Adoptive transfer of chimeric antigen receptor (CAR) T lymphocytes is a powerful technology that has revolutionized the way we conceive immunotherapy. The impressive clinical results of complete and prolonged response in refractory and relapsed diseases have shifted the landscape of treatment for hematological malignancies, particularly those of lymphoid origin, and opens up new possibilities for the treatment of solid neoplasms. However, the widening use of cell therapy is hampered by the accessibility to viral vectors that are commonly used for T cell transfection. In the era of messenger RNA (mRNA) vaccines and CRISPR/Cas  precise genome editing, novel and virus-free methods for T cell engineering are emerging as a more versatile, flexible, and sustainable alternative for next-generation CAR T cell manufacturing. Here, we discuss how the use of non-viral vectors can address some of the limitations of the viral methods of gene transfer and allow us to deliver genetic information in a stable, effective and straightforward manner. In particular, we address the main transposon systems such as Sleeping Beauty (SB) and piggyBac (PB), the utilization of mRNA, and innovative approaches of nanotechnology like Lipid-based and Polymer-based DNA nanocarriers and nanovectors. We also describe the most relevant preclinical data that have recently led to the use of non-viral gene therapy in emerging clinical trials, and the related safety and efficacy aspects. We will also provide practical considerations for future trials to enable successful and safe cell therapy with non-viral methods for CAR T cell generation. Chimeric antigen receptor (CAR) T cell therapy represents a revolutionary therapeutic reality. To unleash T cells against cancer, an artificial receptor has been generated fusing the antigen-binding domain of a monoclonal antibody with a T-cell receptor (TCR)-derived signaling domain, including costimulatory components , 2. CAR-To date, the clinical application that has determined the success of CAR T cells has been conducted mainly targeting the CD19 and CD22 molecules in B cell-Acute Lymphoblastic Leukemia (B-ALL) and B cell lymphoma  and agaide novo cytokine production, activating signaling molecules and pro-inflammatory ligands, checkpoint blockages, increased trafficking with chemokines, receptors and extracellular matrix degrading enzyme, safety switches. Some of the issues associated to the current CAR T cell design that we need to face in the future are summarized in , small interfering RNA (siRNA), and guide RNA (gRNA), can be achieved by non-viral transfection. Transfection relies on the formation of transient pores in the cell membrane or, alternatively, endocytosis through the use of different chemical or physical techniques, such as electroporation, liposomes, and nanoparticles.Gene therapy is an advanced medicine application in which the delivery of genetic material into cells is exploited to confer additional or restore impaired features to treat patients with a wide range of diseases, including genetic disorders, cancer, infectious and immunologic diseases, resulting in long-term therapeutic effects. Gene transfer can be classified as stable or transient depending on whether the genetic material is integrated into the host cell genome. To achieve stable gene transfer, integration of an expression cassette consisting of promoter, leader, transgene and transcriptional termination and polyadenylation sequences is needed. When there is integration into the genome, the transgene is stably expressed, and expression of the inserted gene persists in daughter cells resulting from cell division. In contrast, in the absence of integration, as for mRNA and plasmid vectors, expression will be lost as cells divide. Adoptive cell therapy with CAR T cells utilize gal-pol, rev, env coding sequences into the producer cell line by means of separated plasmids. Separation of the sequences required for virus formation allows the generation of a replication-deficient virus that is capable of infecting mammalian cells and integrate the genetic materials into the cellular genome but does not retain the natural ability to generate new viruses. Integration of DNA into the genome allows stable transduction of the T cell clone and its lineage, leading to long-term expression of the transgene in cells capable of long-term survival, and thus making CAR T cells living drugs.In the case of retroviral vectors, transduction requires the formation of infectious particles containing the transfer plasmid encoding the transgene flanked by Long Terminal Repeat (LTRs) and including the ψ (psi) encapsidation signal. Generally, infectious particles are generated by introducing the necessary viral sequences, i.e. pol gene. Gammaretroviral vectors derived from Moloney murine leukemia virus (MLV) vectors integrate preferentially near transcription start sites (TSS) and in transcriptional regulatory regions, whereas Human Immunodeficiency Virus (HIV)-derived lentiviral vectors have a bias towards transcriptionally active regions  by lentiviral vector integration, as has been reported in patients treated with anti-CD19 and anti-CD22 CAR T cells . Transposase recognizes and binds elements incorporated into ITRs, catalyzes the excision of the transposon element from its original position, and integrates it into another position in the genome. The DNA sequence is inserted without the need for sequence homology. Transposon-based vector systems have been generated by splitting the transposase and the ITRs into two components, so that the transgene cassette lies between the two ITRs in a transposon vector. Throughout the next section, we will focus on the different transposons available for clinical applications, with an emphasis on the most widely used transposon systems, Sleeping Beauty (SB) and piggyBac (PB). Transposase is delivered in a ‘trans’ configuration to better control the system and avoid residual expression, which could then potentially lead to remobilization of the transposon into other genomic compartments which is currently the most prominent safety concern of this type of vector. One of the possibilities to deliver transposon and transposase to generate CAR T cells is through the use of a dual plasmid system, one for the gene of interest and the other for the enzyme, by electroporation of primary T cells, but has some limitations that can be solved by using mRNA and DNA vectors with decreased size compared to conventional plasmids. Anyway, these two-component vector systems are less complex than viral vectors and relies on relatively low costs of goods. Plasmids can be produced in very large quantities, so that the estimated costs are 5 to 10 times lower than the viral process  transposons, long interspersed nuclear elements (LINEs), and short interspersed nuclear elements (SINEs) . DNA tra process . Compare process . The abisequence .PGBD5 gene, apparently derived from PB transposases, has been shown to encode for a transposase capable of mobilizing insect PB transposons in human cell cultures  encoding the transposon , 81. Thegenicity . Unfortuin vivo has been demonstrated in multiple preclinical studies spanning a wide range of fields, ranging from the application in cancer to multiple diseases, reviewed by Hodge and colleagues  , 86. Thein vitro, namely cytokine-induced killer (CIK) cells with different CAR molecules  for the treatment of r/r AML and high-risk myelodysplastic syndrome (MDS). This platform shortened the manufacturing process and allows infusion of the product the day after transduction, obviating the need for g mbIL15 . The samg mbIL15 .The optimized donor-vector architecture of the pT4 vector coupled to the use of the hyperactive SB100X allows the generation CAR T cells engineered with bicistronic vectors. Using this platform, CAR T cells were incorporated with the inducible Caspase 9 (iC9) safety switch and showed anti-leukemic activity in mouse models and were as efficient as CAR T cells generated with a LV vector . Furtherin vivo  cells with anti-CD19 CAR, which showed a safe genomic integration profile and antitumor activity in vivo . Manufacex vivo propagation for approximately 28 days with multiple stimulations using artificial APCs and cytokines. Patients were subsequently enrolled in a long-term follow-up study lasting up to 15 years and the persistence of genetically modified T cells was monitored annually using droplet digital polymerase chain reaction (ddPCR) and flow cytometry. Limited expansion and absence of B-cell aplasia were reported. However, CAR+ T cells were detected up to 4 years after infusion in autologous HSCT recipients and 2 years in allogeneic HSCT recipients , a second-generation approach was developed by reducing to 2 weeks the time require for coculture with feeder cells. A clinical trial was designed (NCT02529813) in which CAR T cells have been infused in combination with a Fludarabine- and Cyclophosphamide-based lymphodepletion regimen in adult and pediatric patients with active CD19+ malignancies  confirm the safety of SB-engineered anti-CD19 CAR T cells in 26 patients with B-ALL and non-Hodgkin’s Lymphoma as adjuvant therapy after autologous or allogeneic hematopoietic stem cells transplant (HSCT) . Cell prcipients . Since lgnancies . This trThanks to the in-house establishment of a clinical-grade platform to obtain non-viral CAR T cells in about 20 days , we desiBesides the reported trials, CAR T studies using the SB platform are currently underway in the USA and Europe.Given the promising preclinical data of UltraCAR T cells, two clinical trials have been launched. Specifically, a Phase 1/1b first-in-human dose escalation/dose expansion study (NCT03927261) is evaluating the safety of PRGN-3006 UltraCAR T co-expressing an anti-CD33 CAR and mbIL15 in adult patients with r/r AML, hypomethylating agents (HMA) failure, high risk MDS and chronic myelomonocytic leukemia (CMML). Preliminary data showed that PRGN-3006 infusion was well tolerated and achieved a 50% response rate in patients treated with lymphodepletion, associated with CAR T cell expansion and persistence .A second study (NCT03907527), evaluating the safety of PRGN-3005 UltraCAR T cells co-expressing an anti-MUC16 CAR, mbIL15 and a kill switch in the treatment of patients with platinum resistant ovarian cancer patients is ongoing .The CARAMBA trial has been recently launched as a joint effort supported by an EU Horizon grant and is using mRNA encoding the hyperactive SB100X transposases in conjunction with CAR transposon supplied as an MC vector. In this Phase I/II clinical trial autologous anti-slam family member 7 (SLAMF7) CAR T cells are being used against MM to investigate the feasibility, safety, and anti-myeloma efficacy .Trichoplusia ni over 30 years ago and has been optimized over the years  flanked by ITRs that in PB are characteristically asymmetric. The transposase recognizes ITRs flanking the transposon and catalyzes transgene excision and integration into genomic DNA by a cut-and-paste mechanism. Specifically, the transposition involves a series of hydrolysis and transesterification reactions with the generation of a DNA intermediate in which DNA hairpins provide exonuclease protection for the transposon ends. One of the peculiar features of PB is its specificity towards TTAA sites for integration in contrast to SB’s preference for TA dinucleotides. Although PB can integrate into any TTAA target site, the epigenetic status may affect integration site preference of PB transposons. Another attractive characteristic of the PB transposase is the lack