The well-orchestrated process of wound healing may be negatively impacted from interrupted or incomplete tissue regenerative processes. The healing potential is further compromised in patients with diabetes mellitus, chronic venous insufficiency, critical limb ischemia, and immunocompromised conditions, with a high health care burden and expenditure. Stem cell-based therapy has shown promising results in clinical studies. Mesenchymal stem cell-derived exosomes (MSC Exos) may favorably impact intercellular signaling and immunomodulation, promoting neoangiogenesis, collagen synthesis, and neoepithelization. This article gives an outline of the biogenesis and mechanism of extracellular vesicles (EVs), particularly exosomes, in the process of tissue regeneration and discusses the use of preconditioned exosomes, platelet-rich plasma-derived exosomes, and engineered exosomes in three-dimensional bioscaffolds such as hydrogels (collagen and chitosan) to prolong the contact time of exosomes at the recipient site within the target tissue. An appropriate antibiotic therapy based on culture-specific guidance coupled with the knowledge of biopolymers helps to fabricate nanotherapeutic materials loaded with MSC Exos to effectively deliver drugs locally and promote novel approaches for the management of chronic wounds. Extracellular vesicles (EVs) are cell-specific lipid-bound organelles that facilitate intercellular communication with their cargo elements, including proteins, nucleic acids, and certain lipids . VariousThe process of wound healing has four phases: (a) hemostasis; (b) inflammatory; (c) proliferative; and (d) remodeling  10,11,1,13,14,15(a) Hemostasis: The earliest phase in wound healing starts with the formation of a platelet plug and the activation of the coagulation cascade to reduce bleeding. Platelets are activated when they come into contact with extracellular collagen, releasing growth factors that cause platelet aggregation and clumping. This is followed by the activation of the coagulation cascade.(b) Inflammation: This phase begins within 24 h of the injury and lasts up to 2 weeks, first with the recruitment of neutrophils and then macrophages. These cells release various cytokines  and growth factors  to activate fibroblasts and epithelial cells. Neutrophils serve as the first line of defense. Macrophages are activated later. The classical proinflammatory pathway of macrophages is activated first, followed by the alternate macrophage pathway (M2).(c) Proliferative phase: This phase is characterized by fibroblast migration, collagen and extracellular matrix production, angiogenesis, the laying of granulation tissue, and epithelialization. Fibroblasts begin moving by first binding to the matrix components (such as fibronectin) via the integrity receptors. The direction of the fibroblast movement is determined by the concentration gradient of cytokines and growth factors. Fibroblasts secrete matrix metalloproteinase, collagenase, and gelatinase to degrade the extracellular matrix, facilitating cell migration and movement. After fibroblast migration, there is an increased production of the extracellular matrix through stimulation by TGF-β and PDGF. Damaged vasculature must be replaced by new blood vessels through angiogenesis, stimulated by VEGF, HIF, and PEGF. In epithelialization, epithelial cells around the margin of the wound lose contact inhibition and epiboly migrate into the wound area . ,17. 4,17In this field, the best-known mechanism is probably the endosomal sorting complex required for transport (ESCRT) ,21. The The preconditioning of MSCs is performed in the first 24 h after harvesting. This period of incubation alters the cell microenvironment by inducing hypoxia, oxidative stress, and inflammation. Four cycles of ultracentrifugation are required to isolate the extracellular vesicles from the preconditioned MSCs. At the end of the first spin, the cell debris is formed; after the second spin, large-sized extracellular vesicles are collected. After the third spin, medium-sized extracellular vesicles are collected and in the last spin, small-sized extracellular vesicles are obtained ,26,27. VPlatelet-derived extracellular vesicles are most abundant in the circulation and help to activate platelets and the formation of fibrin clots. Platelet-derived extracellular vesicles activate both extrinsic and intrinsic pathways . They inNeutrophil-derived extracellular vesicles (NDEVs) show anti-inflammatory and proinflammatory functions, depending on environmental factors ,46. TheyThe TLR4/NF-κB/STAT3/AKT regulatory signaling pathway plays a critical role in the regulation of macrophage plasticity . LPS-preHuman BM-MSC EVs, when administered as an IV injection at the wound site, promote wound healing and the polarization of macrophages to the M2 phenotype. EVs in vitro human monocytes/macrophages promote M2 macrophage polarization through the transfer of miR-223 . MelatonEVs derived from umbilical progenitor cells have proangiogenic effects . They stBM-MSC-derived Exos accelerate wound healing by targeting fibroblasts via the Akt, Erk1/2, and STAT3 signaling pathways . FGF-2, Ren et al. demonstrated that AD-MSC-derived EVs facilitate wound healing via the AKT serine/threonine kinase 1 (AKT) and mitogen-activated protein kinase 1 (ERK) signaling pathways . These AIn dermal fibroblasts and in a keratinocyte-deficient wound model, UC-MSC-derived EVs promoted the proliferation and migration of fibroblasts and keratinocytes through miR-27b, which acts by suppressing E3-ubiquitin ligase ITCH, thereby activating JUNB/IRE1α . UC-MSC-Apoptotic body (AB)-derived EVs accelerate cutaneous wound healing, promote macrophage M2 polarization through downregulated iNOS activity and an upregulated ARG:iNOS ratio, and enhance the functions of fibroblasts and keratinocytes in the wound healing pathway. Given the lack of standardization, cell sources, and retention rate of MSCs after transplantation, AB-derived EVs provide a promising platform to develop a cell-free therapy .EVs facilitate collagen 1 cross-linking and promote collagen gel contraction. A few components of fibrocyte-derived EVs (FDEVs) such as hsp-90 alpha and STAT-3 promote cell motility and re-epithelialization ,87,88. FTSG-6-modified BM-MSC-derived EVs suppress scar formation by suppressing SMAD2/3 signaling and by inhibiting TGF-β1, COL1, COL3, and SMA-α protein synthesis and inflammation in the wound site . A localAD-MSC-derived Exos facilitate