can be due to virus itself or due to administration of QTc prolonging drugs for managing these patients can be a likely predisposing factor for initiation of cardiac arrhythmias in these patients  Fig. 1)1). COVIDrization . QT prolrization . Furtherrization .+-current (IKs) and attenuation of inward-rectifier K+-current (IK1) [COVID-19-induced kick starting of central inflammatory reflexes and peripheral stellation ganglia activation can provoke increased sympathetic outflow to the atrial myocardium . Enhancent (IK1)  Major aberrations instigated by viral infections that predispose to occurrence of A-fib can be classified into impairment of atrial electrophysiology and structural remodeling . In thisIn nonvalvular A-fib secondary to infectious inflammatory etiology, researchers demonstrated higher levels of TLR-2 (Toll-like receptor-2) receptors and IL-6 compared to those with sinus rhythm . InfluenAutoimmunity favors the production of auto-antibodies against myosin, M2 receptors, Na+/K+ pump, heat shock proteins and β-adrenergic receptors –46. ThesPrevious studies had shown that AF is more likely to develop in COVID-19 patients compared to non-COVID-19 patients (Odds > 1.19) and pre-pandemic patients (Odds > 1.57) . Some ofThe clinical presentation of atrial fibrillation can be varied with non-specific signs and symptoms. The following table enumerates the age, clinical symptoms, signs, diagnostic tests, treatment and prognosis in patients presenting with COVID-19-induced atrial fibrillation.Atrial fibrillation (A-fib) is characterized by an irregular and rapid heart rate, which can lead to various symptoms and complications. Case studies suggest that the most common symptoms observed among A-fib patients were palpitations, shortness of breath, and fatigue. Many patients also presented with altered mental status that could be a result of the atrial fibrillation. Other less frequently reported symptoms included dizziness, chest pain and syncope. Patients often presented with elevated heart rates (mean of 120 bpm) and irregular rhythms on physical examination. EKG results showed consistent evidence of A-fib in all cases, with some patients also exhibiting additional abnormalities such as atrial flutter, ST-T wave changes and ventricular ectopy.Atrial fibrillation is regarded as an important risk factor for Major Adverse Cardiovascular events (MACE) such as all point death, heart failure, myocardial infarction and stroke . AccordiEmergent need of catheter ablation is warranted in A-Fib with severe symptoms and hemodynamic instability or collapse, A-Fib refractory to cardioversion/drugs/rate control, refractory A-Fib with several emergency department visits and A-fib with syncope or cardiac arrest , 66. On The medications used to control A-Fib should be used cautiously as they can interact with therapeutic drugs used to neutralize the COVID-19 virus and attenuate the associated systemic inflammation. The interactions can be classified as (a) serious interactions needing to modify the drug regimen, (b) moderate interactions necessitating close monitoring and follow-up, (c) minor interactions that required caution while administering drugs and (d) no interactions where medications can be given with no follow-up. These interactions are summarized in the Additional file The presence of COVID-19 infection along with A-fib substantially increases the thrombogenic risk. The underlying risk factors that increased the risk of thrombogenic risk in COVID-19 patients can range from cytokine storm, microvascular inflammation, increased D-dimer levels, upregulated factor VIII levels, loss of endothelial integrity, platelet activation to deposition of platelets in multiple organs . The iniThese pathological events drive atherosclerotic plaque formation, remodeling and endothelial damage, which ultimately leads to athero-thrombosis and thromboembolism . CHADS2 Patients having A-Fib greater than 48 h are managed with 3 weeks of anticoagulation and transesophageal echocardiography before they are subjected to cardioversion . On the The incidence of stroke in patients with COVID-19-induced A-Fib can range from 28 to 33% , 76, 77.Formation of thrombus in the left atrium (LA) and left atrial appendage (LAA) can be sometimes associated with thrombus formation in the right atrium (RA) and right atrial appendage (RAA) . EchocarPulmonary embolism is more likely to develop in COVID-19 patients with cardiovascular risk factors, and atrial fibrillation is the most common cardiometabolic risk factor encountered in them . ActivatA retrospective cohort study conducted uncovered the fact that the presence of A-fib in patients with pulmonary embolism increased the risk of mortality at 1-month and 6-month interval . On the COVID-19-induced