of a DNA footprint after its excision see Figure 2PB has a higher transposition activity for transposon mobilization than SB in mammalian cells , a largeRecently, the discovery in human genome of the human piggyBac transposable element derived 5 (PGBD5) has raised possible safety concerns in PB-gene transfer application , 118. InTherefore, PB has a series of useful characteristics for genetic engineering: i. a higher transposition activity than SB, ii. its precise excision from an insertion site, restoring the site to its pre-transposon state without DNA footprint, iii. its wide range of applications such as mutagenesis, introduction of reprogramming factors to generate iPSCs, and gene transfer. For the purpose of the review, we focus on the application of PB transposon system in the CAR T production.One of the most challenging issues using transposons is the toxicity of the transduction procedure. In particular, electroporation in the presence of exogenous DNA is toxic and decreases cell survival to less than 40% after 24h from transfection. To improve the efficiency of PB transfection, different approaches have been tested such as the addition of survival-promoting cytokines such as IL-7 or IL-15 that increase the frequency of gene expression and the ability of the transduced cells to expand. Alternatively, T cell expansion was stimulated by the use of feeder cells represented by autologous PBMCs or other sources such as the K562 cells, modified to express costimulatory molecules. Therefore, the quality of the final CAR T product depends on several factors that go from the construct characteristics  to the manufacturing platform. Many preclinical studies exploited PB as a tool to generate CAR T cells for hematological malignancies and solid tumors.in vivo and suggesting this strategy as a possible therapeutic approach for isolated or advanced CNS disease  regardless of the mutation status of the MAPK pathway in tumor cells with an enhanced efficacy of CAR T therapy in the setting of neuroblastoma .CAR T cells directed against granulocyte-macrophage colony-stimulating factor receptor (hGMR or CD116) generated with PB were used in a non-human primate (NHP) model to evaluate their safety . To genein vivo and in vitro  of less than 3 copies of transgene per transduced cell. The linear vector was prepared enzymatically ntiation . Similarin vitro . The manBACH2 and FYN genes. It is not yet clear which event caused the CAR T cell transformation, but it is widely accepted that the probability of insertional oncogenesis increases as the transgene copy number increases, which is why a limit of 5 VCNs is normally required by the regulatory authorities. Furthermore, it is likely that the numerous genomic deletions and insertions observed may have been driven by the use of a single high voltage pulse or excessive transposase activity. Insertional mutagenesis may also have contributed to the process of transformation. Both patients who developed lymphoma have an intronic integration in the BACH2 gene, whose expression is therefore downregulated. BACH2 is a DNA-binding and transcription-regulating protein that plays a key immunoregulatory role and has been previously associated with cutaneous T cells lymphomas  to investigate the efficacy and safety of donor-derived anti-CD19 CAR T cells obtained through the PB transposon system in patients with relapsed and refractory CD19+ B-cell malignancies after HLA-matched sibling HSCT. Early results suggested activity similar to that of anti-CD19 CAR T cells generated with viral vectors with a high response rate. However, two of 10 treated patients developed malignant CAR 19 T cell tumor, one of whom died of disease-related complications while the other patient was successfully treated . Malignaymphomas . BACH2 iagenesis . Hence fTwo phase I studies conducted respectively in Japan  and China  are investigating the feasibility and safety of anti-CD19 CAR T cells manufactured with the PB system. In the Japanese study, three patients with R/R B-ALL were infused with 1x10^5 autologous anti-CD19 CAR T cells per kilogram after lymphodepletion in cohort 1. All patients previously received HSCT. Interestingly, administration of T cells produced by PB was safe and none of the patients showed dose-limiting-toxicities so far. One patients showed a B-cell aplasia lasting 9 months .Results from a phase I trial using PB-generated anti-EGFR CAR T in R/R advanced non-small cell lung carcinoma (NSCLC) were recently published. Nine patients were treated with anti-EGFR CAR T cells, without grade 4 adverse events. Despite most patients showing the presence of circulating CAR T cells, only one patient showed a partial response while the other patients had persistent disease or progressed (NCT03182816) .Although the results of transposon-engineered CAR T cells in clinical trials are preliminary, several early signs of clinical efficacy are emerging. A step forward has been made with anti-BCMA CAR T cells (P-BCMA-101) engineered through the PB platform for patients with R/R MM. To improve transposition, the manufacturing process was changed during the study to include the use of nanoplasmids that allow for the reduction of backbone size and bring ITRs closer. The cellular product showed a high composition of T stem cell memory (TSCM). Ninety patients have been treated with P-BCMA-101 and early results showed an overall response rate (ORR) 57% in the initial dose escalation and 73% in combination with Rituximab with remarkably low toxicity  .RNA has emerged as a versatile therapeutic reagent , 139. SeSuccessful protein expression from RNA depends on its stability and translational efficiency. Those features are determined by cis-acting elements such as a 5’ cap structure, polyA tail, and the composition of the coding sequence as well as untranslated regions that might be present on 5’ and 3’ ends of the molecule. These cis-acting elements in the RNA conspire with trans-acting cellular factors leading to translation and protein production.The sequence of the RNA cassette can be encoded by a linearized DNA plasmid or by a PCR fragment that contains an RNA polymerase binding site or promoter, such as the bacteriophage T7 RNA polymerase to initiate the transcription reaction. Tools are available for coding sequence optimization including codon optimization for changing synonymous codons for enhanced expression in target tissues or cells, reducing secondary structures of the RNA that lower translation levels  or modif® which results in a natural structure of the RNA cap (type 1 cap).The cap structure at the 5’ end of the RNA molecule, required for translation, can be incorporated in a co-transcriptional manner, using the m7G(5’)pppG cap analog , Anti-ReA PolyA tail can be encoded in a transcription template containing a stretch of 64T nucleotides at the 3’ end of the molecule . Some RNAdditionally, both the 5’ and 3’ ends of an RNA molecule can be further modified with flanking untranslated regions (UTRs) to enhance translation –161. Fiin vitro and in vivo preclinical studies to introduce CARs into T cells for testing in model systems for hematological tumors chronic lymphocytic leukemia (CLL), AML, ALL and solid tumors. Cytotoxicity and tumor growth inhibition was demonstrated in these models  followed by SB11 DNA under a PGK promotor (23X) and SB11 RNA using the PGK promotor (9X).A different approach to genetic modification of lymphocytes is to deliver a transgene of interest in the form of DNA together with RNA encoding a transposase enzyme. The first reported demonstration of successful gene transfer using an mRNA-encoded transposase was in the SB11 system . It was There are several advantages to encoding transposase enzymes in the form of mRNA when co-transduced into target cells along with a DNA vector encoding the gene of interest –170. Onevia chromosomal integration, the choice of the vector system should be taken in account to avoid insertion into proto-oncogenes or transcription start sites that can lead to unintended transformation via insertional mutagenesis. We opted for SB system on the basis of the safer, more random integration pattern, as it does not demonstrate preferences to insert the transgene in active genes or TSSs thereby lowering the probability of integration in oncogenic genes. As previously highlighted in 4.3 paragraph, it was recently reported that 2 of 10 patients treated with anti-CD19 CAR T generated with the PB transposase system developed CAR-expressing CD4+ T cell lymphoma  Neon  which use a cuvette and a pipette tip chamber, respectively. Other commercialized instruments are for example the Celetrix electroporation system , and the BTX ECM 830 system . Commercially available electroporation devices for large-scale electroporation are Lonza LV unit and Maxcyte GTx  platform. Lonza LV unit allows for closed electroporation of 1x10^7 to 1x10^9 cells. Maxcyte GTx device is a GMP-compliant, clinical-grade instrument and can electroporate up to 20x10^9 cells using flow electroporation technology.in vivo delivery to correct several diseases such as diabetes type 1 , a common vector due to its broad tropism, large carrying capacity, and optimal efficient transduction regardless the mitotic status of target cells. Ad has non-integrative features and thus results in transient transgene expression. The addition of integrative elements into the viral genome could overcome this limitation. A recombinant Ad vector containing a PB-transposon was shown to allow the integration of the transgene into the genome in presence of PB-transposase, included in the vector design or co-delivered. With these methods, stable expression of a reporter transgene was achieved in 20-40% of mouse liver cells after infusion and lasts for at least 5 months . Similars type 1 , cystic s type 1 , and oths type 1 .ex vivo genetic manipulation. Patients are connected to an apheresis machine for several hours to extract T cells. Manufacturing involves activating and transducing purified T cells, expanding them in vitro for approximately 2 weeks and finally washing and concentrating them prior to administration. Often cells have to be cryopreserved in a central facility and transported to remote treatment centers. Quality controls on final product are mandatory for each batch. Manufacturing must be conducted under GMP conditions, and the entire process is expensive and needs specific resources, facilities and economic capital. In addition, because most CAR T products are currently obtained from an autologous source and thus from the patient’s own cells, there are no economies of scale. In order to overcome the complexity of ex vivo manufacturing, in vivo CAR T cell generation is emerging as a new prospect and exploits the use of liposomal formulations, nanoparticles (NP), cell-penetrating peptides or advanced electroporation methods  mRNA encoding disease specific CAR or TCR encapsulated in poly (beta-amino ester) NPs, coupled to anti-CD8 antibody for specific T cell delivery. IVT-mRNA has the advantages of being directly translated into therapeutic proteins, without the need to enter the nucleus, improving transfection rates and avoiding uncontrolled insertional mutations and promoter dependence. Using this technology, circulating T cells have been reprogrammed with leukemia specific CAR and showed an anti-tumor efficacy when NPs were provided with repetitive infusion  and transiently, resulting in a dose-dependent killing activity similar to virally engineered cells. Administration of CD5-targeted LNP showed a reduction in fibrosis and restoration of cardiac