extracellular matrix remodulation in wound repairs by enhancing and regulating the COL3:1, TGF-β3:TGF-β1, and MMP3:TIMP1 ratios via the ERK/MAPK signaling pathways to mitigate the minimization of scar formation . The proζ protein, which inhibited WNT/β-catenin signal transduction, enhanced collagen deposition, and inhibited excess fibroblast expansion in burn wounds. Such mechanisms have improved tissue remodeling and reduced scar formation in burn wounds [UC-MSC-derived EVs suppressed TGF-β-induced myofibroblast formation in a mouse skin wound model. These EVs were enriched with miR-21, -23a, -125b, and -145, which reduced the TGF-β/SMAD2 signaling in the fibroblasts . An accen wounds . MSC-dern wounds ,108. A sEVs have been classically thought to be vestigial and have been poorly investigated for regenerative medicine purposes. Pluripotent nanovesicles can be retrieved from MSCs of various origins  and endothelial progenitor cells, and only recently have MSC-derived EVs been more intensively studied for their application in tissue-specific regenerative medicine. Intercellular communication and the specific tissue targeting effect mediated by these nanobiomolecules play a role in immunomodulation, angiogenesis, the amplification of the growth potential, and regeneration ,112,113.The wound healing tendency in immunocompromised conditions such as diabetes mellitus and chronic kidney disease is negatively impacted by an impaired local immunity, which leads to a prolonged inflammatory phase and poor vascularity. In such conditions, the priming and recruitment of neutrophils are compromised and innate immunity tends to be defective. In these chronic wounds, engineered EVs have the potential to improve the chemotactic response, activate the respiratory burst of the neutrophils, and facilitate neoangiogenesis and site-specific tissue differentiation, promoting healing . EV therMacrophages are involved in phagocytosis and the process of tissue healing. They are phenotypically classified into M1  and M2  macrophages. Studies have shown improved wound healing after the administration of bone marrow-derived MSCs to a wound site by promoting M2 polarization . PolarizThe activation of the Hippo/YAP (Yes-associated protein) signal pathway is essential for the process of epidermal re-epithelization. Platelet-rich plasma contains various growth factors essential for wound healing. EVs derived from PRP show benefits for tissue regeneration by activating the YAP pathway . A PRP-dMost commonly, EVs are delivered via a direct injection at the desired site. However, this can impair the function because of rapid metabolic clearance. Although MSC-derived exosomes have great potential in disease treatment, issues such as rapid clearance and the maintenance of their inadequate preservation for their viability and function remain to be addressed ,121. To Chronic diabetic wounds require a relatively long time for tissue regeneration and healing. Wang et al. developed a novel stem cell-derived exosome-rich biocompatible scaffold that could serve as a sustained release of growth factors with a local immunomodulation to maintain their bioactivity at the wound site. This is more advantageous than the previously used injection technique, given its enhanced bioavailability and its anchorage to the surrounding tissue . Recent Hydrogels of thiolated chitosan (CSS) and polyethylene glycol-maleimide-modified ε-polylysine (EPL-PEG-MAL) showed enhanced elastic and adhesive properties and no cytotoxicity. These scaffolds with EVs have been developed to accelerate angiogenesis, tissue regeneration, and wound healing . HoweverLi et al. constructed a genipin cross-linked dual-sensitive hydrogel loaded with human umbilical cord-derived MSC (hUC-MSC)-derived EVs to boost cutaneous wound healing. The authors showed accelerated collagen deposition, increased epidermal re-epithelialization rates, and early wound closure . In addi2O2 (H2O2-ADSC Exos) reduced apoptosis and increased the capillary density and blood perfusion unit (BPU) of the graft. Therefore, they significantly increased the flap survival rate following an ischemia/reperfusion (I/R) injury [Recently, researchers constructed a 3D scaffold composed of decellularized small intestinal submucosa (SIS) combined with a mesoporous bioactive glass (MBG), which facilitated the sustained release of BMSC-derived exosomes. They induced diabetes in rats by injecting streptozocin and then produced a 1.5 cm full-thickness skin wound. A cryogenic 3D SIS/MBG@Exos hydrogel scaffold was structured according to the size of the wound and they covered the wound with that scaffolding material appropriately. The result showed neoangiogenesis with patterned collagen deposition, the early formation of granulation tissue, and accelerated full-thickness wound healing in the control group . Split-s) injury .Currently, none of the extracellular vesicle products have been approved by the FDA for therapeutic use in humans. The FDA has only approved umbilical cord-derived stem cells for treating certain hematopoietic disorders and issued a Public Safety Notification in June 2019 to prevent the manufacturing or marketing of illegal “stem cell” products . FurtherMost of the studies to date were preclinical. The results of two clinical trials evaluating the efficacy of serum-derived EVs in wound therapy are still awaited ,138. TheFull-thickness cutaneous wound therapy and regeneration remain major clinical challenges. Wound healing is a highly integrated complex biological process that involves hemostasis, immunomodulation, intercellular communication, and the well-orchestrated release of various growth factors. This combined effect of local molecular homeostasis eventually induces collagen deposition and tissue regeneration. Stem cell-derived EVs can build up a pro-healing environment by activating intercellular signaling, angiogenesis, proliferation, and the regional differentiation of the various cell types in tissue regeneration.In addition, the development of 3D printing technologies can help to fabricate size-specific functional scaffolds to be used in the treatment of chronic non-healing wounds. Continued advances in controlled drug delivery using MSC EVs should allow for the development of new highly effective loco-regional antibiotic delivery strategies. However, the therapeutic benefits of MSC EVs need to be proven in large-scale clinical studies to test the efficacy of such novel treatment modalities.