A-Fib might secondarily precipitate ventricular arrhythmias including ventricular fibrillation (VF) and ventricular tachycardia (VT) via abbreviated AV node refractory period during exercise, sympathetic overstimulation, ventricular myopathy and genetic variations , 90. In Recently, a patient with protracted COVID-19 infection that presented with chest pain, palpitations and shortness of breath was found to have PVC bigeminy and elimination of these with catheter ablation resulted in successful clinical outcomes . In a prNew-onset A-fib is most commonly associated with Pfizer followed by Moderna and Johnson and Johnson vaccines . AccordiDeposition of fibrous tissue, scar formation and generation of atrial substrate in the atrial myocytes are hypothesized to be some of the most critical driving mechanisms for initiating and sustaining A-Fib. Fundamentally, two main types of fibrosis namely reactive fibrosis and replacement fibrosis can potentially occur in atrial chambers in pathological states  Fig. 5)5). ReactIk1) [Ik1 currents and thus provides a potent stimulus for enhanced calcium entry [Previous studies suggest that calcium entry in atrial fibroblast is very important for their survival and activation of fibrogenic pathways . The maiIk1) . Patholoum entry . Extraceum entry , 108. FuIn vitro study performed to assess the interactions between fibroblasts isolated from infarcted hearts and neonatal myocytes, there is decreased conduction velocity, attenuated action potential duration, hyperpolarization of RMP, increased gap junction expression and increased intercellular coupling between myocytes and fibroblasts  Fig. 5)5). AccorMechanical stretch of cardiomyocytes stimulates the secretion of pro-fibrotic signals such as angiotensin-II, transforming growth factor (TGF)-1β, endothelin-1 (ET-1) and platelet-derived growth factor (PDGF)  Fig. 5)5). Pro-fThe relationship between atrial fibrosis and atrial arrhythmogenicity is complex, but few previous studies tried to partially explain it by intracellular calcium overload, oxidative stress, α-SMA (Alpha- Smooth Muscle Actin) over expression and exaggerated myocyte–fibroblast coupling leading to electrical remodeling, enhanced arrhythmogenesis, ectopic impulse generation and abnormal impulse conduction –119 Fig. So, stuInflammation and oxidative stress has been documented to a play a significant role in regulating the role of enzymes and tissue factors that regulate the synthesis and degradation of collagen matrix deposition in the myocardial tissues . PreviouSince COVID-19 infection of atrial cardiomyocytes is associated with cytokine storm  and oxidA Disintegrin And Metalloproteinase) are membrane bound glycoproteins that are expressed in myocardial tissues (Fig. 1 and β3 might lay the foundations for structural remolding and subsequent dilation in fibrillating atria [ADAMs (ues Fig. . Their mng atria . These fA-fib usually occurs acutely in COVID-19 patients admitted to ICU, particularly in those with high-risk factors. On the contrary, some patients presented with atrial fibrillation few months later after complete recovery from COVID-19 infections. These long haulers are known to harbor silent COVID-19 infections in the systemic tissues including myocardium. Clinicians need to be vigilant of this arrhythmia as it can be associated with many life-threatening complications. Prompt management and close monitoring are necessary as it has high chances of recurrence. COVID-19-induced pathological derangements in the myocardium and systemic circulation in combination are accountable for instigation and persistence of this cardiac arrhythmia. This review summarizes the pathophysiology of COVID-19-induced A-fib along with its clinical presentation, diagnosis, management and complications. Understanding the interplay of various risk factors both extra-cardiac and intra-cardiac as well as cellular downstream signaling events is very crucial. Since this is a relatively clinical entity, there is a lot of scope in research to understand its pathophysiology. We summarized the various areas where basic science and clinical research studies need to be concentrated in the near future. Execution of these research studies can generate very useful preliminary data that can help us to comprehend necessary downstream signaling events that happen at the cellular level for initiation and perpetuation of atrial fibrillation in COVID-19 patients. Moreover, the molecular targets that are divulged by these research studies can be exploited for crafting novel diagnostic and therapeutic interventions for reducing morbidity and mortality of this fatal cardiac arrhythmia.Additional file 1. Drug-drug interactions in the management of Atrial fibrillation encountered in COVID-19 patients.