function in a syngeneic model of cardiac injury, proving their ability to reprogram T cells in vivo. Although this platform is not suitable for diseases that require complete elimination of pathological cells, such as some forms of cancer, undoubtedly, for other applications, the ability to generate CAR T in vivo and the inherently transient nature of mRNA have the advantage of limiting toxicities, titrating doses, and offering an off-the-shelf process. in vivo and ex vivo transfection.Building on the dazzling success of the application of lipid NPs encapsulated mRNA (LNP-mRNA) vaccines formulated against SARS-CoV-2, the study by Rurik et al. provides a great proof of concept on the possibility of producing CAR T c injury . By emplin vitro and in vivo. This platform contains no viral components and is capable of replicating extra-chromosomally in the nucleus of dividing cells, leading to persistent transgene expression, in the absence of integration and consequently genotoxicity. Moreover, it also shares all the advantages of non-viral vectors: it seems to be non-immunogenic, easy, simple, versatile and affordable to produce  and RNP aggregates into cells and the endogenous immune response triggered by innate DNA sensor protein pathway. To address the first challenge, Nguyen D. N. et al. used poly-L-glutamic acid, which physically disperse large RNP aggregates into smaller complexes. Additionally, they implemented the incorporation of a truncated Cas9 target sequence (tCTS) at the end of homology arms in order to facilitate the shuttling of the template into the nucleus. The combination of the two systems resulted in high KI efficiency of up to 50% in a wide set of primary human hematopoietic cells . To addre of 68% . Two othe of 68% . Moreovee of 68% .To provide DNA insertion in the absence of DSB and HDR, CRISPR/Cas9 has recently been combined with transposons to increase the efficiency of RNA-guided integration, using a transposase protein to catalyze the integration , 196. ReSuccessful CAR T cell therapies are so far associated with T cells engineered with viral vectors. Yet, the emergence of relapse, the complexity of the manufacturing process, and the application of these technologies to other diseases including solid tumors, require a more complex design and gene transfer technology that fits these challenges. So much progress is being made with non-viral technologies to fill these gaps, and it is certain that a great step forward will be taken soon.Previously discussed non-viral manufacturing methods are briefly summarize in in vitro cell culture ensured the absence of plasmids at the end of culture. Whether the bacterial sequences below the limits of detection is sufficient to protect from an immune response by the host against bacterial sequences, however, is currently unknown. The mean transgene copy number was set at 5 as an upper limit, in order to limit the potential risk of genotoxicity. Others are instead using 8 as release criteria for transposon-generated CAR T cell products. Despite the almost random insertion profile of the transgene into the T cell genome, the study of integrations ensured the monitoring of the clonality of the CAR T population post-infusion. Based on this clinical experience, we therefore recommend the use of vectors and the development of manufacturing platforms designed to contain transposase activity and to limit the number of integrations, and the implementation of a detailed follow-up plan for clonality monitoring by integration site analysis.Non-viral CAR T cells engineered with transposon vectors have already shown some efficacy in early-stage clinical trials, but whether such systems may also show unexpected toxicities and comparable clinical outcomes remains to be seen in additional clinical studies. For first-in-human clinical trials, certain precautions have been implemented to ensure safety given the novelty of the gene transfer method. In our study , given tex vivo production of safer transfected cells. In addition, viral-transposon hybrid vectors and mRNA delivered by polymeric and lipid nanoparticles represent technological platforms that can generate stable or transient CAR T cells in vivo. We are on a learning curve and looking forward to seeing the results of these technologies in future clinical trials.We believe that the main takeaway from the efforts described in this review is the possibility of using novel non-viral engineering approaches in future clinical trials. In addition to non-viral integrating methods, we think that the use of episomal nanovectors may be advantageous for the CM conceptualized, wrote, and edited the manuscript. MP, AM, and IT wrote the manuscript. MP and AM designed the figures and prepared the tables. CN and AB edited the manuscript. All authors contributed to the article and approved the submitted version.CM has received funding from Ministero della Salute, grant number GR-2016-02363491 and Fonds zur förderung des akademischen nachwuchses (FAN) of the University of Zurich (UZH). AB and CM have received funding from AIRC/Cancer Research UK (CRUK)/Spanish Association Against Cancer Scientific Foundation (FC AECC), grant number 22791 (Accelerator award).Authors CM and AB are inventors on a patent related to non-viral CAR-T cell therapy (European patent application 15801344), filed by the Tettamanti Foundation, and licensed to CoImmune. Authors CN and IT are executive officers of CoImmune. Author AB has received fundings from AIRC 5x1000 2018-21147, PRIN 2017-NAZ-0412, and the Ministero della Salute CAR-T project of the “Rete Oncologica”.The remaining authors declare that the research was conducted in the absence of any commercial or financial conflict of interests.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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The prevalence of coronary artery anomalies has been increasing due to the increasing usage of coronary angiography. There is a paucity of literature concerning management of viral-induced myocarditis in patients with anomalous coronary artery. We present a very unusual case of a 44-year-old man with anomalous origin of the left circumflex artery from the proximal ostium of the right coronary artery who was admitted for COVID-19-induced myocarditis. He presented with signs of heart failure and coronary angiography revealed the left circumflex artery with a separate ostium originating from the proximal right coronary artery. He was treated medically with Bisoprolol, Perindopril Arginine, Rivaroxaban, and Furosemide. His condition improved rapidly and he resumed regular life within 1 month. Coexistence of cardiac disease such as viral-induced myocarditis with an underlying anomalous origin of the coronary artery is challenging to spot and can lead to worse outcomes in case of misdiagnosis and inaccurate management. The patient had a history of hypertension, uncontrolled diabetes mellitus type 2 with a most recent HbA1c value of 9.4%, and COVID-19 pneumonia one month before presentation. He had no significant past surgical or family history. The list of home medications was Vildagliptin/Metformin  50/1000 mg once daily, Irbesartan (Aprovel) 300 mg once daily, and Acetylsalicylic Acid (Aspirin) 100 mg once daily.On admission, the patient’s vital signs were all within the normal ranges, except for decreased blood oxygen saturation, at 85% on room air. The patient had orthopnea and was classified as NYHA III according to clinical findings and reported history. The physical exam revealed grade 2+ lower limb pitting edema and bilateral motor limitation. Of note, the patient had a diabetic ulcer on the dorsum of the left foot and reported a previous spontaneous amputation of the left third toe. The rest of the physical examination concerning the gastrointestinal, urinary, and neurological systems did not reveal any additional features and the patient did not report any other complaints.A 12-lead electrocardiogram (ECG) showed T-wave inversion (TWI) in the lateral leads. Computed Tomography (CT) of the chest with intravenous (IV) contrast injection showed bilateral small peripheral emboli in the sub-segmental arteries. It was evident that the patient had moderate to severe bilateral pleural effusion with dilated right heart chambers. Transthoracic echocardiography (TTE) revealed enlarged right heart chambers with pulmonary arterial pressure (PAP) of 90 mmHg. The patient had grade I mitral regurgitation, diastolic dysfunction, and anteroseptal hypokinesia. Ejection fraction (EF) was calculated automatically and was equal to 50%. Arteriography of the lower limbs was normal. Coronary angiography showed that the circumflex artery had an anomalous origin from the proximal ostium of the right coronary artery  and 2.Laboratory workup showed elevated cardiac enzymes, and the patient was diagnosed with COVID-19-induced myocarditis. The patient was treated with Bisoprolol (Concor) 5 mg once daily, Perindopril Arginine (Coversyl) 5 mg once daily, Empagliflozin (Jardiance) 25 mg once daily, Rivaroxaban (Xarelto) 20 mg once daily, and Furosemide (Lasix) 40 mg IV twice daily. The patient’s condition improved rapidly and he resumed his normal life within one month.To date, 1½ years later, the patient has been followed-up twice at the clinic, 1 and 6 months after discharge. Repeated laboratory exams were within normal ranges, especially cardiac enzymes, and we didn’t repeat any imaging exams. The patient is taking the same medications at the same doses and living an ordinary life.In this report, we presented a rare case of a middle-aged man who was admitted to hospital for COVID-19-induced myocarditis in the presence of dyspnea, lower limb edema, and decreased physical activity. An anomalous origin of the left circumflex artery from the right coronary artery was identified later. We will discuss the coronary malformation, of which the attending physician should be aware, to enable correct diagnosis and appropriate treatment.An anomalous coronary artery is a silent and life-threatening risk factor. Depending on the clinical manifestations, it is possible to distinguish between a malignant variant with an interarterial pathway between the pulmonary artery and aorta, and a benign one with no surrounding arteries.,Brothers et al. suggested that the anomalous arterial anatomy is more prone to dynamic compression and may put the heart at risk of myocardial infarction.In the era of COVID-19, Ghori and Ahmed reported a case of a 34-year-old male referred to the hospital for COVID-19 infection and without cardiac symptoms. However, routine workup revealed a high troponin-T level and left ventricular hypertrophy on ECG.It is worth mentioning that a large prospective cohort study developed an algorithm and used it for decision-making on surgical interventions in anomalous coronary arteries. A total of 44 pediatric patients were treated surgically and showed promising results.In summary, the most common coronary anomaly is an ectopic origin of the left circumflex artery. Management of anomalous circumflex artery is still unclear, especially in cases with coexisting heart disease, such as viral-induced myocarditis. In contrast to the previously described literature, our article is the first to report a case of anomalous circumflex artery with COVID-19-myocarditis in a middle-aged man who only required medical treatment and achieved satisfactory results. According to this report, we can postulate that diagnosis of an anomalous coronary artery and choice of treatment modality are not easy tasks and require experienced and skilled physicians.