The scientific, private, and industrial sectors use a wide variety of technological platforms available to achieve protection against SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), including vaccines. However, the virus evolves continually into new highly virulent variants, which might overcome the protection provided by vaccines and may re-expose the population to infections. Mass vaccinations should be continued in combination with more or less mandatory non-pharmaceutical interventions. Therefore, the key questions to be answered are: (i) How to identify the primary and secondary infections of SARS-CoV-2? (ii) Why are neutralizing antibodies not long-lasting in both cases of natural infections and post-vaccinations? (iii) Which are the factors responsible for this decay in neutralizing antibodies? (iv) What strategy could be adapted to develop long-term herd immunity? (v) Is the Spike protein the only vaccine target or is a vaccine cocktail better? Adaptive immune responses triggered by SARS-CoV-2 infections, including B and T lymphocyte cell response, can induce immunological memory during primary infection to prevent or decrease disease severity on repeated exposures to the same pathogen. On reinfection, antibodies produced by short-lived plasma cells derived from B lymphocytes bind to and block the recognized virus, and the long-lasting bone marrow plasma cells (BMPCs) become resident in the bone marrow and retain the ability to produce antibodies for decades .The SARS-CoV-2 Immunity & REinfection EvaluatioN (SIREN) study enrolled 8278 SARS-CoV-2 seropositive and 17,383 seronegative healthcare workers in England between 18 June 2020, and 31 December 2020 [However, this “long-lasting” immunity may not protect against reinfection, and despite huge numbers of infections and deaths worldwide caused by SARS-CoV-2, herd immunity could not be achieved in communities with the previous infection of most of its population. One factor which affects protection against reinfections is the severity of primary infections. In severe SARS-CoV-2 infection, the immune response may be impaired, which will decrease affinity maturation, memory quality, and quantity . In contIt has also been reported that circulating anti-spike IgG antibodies remain detectable one year after hospitalization of COVID-19 patients, and these higher antibody titers and disease severity were associated with increased durability of detectable antibodies . AlthougAnother concern about assessing long-lasting immunity after primary SARS-CoV-2 infection is the difficulty of identifying the precise incidence of SARS-CoV-2 reinfection ,11. ViraNew SARS-CoV-2 variants may be the most important explanation for the immunity loss due to the presence of mutations making them more transmissible, more efficient at avoiding the host immune system and evading immunity elicited by primary infection. In fact, throughout the COVID-19 pandemic, many SARS-CoV-2 variants have appeared. Some are variants of interest (VOI) that may be associated with reduced efficacy of available treatments and vaccinations, and predicted increase in transmissibility or disease severity , B.1.525 (Eta), and B.1.617.1 (Kappa)), and some are variants of concern (VOC) that may cause increase in transmissibility, more severe disease , significant reduction in neutralizing antibodies generated during previous infection or vaccination , Beta (B.1.351), Delta , Gamma (P.1), and most recently Omicron (B1.1.529)). The seriousness of this issue can be illustrated by the case in Manaus, Brazil, where SARS-CoV-2 infected more than 70% of the population by October 2020, which should have provided herd immunity. However, a surge of reinfection occurred in late December 2020, and early January 2021; i.e., about 7 months after the first wave ,13. The Novel mutations will appear as long as the virus continues to spread. This emphasizes the need for precautionary measures to reduce the risk of infection, such as mask-wearing, physical distancing, hand hygiene, and surface sanitation. Obviously, among the very important constituents of the successful strategies to control pandemics are the existing and future anti-SARS-CoV-2 vaccines.Available SARS-CoV-2 vaccines stimulate adaptive immune responses against different SARS-CoV-2 Spike (S) proteins and decrease both symptomatic and asymptomatic disease incidence with the development of immunological memory.The prospective SIREN study among staff working in publicly-funded hospitals in England demonstrated that the BNT162b2 mRNA vaccine could prevent both symptomatic and asymptomatic infection caused by the B1.1.7 (Alpha) variant by 70% (95% CI 55–85) 21 days after the first dose and 85% (74–96) 7 days after the second dose . For theDespite the high efficacy of available COVID-19 vaccines, SARS-CoV-2 infections can still occur in vaccinated individuals due to the decline in neutralizing antibody titers 7–8 months post-vaccination. Furthermore, the emerging new SARS-CoV-2 variants are one of the most important sources of post-vaccination infections . The repConcerns about not acquiring post-vaccination herd immunity also include the uneven distribution of vaccines among countries and within individual countries, and the age limit of vaccination ). These factors make it more difficult to reach the levels of the vaccinated population needed to achieve herd immunity . A studyhttps://www.cdc.gov/coronavirus/2019-ncov/variants/delta-variant.html (accessed on 10 December 2021)). Currently, it is not recommended to test vaccinated people following exposure to infection. However, highly tested groups, such as professional sport teams, demonstrated that asymptomatic breakthrough infections among vaccinated people might be higher than reported.Another thing to consider is the possibility of virus spread by fully vaccinated individuals without manifesting breakthrough disease. Data released by the CDC reported that vaccinated people infected by the SARS-CoV-2 Delta variant could carry viral loads similar to those of unvaccinated people (https://ir.gritstonebio.com/static-files/6a7c26ca-06a6-4295-bf76-83948a341397 (accessed on 10 December 2021)). These observations suggested that the pan-vaccine approach could potentially be a way to outsmart the virus.A crucial question related to vaccine efficacy against current and emerging SARS-CoV-2 is whether we are looking in the wrong place. Most of the current vaccine development focuses on the S protein as a target. Unfortunately, the virus mutates faster than humans engineer vaccines, so it might be a good idea to diversify our efforts. Obviously, the inactivated whole virus vaccines can generate a broad repertoire of antibody responses and the SARS-CoV-2-based vaccine from Sinopharm was approved by the WHO  for emergency use in May 2021. The surprising observation made in several published reports is that the comparison of the inactivated whole virus vaccines with mRNA vaccines revealed that the mRNA-based vaccines were systematically more efficient and durable than the inactivated whole virus vaccines ,36,37,38Many recovered patients have been reinfected worldwide, despite being among the population that had acquired immunity against the virus. However, the best-known example of breakthrough of a population with expected established herd immunity was