The funnel plot showed publication bias, although the sensitivity test using leave-one-out analysis showed that removing any of the study does not remove the significance of the result. The response rate was higher in the Unites States, and meta-regression showed that age, year of publication, and use of concomitant vasodilators did not influence the response rate to iNO. Conclusion: iNO therapy is valuable in the treatment of hypoxemia in COVID-19 patients and may improve systemic oxygenation in patients with COVID-19-ARDS. Future studies should investigate the mechanism of the activity of iNO in COVID-19 patients to provide insight into the unexplored potential of iNO in general ARDS.Background: Severe COVID-19 is associated with hypoxemia and acute respiratory distress syndrome (ARDS), which may predispose multiorgan failure and death. Inhaled nitric oxide (iNO) is a clinical vasodilator used in the management of acute respiratory distress syndrome (ARDS). This study evaluated the response rate to iNO in patients with COVID-19-ARDS. Method: We searched Medline and Embase databases in May 2022, and data on the use of iNO in the treatment of ARDS in COVID-19 patients were synthesized from studies that satisfied predefined inclusion criteria. A systematic synthesis of data was performed followed by meta-analysis. We performed the funnel plot and leave-one-out sensitivity test on the included studies to assess publication bias and possible exaggerated effect size. We compared the effect size of the studies from the Unites States with those from other countries and performed meta-regression to assess the effect of age, year of publication, and concomitant vasodilator use on the effect size. Results: A total of 17 studies (including 712 COVID-19 patients) were included in this systematic review of which 8 studies (involving 265 COVID-19 patients) were subjected to meta-analysis. The overall response rate was 66%  with significantly high between-studies heterogeneity (I2 = 94%,  Coronavirus disease COVID-19), as defined by the World Health Organization (WHO), is an infectious illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 9, as def,4, and tNitric oxide (NO) is a systemic vasodilator endogenously produced in the endothelium . The sigAlthough various studies have previously reported significant response to iNO in patients with hypoxemia or ARDS due to COVID-19 and early systematic reviews have been performed , there ihttp://rayyan.qcri.org, accessed on 22 August 2022) [This systematic review was performed based on the Preferred Reporting in Systematic Reviews and Meta-Analyses (PRISMA) guidelines , and thest 2022) .Inclusion eligibility include studies that reported the use of inhaled nitric oxide (iNO) either alone or with concomitant vasodilators as an intervention in patients with COVID-19 irrespective of treatment settings  or severity. Studies that used iNO as a potential preventative measure against SARS-CoV-2 infection, case reports, studies not published in the English language, and studies that are not available as open access or in an institutional database were excluded.2 statistic. The response rate is defined as the proportion of patients who responded to iNO administration in terms of improved systemic oxygenation as defined by the authors. The "metaprop" algorithm on Stata/SE 16 was used, and the result was displayed as a forest plot. Response to iNO was defined as improvement (≥20%) in oxygenation following administration and was characterized by patients’ systemic oxygen saturation measured by the ratio of arterial oxygen partial pressure (PaO2 in mmHg) to fractional inspired oxygen (FiO2) expressed as a fraction (P/F ratio), and SpO2, among others. Small study effect was visually assessed by computing a funnel plot. The response rate is computed as the proportion of patients treated with iNO who were classified as “responders” after follow-up, i.e., response ratio = number of responders/treatment population, while response rate = response ratio × 100.Screening of the titles, abstracts, and full texts was performed by two authors (TO and JSA) with resolution of conflict resolved via online meetings. Once the studies were collectively agreed upon by the screening group, the extraction of relevant data was carried out based on a predesigned narrative table. A meta-analysis was performed to pool the reported response rates to iNO in COVID-19 patients using a random- or fixed-effect model based on the between-studies heterogeneity measured by the ISensitivity test was also performed to assess whether any of the analyzed studies disproportionately influenced the output using the "leave-one-out" algorithm. Furthermore, we performed meta-regression based on the DerSimonian–Laird (random-effect) model on the year of publication, mean age of populations that were studied, and administration of concurrent vasodilators to understand the effect on the pooled proportion that these possible confounders may have. In terms of locations of studies, we also performed further analysis comparing the response rate to iNO reported in the United States of America with other countries.To assess the quality of studies included in this systematic review, a modified version of the Newcastle-Ottawa Scale (NOS)  was usedSearch of databases generated a total of 512 studies, which included 157 duplicates. Title and abstract screening of the remaining 355 studies was performed resulting in further exclusion of 315 studies to give a total of 40 potentially suitable studies based on the inclusion criteria. The 40 studies were sorted for full-text retrieval with 2 not retrievable. A final 38 studies were then subjected to full-text screening resulting in the exclusion of 21 studies to give a total of 17 studies that satisfied the inclusion and exclusion criteria . The NOSThe dosage of iNO in patients with COVID-19 ranged between 9 and 160 ppm over a duration between 30 min and 5 days. The most popular mode of delivery of iNO was via mechanical ventilators 71%) in intubated patients suffering from ARDS due to exacerbation of COVID-19. In six of the included studies, iNO was administered in combination with other vasodilators such as almitrine , inhaled1% in int2 = 118.91, I2 = 94.11%, p < 0.001).Eight (47%) of the included studies reported response to iNO in COVID-19 patients suffering from ARDS ,14,16,17The funnel plot shows the possibility of small study effect or publication bias , which rp = 0.256), year of publication , nor the administration of concomitant vasodilators  has significant effect on the standard error of the effect sizes of the studies  for patients in terms oxygenation levels following iNO therapy with or without concomitant vasodilators. Furthermore, the included studies show significant variability. However, the random-effect model that was used is robust to high between-studies heterogeneity.Our findings contradict some previous studies. For instance, Tavazzi et al.  reported2/FiO2 ratio after receiving iNO with almitrine . Although both studies did not report a response rate and involved a small sample size, their findings were consistent with another similar study conducted by Caplan et al. [2/FiO2 ratio in both responders compared and nonresponders to iNO combined with almitrine therapy (p < 0.0001). The same pattern was found in our study, suggesting that iNO with or without vasodilators is effective in the management of hypoxic patients with COVID-19. Thus, it could be hypothesized that the combined use of iNO and vasodilators can boost oxygen levels by increasing ventilation and perfusion, therefore enhancing ventilation/perfusion (V/Q) matching, reducing shunt, and improving arterial oxygenation. However, the mechanism by which this occurs requires further investigation. Furthermore, chronic lung diseases are associated with pulmonary microvascular dysfunction, contributing to the increased burden caused by such diseases [The use of iNO in conjunction with pharmaceutical vasodilators, such as almitrine and prostaglandin, has shown a positive clinical value as a rescue therapy to enhance oxygen levels in patients with COVID-19. Two studies by Bagate et al.  and Laghdiseases . The usediseases . This isdiseases .2/FiO2 (from median 136 (77–168) to 170 (138–213) mm Hg, p = 0.003), which increased even further after placing the patients in the prone position (from 145 (122–183) to 205 (150–232) mm Hg, p = 0.017). Another study [2/FiO2 was substantially increased  after receiving both therapies. These findings along with our results make a strong case for the integration of iNO with prone positioning as a routine care to enhance arterial oxygenation in patients with COVID-19-ARDS.Other clinical approaches to increase oxygenation levels in the management of COVID-19-ARDS patients have been described and include prone positioning ,24 and rer study  that useThis study is limited by several factors. Firstly, due to underreporting, the total number of studies and, by extension, the number of patients included in our meta-analysis are low. Thus, as more data become available on the use of iNO in the clinical management of COVID-19-ARDS, future studies will further clarify this topic. However, the quality of the included studies as measured by the modified NOS assessment showed that the included studies are mainly of high quality. Secondly, considering the novelty of the SARS-CoV-2, high heterogeneity exists in the clinical management of COVID-19 patients within and across countries and continents, which results in significant between-studies variability. This is further supported by the stratified analysis (USA vs other countries) that showed that patients in the USA may have a higher response rate compared with those of their counterparts from other countries. However, we have combined a random-effect model with the measurement of publication bias; sensitivity test; as well as meta-regression controlling for age, years of publication, and administration of concomitant vasodilators to improve the interpretation and extrapolation value of our result.In sum, this review involved a thorough and comprehensive search of the literature followed by a systematic analysis of data including a total of 265 patients diagnosed with COVID-19 who received iNO therapy. For the first time, we have reported the potential clinical value of using iNO for the management of COVID-19 patients, which suggests a relatively high benefit in terms of improved arterial oxygenation. As COVID-19 patients are at high and consistent risk of suffering from refractory hypoxemia, it is recommended that clinical guidelines and practices consider the integration of iNO in the routine clinical management plan, particularly those who are critically ill due to ARDS. Future studies should investigate whether the combined use of iNO and systemic vasodilators, or other clinical supportive means, such as prone positioning, has superior effect on the response rate and arterial oxygenation on COVID-19-ARDS population as more data become available.