observed in Manaus city , where from June 2020 to October 2020 the SARS-CoV-2 prevalence increased from 60% to more than 70%, a condition which may mirror achieving herd immunity . By Januhttps://www.wcvb.com/article/massachusetts-covid-breakthrough-cases-delta-variant-pandemic-vaccine-data-charts-maps/37089843 (accessed on 10 December 2021)). However, the Department of Public Health of Massachusetts reported 7737 total COVID-19 breakthrough cases in fully vaccinated individuals as of 3 August 2021. Among the breakthrough cases, 395 patients have been hospitalized, and 100 have died. Genomic sequencing of specimens from 133 patients revealed that this breakthrough was caused by the newly identified and highly transmissible SARS-CoV-2 variants. The B.1.617.2 (Delta) variant was identified in 119 (89%) patients, and one person was infected by the Delta AY.3 sublineage (1%). Since the youngest age group (12–15) showed the lowest coverage by one and/or two vaccine doses (43% vs. >60% in other age groups), these data indicated the need for higher vaccination levels in this group. Although many factors, such as hesitancy and shortage of vaccine supply  [On a personal level, recovered patients can be reinfected, and the vaccinated persons can contract new infections (despite being previously infected or not). For example, the CDC reported that Massachusetts (USA) has already fully vaccinated more than 4 million people  (56.899%). The total rate of fully vaccinated people in the Barnstable County is 76% , with thThe emergence of breakthrough infections has urged many developed countries to call their citizens for additional vaccine booster doses  from the same or newly approved vaccines (combined vaccines). However, it is not clear whether such boosting will help overcoming the viral infectivity and/or transmissibility, especially related to emerging new variants. The COVID-19 pandemic incidences worldwide clearly indicate that it might not. Overall, it seems that this virus can overcome herd immunity , suggesting that it can prevail in the general population for a long time. Of note, young individuals may represent a live source for new infections, as most of them, when infected, are asymptomatic or show light to moderate symptoms.https://apps.who.int/iris/handle/10665/341241 (accessed on 28 April 2022)), the levels of which decline during 6–12 months post-infection. Of note, the acquired herd immunity would not protect from reinfection but would significantly enhance the chance of building up the hybrid immunity post vaccination with any type of vaccine [As the WHO has emphasized, 90–99% of individuals infected with the SARS-CoV-2 virus develop detectable neutralizing antibodies within four weeks after infection ( vaccine . In paraThe SARS-CoV-2-specific antibody titer kinetics and decay have been evaluated in individuals vaccinated with the BNT162b2 mRNA vaccine and in convalescent COVID-19 patients ,42. AlthTaking all these facts into account, it is clear that the war against COVID-19 should be conducted at multiple levels see , and ourIn light of these considerations, we also need to place greater emphasis on the various hygienic precautions. Therefore, non-pharmaceutical interventions and protective precautionary actions such as use of protective masks, frequent disinfection of public areas, and social distancing, especially indoors, should continue to remain an important and mandatory part of our daily life. One should keep in mind that when the non-pharmaceutical interventions are relaxed when the majority of the population has already been vaccinated, the probability of the emergence of a new resistant strain is greatly increased. Therefore, individuals should be encouraged to maintain the non-pharmaceutical interventions and transmission-reducing behaviors throughout the entire vaccination period . Obvious


Nevertheless, samples from all analyzed persons contained MBCs with neutralizing antibodies against Omicron Spike receptor-binding domain (RBD), which provide protection against this SARS-CoV-2 variant to some degree.Initiation of humoral immunity occurs in several steps Fig. . While l2 During 6 months following infection RBD-specific MBCs cells expanded and matured. They progressively accumulated mutations in variable regions but retained strong neutralizing potential against the ancestral SARS-CoV-2 strain (Hu-1).3 Importantly, mRNA vaccination boosted those cells, inducing a novel round of GC reaction and further mutation events in the variable regions of heavy chain (VH) domains.3 Almost all vaccine-induced MBCs containing >10 mutations within the VH and produced mid- and high-affinity antibodies directed against the Hu-1 strain. Although some MBCs increased their antibody affinity, the overall diversity of the RBD-specific MBC repertoire was maintained and was similar to the repertoire of MBCs from virus-naive, vaccinated individuals. Depending on the donor, 5–60% of this broad MBC repertoire had high-affinity clones against Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2.3 These data suggest that infection- or vaccination-induced MBCs may still provide neutralizing antibodies upon vaccination boost or reinfection with novel virus variants.The word wide onset of COVID-19 vaccination campaigns allowed researchers to study the de novo formation of humoral immunity against SARS-CoV-2 and to compare the one found in vaccinated and non-vaccinated COVID-19 convalescent individuals. While many studies focused on the production of SARS-CoV-2-specific antibodies in serum, the group of Pascal Chappert and Matthieu Mahévas focused on virus-specific MBCs. Previously, they showed that SARS-CoV-2 infection induces not only high serum titers of anti-SARS-CoV-2 spike antibodies but also differentiation of circulating anti-spike MBCs.1 However, Sokal et al. now provide evidence of incomplete immune evasion of Omicron to circulating MBCs.1 The authors shown that although approximately 70% of the analyzed MBC clones showed no or low affinity to Omicron RBD, approximately 30% of the antibodies encoded by the MBCs of convalescent and/or vaccinated individuals still retained high affinity. The reason for such a drastic drop in affinity lies in the fact that, compared to other variants, Omicron-specific mutations seem to affect a more diverse range of RBD-specific MBCs. Of note, potent neutralizers of other SARS-CoV-2 variants had the highest loss of affinity against Omicron RBD. Furthermore, the loss of neutralization potency was even more pronounced and only 13% of the high-affinity MBCs could still produce anti-Omicron-RBD neutralizing antibodies infection. A significant hurdle for their reactivation is immune imprinting . In this phenomenon when adaptive immunity to a particular antigen is boosted with a second, cross-reacting antigen, the ensuing recall responses react faster to the initial priming antigen, hampering the creation of de novo responses. Recent report supported the antigenic sin model in the context of SARS-CoV-2 vaccination. In that study, the authors boosted animals that had been vaccinated twice before with a Hu-1-based mRNA vaccine with either original vaccine or an Omicron-adapted version. When testing the sera collected sera 14 days after the booster injection for their ability to neutralize Omicron no differences could be observed.