The WHO Strategic Advisory Group of Experts recommended that an extended interval of 3–5 years between the two doses of the human papillomavirus (HPV) vaccine could be considered to alleviate vaccine supply shortages. However, three concerns have limited the introduction of extended schedules: girls could be infected between the two doses, the vaccination coverage for the second dose could be lower at ages 13–14 years than at ages 9–10 years, and identifying girls vaccinated with a first dose to give them the second dose could be difficult. Using mathematical modelling, we examined the potential effect of these concerns on the population-level impact and efficiency of extended dose HPV vaccination schedules.We used HPV-ADVISE, an individual-based, transmission-dynamic model of multitype HPV infection and disease, calibrated to country-specific data for four low-income and middle-income countries . For the extended dose scenarios, we varied the vaccination coverage of the second dose among girls previously vaccinated, the one-dose vaccine efficacy, and the one-dose vaccine duration of protection. We also examined a strategy in which girls aged 14 years were vaccinated irrespective of their previous vaccination status. We used a scenario of girls-only two-dose vaccination at age 9 years  as our comparator. We estimated two outcomes: the relative reduction in the age-standardised cervical cancer incidence (population-level impact) and the number of cervical cancers averted per 100 000 doses (efficiency).Our model projected substantial reductions in cervical cancer incidence over 100 years with the two-dose schedule (79–86% depending on the country), compared with no vaccination. Projections for the 5-year extended schedule, in which the second dose is given only to girls previously vaccinated at age 9 years, were similar to the current two-dose schedule, unless vaccination coverage of the second dose is very low . However, when the dose at age 14 years is given to girls irrespective of vaccination status and assuming high vaccination coverage, the model projected a substantially greater reduction in cervical cancer incidence compared with the current two-dose schedule . Efficiency of the extended schedule was greater than the two-dose schedule, even with a drop in vaccination coverage.The three concerns are unlikely to have a substantial effect on the population-level impact of extended dose schedules. Hence, extended dose schedules will likely provide similar cervical cancer reductions as two-dose schedules, while reducing the number of doses required in the short-term, providing a more efficient use of scarce resources, and offering a 5-year time window to reassess the necessity of the second dose.WHO, Canadian Institute of Health Research Foundation, Fonds de recherche du Québec–Santé, Digital Research Alliance of Canada, and Bill & Melinda Gates Foundation. In 2019, the WHO Strategic Advisory Group of Experts recommended that an extended interval of 3–5 years between the two doses of the human papillomavirus (HPV) vaccine (first dose given around ages 9–10 years and the second dose around ages 13–14 years) could be considered when introducing HPV vaccination in a country to alleviate HPV vaccine supply shortage.Evidence before this studyWe have previously shown, using mathematical modelling, that a 5-year extended schedule is likely to be highly efficient and cost-effective in low-income and middle-income countries. In 2019, partly based on these results, the WHO Strategic Advisory Group of Experts recommended that an extended interval of 3–5 years between the two doses of the human papillomavirus (HPV) vaccine  could be considered when introducing HPV vaccination in a country. This recommendation is particularly relevant during the COVID-19 pandemic, because organising HPV routine vaccination with two doses in the same year might represent a major challenge given resource constraints, which have been exacerbated by the COVID-19 pandemic. Moreover, there is increasing evidence of the high efficacy and durability of a single dose of HPV vaccines. However, three concerns have limited the widespread introduction of HPV vaccination extended schedules: (1) girls could be infected between the two doses, if they become sexually active and one-dose vaccine efficacy is limited; (2) vaccination coverage for the second dose could be much lower if scheduled at ages 13 or 14 years in countries where school attendance substantially drops with age; (3) and implementation challenges of finding the girls to administer the second dose several years after the first dose, although there might be alternative strategies, such as catch-up campaigns irrespective of vaccine status. We searched PubMed (no date restriction) and found no modelling study that have examined the potential impact of extended schedules considering these concerns.Added value of this studyIn this modelling analysis, we showed that: girls becoming sexually active between the two doses should be, at the very least, partially protected by one dose; (2) reductions in coverage for the second dose in an extended dose schedule at ages 13–14 years is unlikely to have a substantial effect on the number of cases of cervical cancer prevented, unless vaccination coverage of the second dose is very low  or one-dose vaccine efficacy is of short duration; and (3) alternative strategies to finding previously vaccinated girls, such as catch-up campaigns in which girls are vaccinated at age 14 years irrespective of their vaccination status, could provide an opportunity to vaccinate girls who were missed at age 9 years, thus increasing population-level impact. Hence, extended dose schedules would likely provide similar cervical cancer reductions as two-dose schedules, while reducing the number of doses required in the short-term and providing a more efficient use of scarce vaccine resources.Implications of all the available evidenceThese findings have important policy implications because they show that the concerns about extended schedules should not prevent countries from introducing such strategies, particularly in the context of the COVID-19 pandemic. An extended dose schedule could offer an effective and efficient strategy of HPV vaccination while providing time to reassess whether it is necessary to give the second dose. In fact, with the increasing evidence showing high efficacy and durability of a single dose, the province of Québec (Canada) and England (UK) have adopted such a strategy. Moreover, an extended schedule with a catch-up campaign at ages 13 or 14 years, in which girls are vaccinated with one dose, irrespective of whether they were previously vaccinated at age 9 years, could provide the opportunity to vaccinate a greater number of girls with at least one dose and therefore increase the population-level impact of HPV vaccination. Finally, an extended schedule might be easier to implement with the WHO's 2022 recommendation that one-dose or two-dose schedules could be considered for girls aged 9–14 years, as it could provide a security net for countries who are uncertain about starting directly with or switching to a one-dose strategy.Although we have previously shown that a 5-year extended HPV vaccination schedule was likely to be highly efficient and cost effective in low-income and middle-income countries (LMICs),The objective of this modelling study was to examine the concerns about the extended schedules of HPV vaccinations and their potential effect on population-level impact  and vaccination efficiency . To do so, we used a transmission-dynamic model to compare the population-level impact and efficiency of 5-year extended HPV vaccination schedules to the current two-dose recommended schedule at age 9 years, for different assumptions of vaccination coverage and one-dose vaccine efficacy and duration of protection, using India, Viet Nam, Uganda, and Nigeria as examples.We used HPV-ADVISE LMIC, an individual-based, transmission-dynamic model of multitype HPV infection and diseases .7 The moWe modelled the impact of the HPV vaccination in two Asian countries (India and Viet Nam) and two African countries (Uganda and Nigeria) to represent different profiles of sexual activity and HPV-related burden .7 We parWe reproduced three different girls-only vaccination scenarios to examine the main concerns of extended schedules using the 9-valent HPV vaccine: (1) a current two-dose vaccination schedule given at age 9 years ; (2) a 5For all scenarios, vaccination coverage at age 9 years was assumed to be 80% on the basis of estimates of the median vaccination coverage for the first dose observed in LMICs in 2019.14In our sensitivity analysis, we chose 20 years of duration of protection following one-dose vaccination as a pessimistic scenario, as results from the India IARC Trial show sustained protection of one dose through 10 years.We used two main outcomes. To examine the population-level impact of the different HPV vaccination scenarios, we used the relative reduction in cervical cancer incidence compared with no vaccination. To examine the efficiency of the vaccination scenarios, we calculated the number of cervical cancers averted over time per 100 000 doses given. For both outcomes, we present the mean of the ten best fitting parameter sets for cervical cancer incidence from 2020 from the Global Cancer ObservatoryThe funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.Under base-case assumptions for the current two-dose strategy , the model projected that vaccinating girls at age 9 years would produce substantial reductions in cervical cancer incidence by 85% in India, 86% in Viet Nam, 80% in Uganda, and 79% in Nigeria, after 100 years . As expeWhen assuming lower vaccine efficacy for one dose  and a high vaccination coverage for both doses (80% vaccination coverage), the model projected that a 5-year extended schedule would produce similar reductions in cervical cancer incidence in the four countries compared with the current two-dose strategy  and a 70% vaccination coverage for the second dose at age 14 years (80% for the first dose), the model projected that a 5-year extended schedule with the second dose given to previously vaccinated girls only would result in slightly more cervical cancer cases in the four countries than the current two-dose strategy with 80% vaccination coverage , 80% vaccination coverage at age 9 years and 70% at age 14 years, irrespective of the vaccination status, the model projected that a 5-year extended schedule would result in substantially less cervical cancer cases in the four countries than the current two-dose schedule  is estimated to be substantially greater than the incremental impact of the second dose with 100% vaccine efficacy (vs the first dose). In other words, when the efficacy of the first dose is high (≥85%), giving the second dose does not provide substantial additional