In patients with severe coronavirus disease 2019 (COVID-19), acute kidney injury (AKI) is common 24-57%), particularly in those treated in intensive care unit (ICU) (61-78%), and is associated with poor outcomes.-57%, par5In a recent edition of the journal, (Pak J Med Sci) Anees et al.6However, although the incidence and clinical predictors of AKI during acute phase of COVID-19, and impact on prognosis are now well-established, it is important to direct focus towards those patients who survive severe COVID-19 and develop post-acute sequelae. These are defined as signs and symptoms lasting beyond 30 days after initial infection with SARS-CoV-2 virus. This involves pulmonary and extra-pulmonary organ manifestations, such as incident diabetes, arterial damage  and subclinical myocardial dysfunction assessed by speckle-tracking echocardiography or cardiac magnetic resonance imaging with and without persistent myocardial inflammation and edema, elevated blood pressure and resting heart rate and anemia .8-11 In The incidence and clinical predictors of AKI during acute phase of COVID-19, and impact on prognosis are well-established. Future studies should direct their attention towards the late complications, particularly decline in renal function as post-acute COVID-19 syndrome sequelae, also referred to as long COVID. These late clinical outcomes require careful medical attention with earlier and more frequent follow-up strategies to avoid long-term cardiovascular complications.SS prepared the first draft of the article which was edited by DG.Both authors approved the final submission.


Immunoglobulin G4-related disease (IgG4-RD) is a systemic immune-mediated fibro-inflammatory disorder. Coronary IgG4-RD has been scarcely reported and may present as “tumor-like” lesions. These pseudo-masses may be underdiagnosed mainly due to a vague clinical picture that can vary from complete lack of symptoms to acute coronary syndrome or sudden cardiac death. Early recognition of coronary IgG4-RD is essential to monitor disease activity and prevent life-threatening complications. We report a comprehensive non-invasive imaging evaluation of a patient affected by coronary IgG4-RD, which was diagnosed as an incidental finding during routine pre-laparoscopic cholecystectomy checkup. Non-invasive imaging revealed the presence of a peri-coronary soft-tissue mass that was stable at 12 months follow-up. Immunoglobulin G4-related disease (IgG4-RD) is an emerging fibro-inflammatory condition with unknown etiology, characterized by lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells, storiform fibrosis, tumor-like lesions and elevated serum IgG4 concentrations in 60–70% of patients . RecentlPeriarteritis, with involvement of large-to-medium-sized vessels, may also be a manifestation of IgG4-RD as well as inflammatory aneurysm formation. More rarely, this condition has been reported to affect small-sized vessels such as coronary arteries, leading to potentially life-threatening complications, such as acute coronary syndrome or sudden cardiac death. Histopathologic assessment is considered the method of choice to confirm the diagnosis of IgG4-RD; however, it can be challenging to perform biopsy or to obtain surgical samples when this disease involves vital structures. In these cases, non-invasive imaging evaluation of coronary IgG4-RD may play a crucial role both for the diagnosis and management of this condition .Since IgG4-RD involvement of coronary arteries has been scarcely described, our aim is to report multimodality non-invasive imaging findings of this rare entity and to present a review of the literature.A 63-year-old man came to our observation in May 2021 to perform a chest CT study for suspected pulmonary nodule previously detected on a chest X-ray during a routine pre-laparoscopic cholecystectomy checkup. The patient was asymptomatic for angor and dyspnea. Past medical history was significant for multiple comorbidities including obesity, dyslipidemia, arterial hypertension, gouty arthritis, multi-district arthrosis, chronic renal failure, phlebopathy of the lower limbs with dyschromia and trophic ulcerations and multinodular thyroid goiter. The patient reported remote tobacco use, past episodes of chest pain and no history of coronary artery disease.Chest CT did not reveal lung nodules but a peri-coronary “pseudotumor” as an incidental finding. Dual-energy coronary computed tomography angiography (DE-CCTA) was subsequently performed, and showed a soft-tissue mass within the anterior interventricular sulcus, which encased the distal tract of left main stem (LM), the proximal and the middle segment of the left anterior descending artery (LAD), the first diagonal (D1) and the proximal segment of the left circumflex artery (LCx), without causing any stenosis of the involved coronary segments and configuring the typical “pigs-in-a-blanket” sign . All corThe pseudo-mass did not show early enhancement or iodine uptake during arterial phase but had a tenuous late-enhancement at delayed phase A,B. Extended laboratory tests revealed mildly elevated IgG  and IgG4 levels  at turbidimetric assays, normal erythrocyte sedimentation rate, normal C-reactive protein levels, normocomplementemia, negative VDRL test and negative rheumatoid factor (RF) and antineutrophil cytoplasmic antibodies (ANCAs). Slightly high serum IgG4 levels raised suspicions of an immune-mediated disease.Cardiac MRI (CMR) was subsequently performed for a non-invasive characterization of the pseudo-mass. The lesion was isointense to myocardium in bSSFP cine-images, heterogeneously hypointense in T2-weighted sequences, and hypointense in T1-weighted sequences. The pseudo-mass did not present early enhancement during arterial first-pass perfusion imaging, but it showed late gadolinium enhancement (LGE), supporting the provisional diagnosis of a fibrotic/granulomatous lesion .In the suspicion of coronary IgG4-RD, the patient underwent a 18F-fluorodeoxyglucose (FDG) positron-emission tomography (PET) scan, which did not show an increased FDG uptake of the cardiac pseudo-mass C.In consideration of the complete absence of symptoms, the patency of coronary vessels and the good cardiac function, a biopsy was contraindicated because of the high procedural risk. Moreover, since the non-invasive imaging characteristics of the pseudo-mass were not consistent with active inflammation or malignancy, no surgical or pharmacological treatment was planned.The patient underwent DE-CCTA follow-up studies at 6 months and 12 months, which demonstrated substantial stability of radiological findings and good clinical condition .IgG4-RD is a systemic immune-mediated inflammatory disease typically associated with high serum IgG4 concentration and IgG4+ plasma cell infiltration that can involve several organs. The most common sites are the pancreas, salivary glands, retroperitoneal organs, and liver ,3.Cardiovascular system involvement may manifest with arteritis or periarteritis and inflammatory aneurysms of large-to-middle-size vessels. Coronary artery involvement  has rarely been reported in the literature, but over the past years its frequency has increased, probably due to the increasing availability of advanced cardiovascular imaging techniques. When this occurs, clinical presentation may vary from complete lack of symptoms to potentially fatal conditions such as acute coronary syndrome or sudden cardiac death .Currently, IgG4-RD diagnosis is defined by the Comprehensive Diagnostic Criteria proposed in 2012 by Umehara et al. and revised in 2020, which refer to specific clinical, radiological, serological and pathological findings [Serological findings imply detection of IgG4 serum levels > 135 mg/dL, which were considered critical for the diagnosis of IgG4-RD. Nonetheless, almost 30% of patients shows normal or mildly elevated IgG4 serum and their increase has shown to depend on the number of involved organs, and it is more common in case of pancreatic disease. Increased IgG4 concentration is also not specific for IgG4-RD, since other entities have also been associated with this laboratory finding  .Biopsy has always been considered the gold standard for the diagnosis of IgG4-RD, since it allows confirmation of the presence of the typical histological features characterized by lymphoplasmacytic infiltrate rich in IgG4-positive plasma cells and storiform fibrosis . AccordiIn our case, the lack of clinical symptoms and the benignity of the lesion assessed by comprehensive multimodality imaging suggested a biopsy should not be performed in such a critical anatomical site. As matter of fact, 2019 ACR/EULAR IgG4-RD classification criteria do not require confirmation by biopsy when there is a strong suspicion of IgG4-RD by clinical, serological and radiological findings [In our case, coronary CTA allowed for the direct visualization of the soft-tissue mass completely encasing the coronary vessels without causing their luminal narrowing. This finding configures the typical “pigs-in-a-blanket” sign, which is considered to be specific for coronary IgG4-RD .Dual-energy CCTA also provides additional parameters for non-invasive characterization of cardiac lesions ,13,14,15CMR findings in coronary IgG4-RD have been scarcely described , despiteConversely, in case of active perivascular inflammation, increased iodine density during arterial first-pass in DECT or increased signal intensity in T2-weighted sequences and early gadolinium enhancement in CMR would have been expected findings . Moreove18F-FDG PET/CT represents a remarkable tool to evaluate a systemic inflammatory disease and to highlight IgG4-RD localization for guiding the biopsy, or to monitor treatment response . Its maiThe first-line therapy in patients with symptomatic active IgG4-RD is glucocorticoids, which are extensively used for remission induction and disease relapse, but they can also be used for maintenance therapy. Steroid-sparing agents can also be used for remission maintenance when long-term therapies expose patients to glucocorticoid toxicities. A “watchful waiting” strategy is also suitable in asymptomatic patients and in case of highly fibrotic inactive lesions that are weakly responsive to pharmacologic treatments. In our case, the lack of clinical symptoms, together with laboratory and imaging findings, excluded the presence of active inflammation, and the patient did not require any pharmacological treatment. Moreover, in these patients, the risk/benefit balance may not encourage any therapeutic courses .Coronary IgG4-RD diagnosis is a rare and potentially fatal condition whose diagnosis is generally challenging. Non-invasive multimodality imaging plays a pivotal role for its diagnosis, since performing a biopsy might not always be an option due to the critical location of these lesions. Finally, non-invasive imaging is crucial to monitor disease activity and guide treatment.