benefits.Efficiency of vaccination  was greater for the 5-year extended schedule than the current two-dose schedule, even with a drop in vaccination coverage for the second dose . Under bWhen assuming a shorter duration of vaccine protection with one dose  and 70% vaccination coverage for the second dose at age 14 years, the model projected slightly more cervical cancer cases than the current two-dose schedule assuming 80% vaccination coverage for both doses ; (2) the prevalence of a high-risk HPV infection is also relatively low before age 14 years in those countries  the proportion of girls becoming sexually active before age 15 years is relatively low in the four countries examined  would still produce very similar reductions in the number of cervical cancer cases prevented compared with the current two-dose schedule. However, a larger drop  could substantially reduce the number of cervical cancer cases prevented compared with the current two-dose schedule, especially if the one-dose duration of protection is short . However, a duration of protection of 20 years or less after one dose would be unlikely given that a recent study in India showed sustained protection of one dose through 10 years.The third concern is that there could be implementation challenges of finding the girls to administer the second dose several years after the first dose. However, instead of finding these girls to provide a second dose, vaccinating girls at age 14 years, irrespective of their vaccination status, during routine catch-up campaigns could provide the opportunity to vaccinate girls who missed the first dose at age 9 years. By catching up girls at age 14 years and therefore increasing the number of girls vaccinated with at least one dose, our model projected that more cervical cancer cases would be prevented compared with the current two-dose schedule or the five-year extended schedule with a high coverage at age 14 years of previously vaccinated girls .Finally, an extended schedule might be easier to implement with WHO's 2022 recommendation that one or two-dose schedules could be considered for girls aged 9–14 years,Our model projections also suggest that almost all extended scenarios examined are more efficient than the current two-dose schedule, even when assuming a lower efficacy of one dose, lower duration of protection of one dose, or lower vaccination coverage at age 14 years. If both the vaccine efficacy of the first dose and vaccination coverage for the second dose are lower for the extended schedules  compared with the current two-dose schedule , the number of cervical cancer cases prevented would be slightly reduced, but the efficiency per dose would be greater. Indeed, when the first dose is highly effective , the additional benefit of providing a second dose is reduced.These results showing high population-level impact and efficiency of the two-dose extended schedules have important implications, particularly in the context of the COVID-19 pandemic. The pandemic has affected public health programmes worldwide. Significant decreases of vaccination coverage for recommended vaccines for children and adolescents, including the HPV vaccine, have been documented in several countries.To our knowledge, this is the first study to comprehensively examine the population-level impact and efficiency of a 5-year HPV vaccination extended dose schedule in LMICs, with varying one-dose efficacy and duration, and vaccination coverage for the second dose. Our study has major strengths. First, we used an individual-based transmission-dynamic model calibrated to LMIC-specific behavioural and epidemiological data from four countries (HPV-ADVISE LMIC). Second, despite differences in sexual activity and cervical cancer burden between the four LMICs included in this study, the population-level impact and efficiency of an extended vaccination schedule were consistent across the four countries. Our results are most likely generalisable to other LMICs with similar HPV and cervical cancer epidemiological profiles and should be considered as general principles guiding HPV vaccination policies in different countries. Third, our model projections were based on 1000 runs simulated from 50 parameter sets that capture uncertainty and variability in sexual behaviour, HPV transmission, and natural history of HPV-related diseases within a country.Our study also has some limitations. First, although we modelled several scenarios of extended schedules by varying vaccine efficacy and duration of protection after one dose, and vaccination coverage at age 14 years, we did not model the complete range of potential scenarios . However, our results were robust to decreases in one-dose duration of protection (lifelong to 20 years) and efficacy (100% to 85%) and recent results suggested that one-dose efficacy should be higher than 85%.In summary, a 5-year extended HPV vaccination schedule represents an effective and efficient HPV vaccination strategy, particularly in the context of HPV vaccine supply shortage and scarce human and financial resources, which has been exacerbated by the COVID-19 pandemic in many countries. This strategy could allow countries to gradually introduce or reintroduce HPV vaccination while providing a 5-year window to reassess the necessity of giving a second dose.https://marc-brisson.net/HPVadvise-LMIC.pdf).No individual participant-level data were used in this study. Descriptions of the model structure, the parameters included in the model, and the empirical data used for calibration and validation are available in MB, MD, and MJ are members of the Single-Dose HPV Vaccine Evaluation Consortium. EB, J-FL, KP, and M-CB declare no competing interests.


The application of higher-resolution scanning electron microscopy (HR-SEM) illuminates the heterogenous, morphological profile that exists on the surface of BEC membranes and the relationship between these ultrastructures during the molecular construction of the PC space between adjacent BECs. In this study developing BEC monolayers were grown on mixed, cellulose esters insert membranes in a bicameral system. BEC monolayers were fixed in 2.5% glutaraldehyde, hydrated, critically dried, and sputter-coated, for imaging utilizing HR-SEM. This study, for the first time, showed membrane-bound exosomes were attached to the plasma membrane surfaces of the BECs. The exosomes were characterized as small membrane-bound, nano-sized exosomes (30–300 nm). Based on their membrane morphology and anatomical structure, exosomes appear to possess two distinct functions, namely: paracrine secretion and nanotube construction between adjacent BECs, during in vitro barrier genesis. The HR-SEM micrographs in conjunction with the Tipifarnib inhibition of exosome formation, suggests that brain capillary endothelial exosomes play a prominent role in the bilateral signaling, which contribute to the regulation of the permeability of the BBB. Given that blood-brain barrier permeability has been implicated in the progression of many neurodegenerative pathologies, the role of these exosomes and TUNTs posits the capacity of these structures to exacerbate neuropathologies that implicate BBB permeability. These findings could lead to the development of novel treatment interventions and moreover, the characterization of BBB exosomes may be a reliable target for identifying therapeutic biomarkers in neurodegenerative disease. Conversely, the presence of BBB exosomes raises a critical enterprise to target the exosome-induced nanotubes as a vehicle for transferring therapeutic treatments across the BBB.The blood-brain barrier (BBB) is a robust interface between the blood and the central nervous system. Barrier type endothelium is able to limit paracellular (PC) movement, relegating molecular flux to the transendothelial pathways of brain endothelial cells (BECs). It is, therefore, apparent that any leakage  It, therefore, becomes naturally prudent to understand the physical functionality of BBB endothelium and to explore the possibility of molecular intervention. To date, the nanoscopic landscape of the BBB is unchartered territory. High-resolution scanning electron microscopy (HR-SEM) enables the observation of the ultrastructural, anatomical exploration of the BBB endothelium on a nanoscale. This study illuminates the complex, interactive nature of barrier genesis by describing the exosome landscape of the BEC as the nexus to the novel development of tunneling nanotubules (TUNTs) that facilitate BECs engagement and ultimately BBB formation. TUNTs are nanosized hollow membrane tubes which extend from cells and can interact with distal cells. The TUNT is a hovering apicolateral structure, with 50–200 nm diameters  is a dynamic interface, which regulates the movement of substances and ions between the blood circulation and the brain’s microenvironment. It is comprised of highly specialized brain endothelial cells (BECs) which are tightly regulated by the interaction of intercellular tight junction (TJ) protein complexes. BECs, thus form the anatomical basis of the BBB. Although the BBB functions to maintain a well-regulated neuronal in vitro BBB at high-resolution (HR) suggests it plays a key role in the alignment of TJ zones on the lateral membranes of BECs while forming the impermeable capillaries of the brain  ranging between 40 and 1,000 nm in diameter and processing biomolecular contents, whereas TUNTs synergistically establish bi-directional, long-range cross-talk between cells . Due to the heterogeneous nature of the NV, an interchangeable nomenclature relating to extracellular vesicles exist. In the extracellular Although, our study does not focus on the intracellular mechanisms of exosome formation, for the context of this study it is important to briefly summarise the process. Exosome production begins through the process of endocytosis with the formation of the endosome, normally encapsulating extracellular cargos at the plasma membrane. This early endosome matures into a late endosome. Thereafter, the late endosome matures into intraluminal vesicles (ILVs), which generate multi-vesicular bodies (MVBs). Exosome biogenesis is modulated by two mechanisms: (i) endosomal sorting complexes required for transport (ESCRT)-dependent or ESCRT independent mechanisms. MVBs are then transferred to the cell membrane, facilitated by proteins coactin, Rab, and GTPases to fuse with the plasma membrane, resulting in the exocytotic release of exosomes , ESCRT, syntenin, ALIX, tetraspanins, and phospholipase D2 (PLD2). The vesicles derived from the Golgi apparatus fuse with endosomes and are incorporated into MVBs. The MVBs fuse with the plasma membrane releasing their contents. Ras-related proteins  are proteins involved in GTPase-mediated signal transduction and protein transport, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), cortactin, and coronin 1b are involved in membrane docking