Before emergence in late 2021 of the highly transmissible B.1.1.529 (Omicron) variant of SARS-CoV-2, the virus that causes COVID-19 (N) and envelope protein (E) genesAs part of an ongoing longitudinal cohort study, persons with documented SARS-CoV-2 infection  and their household members were recruited within 5 days of the first symptom onset in the household (or first RNA-positive test result if the infected person was asymptomatic). All participants self-collected nasal swab specimens once daily for 2 weeks from the first onset in the household; some participants also provided a serum specimen at enrollment to identify evidence of previous infection.N and E) were included, and the analysis was limited to specimens collected within 14 days of onset for each participant . Participants aged ≥18 years were classified as adults, and those aged <18 years were classified as children and adolescents. Vaccination status was classified as fully vaccinatedE-specific Ct value as the main outcome and variant group (Omicron versus pre-Omicron) as the main exposure, mixed linear regression models were used to account for clustering of multiple specimens per participant, and to control for potential confounding by age group and vaccination status. When Ct values among all or RNA-positive specimens were compared, an interaction term of the product of variant and infectiousness  was included; this interaction term was excluded when Ct values within virus-positive specimens were assessed. Longitudinal sampling of infected participants resulted in some subsequently negative real-time RT-PCR specimens (no target detected); these were included in the all-specimen models and were assigned a Ct value of 40 for analysis. Sensitivity analyses were conducted with comparable models using N-specific Ct values as the outcome. All statistical analyses were performed using Stata Software . This activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.Participants with confirmed infection  and 107 infected with pre-Omicron variants,E-specific Ct values in all specimens were significantly higher in Omicron specimens than in pre-Omicron specimens .E-specific Ct values were significantly higher in Omicron specimens than pre-Omicron specimens .E-specific Ct values than were virus-positive pre-Omicron specimens (p<0.01). Similar findings were observed in the N-specific analysis (p<0.001).Accounting for age group and vaccination status, In this study, and consistent with other published findings  specimens from participants infected with SARS-CoV-2 Omicron variants had significantly higher Ct values than did virus-positive specimens from participants infected with pre-Omicron variants. Supporting CDC guidance (Before emergence of the SARS-CoV-2 B.1.1.529 (Omicron) variant, infectious SARS-CoV-2 was unlikely to be cultured at high cycle threshold (Ct) values. Based on this, low Ct values, which are suggestive of high RNA levels, are sometimes used as surrogate markers for infectiousness.In a longitudinal study including daily nasal swabbing, although Omicron BA.1 sublineage infections exhibited higher Ct values than did pre-Omicron infections, culturable Omicron virus was still detected. Among virus-positive specimens, Ct values were higher for Omicron than for pre-Omicron specimens, especially during the first week of illness.Supporting CDC guidance, these data show that Ct values likely do not provide a consistent proxy for infectiousness across SARS-CoV-2 variants.


Diseases caused by microbial infections continue to pose major public health challenges despite the enormous modernization of therapeutic strategies to alleviate human health disorders. From time to time, numerous such afflictions due to HIV, hepatitis B & C, herpes simplex, dengue, Ebola, and influenza viruses raised universal concern, underscored by the recent COVID-19 pandemic caused by SARS-CoV-2 . AlthougPlant products for antiviral therapeutics was published, comprising eight Original Research articles and seven Reviews/Mini-reviews, which will be briefly introduced in this editorial.Time-honoured herbal prescriptions practised worldwide, such as Traditional Chinese Medicines (TCM), Indian Ayurvedic and Middle-eastern Unani systems, and various regional folkloric treatments in Africa and elsewhere continue to fulfil basic healthcare requirements in developing countries . As for Ping et al. explored the efficacy of Jing Guan Fang (JGF) to prevent SARS-CoV-2 infection. The authors showed that JGF, a TCM formula comprising five commonly used herbs, effectively blocked syncytium formation and inhibited SARS-CoV-2 plaque formation. They demonstrated the underlying mechanism of JGF working via induction of lysosome-dependent ACE2 degradation and inhibition of TMPRSS2 expression in the lung tissue of mice. Additionally, a clinical study conducted on human volunteers recommended that JGF would be a useful preventative measure for frontline medical staff or people who have had high-risk exposure to COVID-19 cases.TCM is one of the world’s classical therapeutic modalities, gaining substantial popularity worldwide. Actually, TCM is known to utilize the characteristics of its multiple components and produces a cocktail-like effect through integrated regulation of multiple biochemical targets to block viral infections . Ping etDuan et al. had explored a prescription of JieZe-1 composed of authenticated botanical ingredients as specified in ancient Chinese scripture and reported its preventive and therapeutic efficacy on HSV-2 patients in Chinese hospitals . Since many years, a TCM called JieZe-1 had been used for the treatment of female lower reproductive tract infections. Hence, ospitals . PresentLai et al. explored a standardized composition of Ganghuo Kanggan Decoction (GHKGD) and its components for prospective activity against the influenza virus, using a series of network pharmacology involving compound screening, target prediction, and pathway enrichment analysis. Hence, a compound-target-disease network was constructed and analyzed to explore the relevant mechanism. Thus, they could identify 116 active compounds and 17 potential therapeutic targets of GHKGD. Further, they established a mouse model to study the therapeutic efficacy of this TCM against viral pneumonia caused by the H1N1 influenza virus. Thus, GHKGD treatment was found to balance the pro- and anti-inflammatory cytokine levels, thereby alleviating mouse lung inflammation due to influenza virus infection. Furthermore, they performed in vivo experiments to reveal the synergistic action of multi-component and multi-target characteristics of GHKGD and offered a theoretical basis for prospective drug development against this contagious respiratory viral disease.For using a complex mixture of natural products, it is crucial to identify the target proteins of TCM and evaluate the mechanism of action. Wei et al. utilized this approach to precisely clarify the efficacy of Hou Yan Qing (HYQ) oral liquid, subsequent to its validation in a mouse model of pneumonia caused by influenza A virus (H1N1), in vivo. Experimental results indicated that HYQ treatment would cause elevation of galectin-3-binding protein and glutathione peroxidase 3 to produce distinctive effects on viral pneumonia patients. This TCM composition of four authentic plant samples as per Chinese Pharmacopoeia 2020, showed multiple effects on the complement system and inflammatory processes; hence, it could be a promising