of the vesicles  were seeded on the upper side of mixed cellulose esters insert membranes, placed in a 24-well culture plate, creating a bicameral system. A bicameral system utilizes an insert membrane placed within a 24-well plate-BEC monolayer  grown on a mixed cellulose insert membrane  and were allowed 24 h to attain cell confluence before processing the biological sample for HR-SEM imaging.BECs  prepared in standard cultured buffer ; Faso et al., 2O and placed in a series of graded ethanol (EtOH) solutions: 50%, 70%, 90%, 95%, and twice in 100% EtOH for 10 min each. All EtOH solutions were prepared by diluting 99.9% absolute EtOH in de-ionized H2O v/v.Biological specimens were removed from the de-ionized H2, which functions as a “transitional fluid”. Since H2O is not miscible with CO2, thus, EtOH was used as the “intermediate fluid”/“dehydration fluid.” Following the dehydration of the fixed samples, it is required that the sample be dry before further processing could occur, this was performed using the Hitachi HCP-2 CPD (2). Dehydrated samples were placed inside the CPD chamber and filled with liquid CO2. The chamber would not reach the critical point (CP) with inadequate liquid CO2. The temperature was set to 20°C for 15 min and then 38°C for 5 min until the critical pressure was reached (73 kg/cm2).Critical point drying (CPD) was conducted for the phased drying of wet, delicate samples from liquid to gas form, by using liquid COCP-2 CPD . EvaporaCP-2 CPD . CPD wasBEC monolayers were coated using a Gold: Palladium (Au: Pd) alloy, at ratios of (60:40). The sample was imaged using a Zeiss Auriga HR field-emission gun SEM, which was operated at an electron beam energy of 5 keV and a nominal working distance of 5 mm.HR micrographs were collected utilizing an in-lens secondary electron detector, which allowed for HR image generation. The use of Au:Pd metal alloys as a routine coating method reduces damage to bioorganic samples , and functions by inhibiting exosome biogenesis and secretion 5 bEnd5 cells/insert/well on membrane inserts , in 24-well plates, they reached confluence and the cultured medium was replaced by selected concentrations of the exosome inhibitory agent: Tipifarnib at 1 μM, 5 μM, and 10 μM, relative to control samples. A humidified atmosphere of 5% CO2 at 37°C. Transendothelial electrical resistance (TEER) was carried out for 12 h, 24 h, and 48 h using a Millicell electrical resistance system . Quantitative analysis of the recorded TEER readings employs the parameters of an appropriate equivalent circuit, which represents the electrical parameters across the in vitro BEC monolayer under investigation.Twenty-four hours (h) after seeding 5 × 10All morphological experiments utilized thousands of micrographs repeated over a 4-year period. All cell culture experiments were performed in triplicate or repeated a minimum of three times . Statistical calculations were performed using the One-Way ANOVA. Furthermore, the Tukey multiple comparsion test was utilized to compare the means between not normally ditributed, independent samples. Statistical significance was designated at The resolution of HR-SEM allows the observation of not only the plasma membrane products but also allows limited observation of the molecular make of these anatomical structures  were observed to have a distinctly different texture to “non-TUNT forming” exosomes (NTF-exosomes). These TF-exosomes had characteristically smoother membranes but were also porous. It appears that these TF-exosomes were formed Exosome-exosome interaction is evident as the exosome-induced TUNT scaffolding network is formed at the apicolateral domain of the PC space see  and the TF-exosomes appear to be able to fuse at their elliptical ends to promote the generation of TUNTs extensions along the apicolateral membrane borders and also across the PC space. TF-exosomal trafficking to form TUNTs occurs bi-directionally on both adjacent BECs. The TUNT emanates from the serial fusing of these TF-exosomes. The contact of the newly formed TUNTs extends from the apicolateral membranes of the BEC membrane across the PC space to form a TUNT-membrane interaction between the adjacent BECs , 6.The distal extremities of these TUNTs display foot-like projections, which attach to the cell membrane on the target cell M2—. MoreoveTUNTs typically extend across the PC spaces resembling intercellular, cross-bridges between adjacent BEC, which form a tunneling scaffold during BEC monolayer development, and possess a porous membrane filled with yet unidentified substances . It mustTUNT development is an event that occurs, once a substantial amount of TF-exosomes accrue on a particular BEC. The TUNT extensions are bi-directional and are generated by the fusing of TF-exosomes into an intercellular nano-sized tube, hence the name tunneling nanotubule (TUNT). Fusing TF-exosomes begin to form primordial tunnels, which develop from a cell from which a mature TUNT originates and that traverses the PC space from M1 (on which TUNT originates) and aims to lodge itself onto M2, which denotes a target cell (M2) growing in close proximity  below.in vitro. The cross-bridge TUNTs are both dynamic and transient phenomena, forming within 24–72 h Treatment with Tipifarnib resulted in the inhibition of the exosome formation. The compound has a direct effect on the rate of release of the exosome from the BEC , relativP < 0.001). At 24 h a significant increase in TEER was observed between the controls and 5–10 μM), however, at 48 h the continued exposure to Tipifarnib resulted in a significant decrease in TEER across the bEnd5 monolayer (P < 0.001).The impetus of the exosome to generate tunneling nanotubes between adjacent BECs at a steady rate is highly inhibited upon treatment with Tipifarnib. via regulated channels and molecular transporters. Although this robust barrier is crucial for homeostatic regulation of the neuronal environment, it is also a fundamental obstacle in treating neurodegenerative diseases. It is, therefore, rational to understand the functional anatomical endothelium of the BBB and to investigate if molecular intervention may be utilized to modulate exosome function. Although the micro-anatomical and molecular structures have been well documented in the scientific literature, its nanoscopic landscape remains largely unexplored. Recently the scientific focus has been on how cells nanoscopically interact with each other under normal and pathological conditions. HR-SEM enables the observation of the nanoscopic anatomical complexity of the BBB endothelium. This study provides a glimpse of that complexity by describing the exosome landscape on the BEC and the novel development of TUNTs that facilitate the interaction of adjacent BECs.The BBB is essentially a physical anatomical barrier that seals its paracellular (PC) spaces with TJs only allowing for molecular and ionic transendothelial transport in vitro BEC monolayer. Once cells divided to the point where they were semi-confluent, exosomes were formed in abundance on the BEC surface. We could easily characterize two main types of exosomes, namely, those involved in the formation of TUNTs (TF-exosomes) and those that were not (NTF-exosomes). The formation of TUNTs from extracellular exosomes is novel for the endothelium of the BBB. We have not been able to currently find TF-exosomes in other epithelia or tissue. This may be that they are yet to be discovered or that TF-exosomes are peculiar to the endothelium of the BBB.Exosomes are only found on bEnd5 cells once they grow closer to each other. The initially seeded bEnd5 cell displays a fairly denuded exosomal and NT landscape during the formation of the However, the documentation of extracellular vesicles (EVs) has been sighted in many bacterial species -dependent and ESCT-independent mechanistic pathways, that attenuates the production and secretion of the exosome, in conjunction with the down-regulation of Programmed death-ligand 1(PD-L1) expression on sub-unit sensitive exosome . The impermeability of the BBB is crucial to the homeostasis of the neuronal environment and also ensures that toxic substances that cause neurodegeneration are transported out of the brain, while toxic blood-borne substances are kept out of the brain.via extraneous exosomes, which could be designed to increase permeability and access to metastatic cancer cells and other predatory blood-borne cells. Furthermore, it is well known that exosomes are released in colossal amounts and may be involved in both an immune response to prime tumor progression and survival via tumoral angiogenesis  have been reported to utilize the oncosome secretions from the site of the primary tumor into the blood to prepare distal anatomical sites for metastatic access of cancer cells  and multiple sclerosis (MS) are all neurodegenerative diseases characterized by BBB disruption caused by an increase in transendothelial permeability  and hyperphosphorylated tau in the brain leading to neuronal damage  NTF-exosomes that have the propensity to mediate cell-cell interaction and function by creating a chemo attractant gradient within the PC space; and (ii) TF-exosomes that are formed repetitively and asynchronously, which fuse with each other to form TUNTs during BEC barrier genesis.via the intercellular, cross-bridge TUNT formation in BECs, which could be implicated in the spread of molecular signaling during tissue formation and in the progression of pathology. Moreover, the exosome displays the potential to play a critical role in drug delivery across the BBB. At this stage mechanism of exosome extrusion from the BEC and its ability to transform into TUNTs crucial for functional BBB genesis, suggest its propensity as a promising target for disease intervention.Based on the snap-shot development of BEC TUNT formation, we can infer that exosomes have signaling potential The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.SM shares first authorship. DF shares last authorship. All authors contributed to the article and approved the submitted version.This research received no external funding. Funding was received from the University of the Western Cape (UWC) Senate funds.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The primary objective of this observational study was to evaluate the prevalence of contamination from independently collected quarter-level milk samples pooled in a laboratory and subjected to bacterial culture. To address this objective, weekly quarter-level milk samples were collected longitudinally from a cohort of 503 primiparous cows from five organic dairy farms during the first 5 weeks after calving. Individual quarter milk samples were pooled in a laboratory using aseptic technique (“lab-pooled”) and subjected to bacterial culture. In the sample set of 2,006 lab-pooled milk samples, 207 (10.3%) were classified as contaminated using a standard definition . Subsequent culturing of corresponding quarter-level milk samples revealed that many of the contaminated lab-pooled sample results  were the result of intramammary infections with different pathogens across the quarters, rather than actual contamination within any single quarter . The odds of true contamination were lower when the lab-pooled sample exhibited growth of three microorganisms compared to more than 3 microorganisms. Our findings suggest that pooling of quarter samples within a laboratory setting may yield lower rates of contamination compared to those previously reported from samples composited on-farm, but that current cut-offs to define contamination may need to be evaluated for use with lab-pooled samples. Further investigation of use of lab-pooled samples may be warranted to reduce costs while still providing useful scientific insight. Staphylococcus aureus, particularly in large herds when the prevalence is low and thus the negative predictive value would be high despite potential low sensitivity from pooled samples. Previous work has shown that subjecting pooled milk samples to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can provide additional benefit by allowing for the identification of different bacterial species, which more traditional on-farm culture does not typically provide , Minnesota , New Mexico (n = 122), and Texas (n = 130) based on convenience sampling. Herd-level characteristics of each farm are presented in The samples used in this study were collected as part of a larger observational study investigating associations between the cow udder microbiome and mastitis. The study was approved by the University of Minnesota Institutional Animal Care and Use Committee (Protocol Number: 1807: 36109A). In this longitudinal study, 503 primiparous Holstein cows were enrolled from 5 certified organic dairy farms in: Colorado  and subjected to 15 min of ultraviolet light exposure. Thawed milk samples were homogenized by shaking them back and forth. Next, 2 mL of milk was extracted from each quarter-level vial and dispensed into a single sterile plastic vial (“lab-pooled sample”). Quarter milk samples were then placed back inside −20°C. The lab-pooled samples were then submitted to the Udder Health Lab at the University of Minnesota  for culture. Lab-pooled milk samples classified as contaminated were resubmitted at the quarter-level for culture, using the same overnight thawing and homogenization protocols as described above. A total of 207 lab-pooled samples were classified as contaminated, however 57 samples did not contain enough milk across at least 4 quarters, typically because the cow was difficult to sample on that day, or the quarters did not contain enough milk to obtain a full 10 mL. Of the remaining 165 samples, 150 had sufficient milk across all 4 quarters, resulting in a total of 600 quarter-level results. A total of five lab technicians and graduate students were involved in the pooling procedure, and each received hands-on training in aseptic technique by a PhD-level molecular biologist before performing pooling on the study samples.For both pooled and quarter level samples, approximately 100 μL of milk was plated onto blood agar using a sterile cotton tip swab. Cultivation was performed under aerobic conditions by a single trained technician who regularly performs these procedures on submitted milk samples. Samples were incubated for 42–48 h at 37°C, as described previously , 10. A sContamination of a lab-pooled milk sample was defined as growth of three or more distinct microorganisms . Contamipost-hoc sample size calculations were performed to estimate the minimum sample size needed for an alpha (type I error) of 0.05; prevalence of true contamination of 46.7% ; and minimum difference of 20%. The resulting sample size was then multiplied by 1.2 to account for non-independence of observations within each farm, based on a previous estimate . Summary statistics for animal and farm characteristics were generated to assess the integrity and accuracy of the data, and electronic and paper records were utilized to correct for any discrepancies identified . Two outcomes were modeled using the resulting data: 1) odds of contamination in the lab-pooled samples and 2) odds of true contamination in the lab-pooled samples. Both models were constructed using a mixed logistic regression modeling approach as implemented in the glmer function in the “lme4” package  as compared to the results that would have been obtained from the corresponding quarter-level samples , as implemented in the “fmsb” package .Staphylococcus aureus and Streptococcus spp. and Streptococcus-like organisms , the most common microorganisms included non-aureus Staphylococci, rganisms , though Based on the standard definition of contamination , 207 of the 2,006 cultured lab-pooled samples (10.3%) were classified as contaminated, with 43 of the 207 (20.8%) exhibiting growth of exactly three different microorganisms and 164 of the 207 (79.2%) more than 3 different microorganisms . Of thesP < 0.05, P < 0.05, Among the 80 lab-pooled samples with no contaminated quarters, 60.0% exhibited growth of 3 or more distinct microorganisms across all quarters comprising the lab-pooled sample, while 15.0, 13.8, and 11.3% exhibited growth of two, one or no microorganisms, respectively . The proP = 0.02, The number of microorganisms detected in the contaminated lab-pooled milk samples was associated with true contamination status  for quarter milk samples and 49 days  for pooled milk samples. We observed a statistically significant association between storage duration and contamination in lab-pooled samples, with the odds of contamination decreasing with increased storage duration (OR (95% CI) = 0.76 (0.63–0.91), A unique feature of this study was the aseptic pooling of milk from individually collected quarter milk samples. Using this approach, we reported an overall contamination prevalence of 10% , which iDespite the relatively low prevalence of contamination in lab-pooled samples, we wanted to understand whether such results stemmed from true contamination or presence of distinct microorganisms across quarters. Therefore, we decided to perform culture on the quarter-level milk samples that corresponded to the contaminated lab-pooled samples. These quarter-level results revealed that only 46.7% of contaminated lab-pooled samples were actually contaminated in at least one quarter . To explStaphlococcus spp. , alFuture studies may wish to investigate the economic viability and practicality of the sample collection and screening procedures described in this study. Although the pooling of quarter samples in a laboratory may not be as efficient compared to collecting quarter milk into a single vial on-farm, it may represent an acceptable compromise between contamination and practicality. Furthermore, it could be hypothesized that pooling quarter level samples in the lab would result in significantly lower contamination rates than those pooled on-farm, with a much lower cost for bacterial culture. On a well-managed farm, many of the cultures would yield no-growth results, and if pooled, would equal the direct cost of a single culture; if not pooled, this would equal the direct cost of four cultures. In such cases, pooling could significantly reduce diagnostic costs, even if follow-up cultures were necessary for positive cows. However, it should be noted that direct diagnostic costs are only a small proportion of all costs associated with mastitis , and indThe farms in this study represented a unique population of farms given their organic-certified status; additionally, they represented a range of herd sizes and management strategies . Based oMilk collected at the quarter level on farm and then aseptically pooled in a laboratory could be a cost-effective and robust method to screen for zero-tolerance pathogens at the herd-level, as it obviates the limitations introduced by collecting quarter milk into a single vial; and allows for the retention of quarter-level data that might otherwise be lost or discarded using an on-farm pooling approach. However, further research is needed to understand the expected level of contamination in lab-pooled samples, particularly when collected and processed by different personnel. One of the strengths of this study was that samples were collected and processed by highly trained personnel, however this also means that the results may not be directly applicable to samples collected and processed under more typical conditions. In addition to applied uses, the lab-based milk pooling approach may have application within research studies that investigate epidemiological or biological questions focused on the cow level, and when funds are limited and information retention is critical.In this study, we described the prevalence of bacterial contamination in milk samples collected from individual quarters that were then pooled in a laboratory prior to culture. Our results, based on samples collected from first lactation Holstein heifers on five organic U.S. dairies, indicate that rates of contamination comparable to those of quarter milk samples are achievable when culturing composite milk samples with proper sampling and pooling techniques. Under these circumstances , pooled samples may provide useful information while reducing total cost. The short-term retention of quarter-level samples also provides an opportunity to retrospectively identify quarter-level intramammary infections if the corresponding pooled sample is found to exhibit bacterial growth. To maximize the utility of lab-pooled samples in large screening programs or research studies, further evaluation is needed to understand biases across pathogens, farms and sampling procedures; and to understand how different cutoffs for contamination in the lab-pooled sample impact diagnostic sensitivity and specificity and potential for misclassification.https://github.com/TheNoyesLab/Comparing-lab-pooled-and-quarter-level-milk-cultureThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by University of Minnesota Institutional Animal Care and Use Committee. Written informed consent was obtained from the owners for the participation of their animals in this study.CD and FP-M wrote initial and final drafts of the manuscript, conducted statistical analysis, and collected milk samples. TR prepared milk samples for bacterial culture. CD, FP-M, NN, LC, BH, VM, and PP collected milk samples, enrolled farms and animals, and revised initial and final drafts of the manuscript. LC and NN obtained grant funding, designed the study, and revised initial and final drafts of the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by the Organic Agriculture Research and Extension Initiative (OREI) from the National Institute of Food and Agriculture (Grant Number: 2018-51300-28563).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.