strategy for personalized management of viral pneumonia.Viral pneumonia is one of the most pernicious respiratory ailments; TCM treatments are documented to manage infected patients. The multi-target action of TCMs could be elucidated by way of proteomics . Hence, Balkrishna et al. explored a prospective Ayurvedic medicine called Giloy Ghanvati (GG), an oral tablet prepared from Tinospora cordifolia (Willd.) Hook. f. & Thomson, a well-known tropical plant widely used against various diseases in the Indian subcontinent. GG is a time-tested formulation recommended as a natural immunity booster to improve overall health, particularly to inhibit SARS-CoV-2 infection during the Covid-19 pandemic. Balkrishna et al. developed the SARS-CoV-2 spike-protein induced disease phenotype in a humanized zebrafish model. They studied several parameters to prove that the disease symptoms due to SARS-CoV-2 spike protein expression were substantially ameliorated by treatment of the afflicted fishes with aqueous extract of GG. Also, HPLC analysis of GG extract could detect the major phytoconstituents as cordifolioside A, magnoflorine, β-ecdysone, and palmatine, already known for anti-inflammatory and antiviral activities. Thus, the findings correlated well with the potential role of these constituents of GG against SARS-CoV-2 viral symptoms.Indian Ayurveda is a well-documented traditional system of complementary herbal medicines recommended for chronic ailments and general wellness improvement . Since SVigna radiata (L.) R. Wilczek (mung bean legume), a common functional food in Oriental culture, is sparsely studied for its antiviral potential. Using appropriate techniques, Lo et al. prepared Vigna radiata extract (VRE) from seed coats of mung beans and investigated the antiviral activity and mechanism of action of VRE against the influenza virus. They found that VRE mainly targets viral entry and release by interfering with hemagglutinin and neuraminidase activity and significantly inhibited viral replication in a concentration-dependent manner. The authors concluded that VRE is a multi-step inhibitor for both mammalian H1N1 and avian H6N1 subtypes of influenza virus and demonstrated its broad-spectrum potential as a preventive and therapeutic agent against influenza virus.Buddleja indica Lam. is an evergreen shrub native to Madagascar, locally known as a topical antiseptic. The investigation by Youssef et al. is a rare study to establish the traditional medicinal claims on Buddleja spp. of plants, probably owing to its richness of phenolic acids and flavonoids with antioxidant and hepatoprotective activity. Youssef et al. prepared Buddleja indica Lam. leaf extract with credible anti-tubercular and anti- Helicobacter pylori activity and isolated caffeic acid, quercetin 7-O-β-D-glucoside and kaempferol. Further, in silico study on molecular docking of these compounds showed the highest fitting score in proteins implicated for bacterial infection and the occurrence and progression of SARS-CoV-2 virus.Saha et al. used in silico tools to identify RNA dependent RNA polymerase (RdRp) inhibitors as prospective drug candidates against COVID-19. A collection of 248 plant-derived molecules with strong antiviral activity were subjected to molecular docking against the catalytic sub-unit of RdRp. Pharmacokinetics analysis and molecular dynamics simulation of the best-docked compounds showed that tellimagrandin I, saikosaponinB2, hesperidin & (-)-epigallocatechin gallate were the most prominent ones with a strong binding affinity towards RdRp. Overall, the study unveiled saikosaponin B2 to serve as a prospective molecule for the development of effective therapy against COVID-19, as it was one of the top inhibitors of RdRp, the crucial replication protein. Such an approach combined with biological testing can substantially speed up the drug discovery process from plant products.Huang et al. reviewed an updated selection of phytochemicals active against MERS-CoV, SARS-CoV, or SARS-CoV-2, along with their specific molecular targets and mechanisms of action. Omrani et al. focussed on phylogenetic and taxonomic similarities of viruses, like influenza, MERS-CoV, SARS-CoV, and SARS-CoV-2, and pertinent mechanisms of action. Accordingly, they presented 130 plant-derived compounds with therapeutic potential to combat the currently relevant infections caused by these respiratory viruses. Alam et al. reviewed prospective anti-COVID plant products undergoing clinical trials to address a pandemic-like situation happening in the future. Anywar et al. enlisted medicinal plant species indigenous to Africa and Asia as potential antiviral agents, especially for HIV and SARS-CoV-2. Abubakar et al. reviewed selected medicinal plants and compounds implicated in the mitigation of viral invasion either via direct or indirect modulation of ACE2 activity to ameliorate COVID-19. The review article by Hu et al. focused on herbal products with reported therapeutic efficacy against experimental models of influenza, respiratory syncytial virus, and coronavirus - the three types of viruses eliciting pathologic manifestations of viral pneumonia. Severe respiratory distress and associated lung problems due to SARS-CoV-2 infection often create cardiovascular complications, leading to severe myocardial injuries. Hence, Beura et al. dealt with the significance of inhibiting platelet-mediated thrombus formation and discussed the role of anti-platelet and anti-thrombotic phytochemicals available from medicinal plants.Taken together, it remains to carry on further research and clinical trials on plenty of plant products to get novel therapeutics suitable for clinical application against viral diseases.


Three-dimensional co-cultures of the supporting cell types with either HRE or HUVEC were used as controls. Mucociliary differentiation and formation of vascular-like structures were analyzed by scanning electron microscopy (SEM), periodic acid Schiff’s reaction (PAS reaction), two-photon laser scanning microscopy (TPLSM) and immunohistochemistry. Cytokine levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-6 (IL6), interleukin-8 (IL8), angiopoietin 1, angiopoietin 2, fibroblast growth factor basic (FGF-b) and placenta growth factor (PIGF) in media supernatant were investigated using LEGENDplex™ bead-based immunoassay. Epithelial morphology of tri-cultures with BM-MSC most closely resembled native respiratory epithelium with respect to ciliation, mucus production as well as expression and localization of epithelial cell markers pan-cytokeratin, claudin-1, α-tubulin and mucin5AC. This was followed by tri-cultures with HNF, while ASC-supported tri-cultures lacked mucociliary differentiation. For all supporting cell types, a reduced ciliation was observed in tri-cultures compared to the corresponding co-cultures. Although formation of vascular-like structures was confirmed in all cultures, vascular networks in BM-MSC-tri-cultures were found to be more branched and extended. Concentrations of pro-angiogenic and inflammatory cytokines, in particular VEGF and angiopoietin 2, revealed to be reduced in tri-cultures compared to co-cultures. With these results, our study provides an important step towards a vascularized and ciliated tissue-engineered tracheal replacement. Additionally, our tri-culture model may in the future contribute to an improved understanding of cell-cell interactions in diseases associated with impaired mucosal function.Patients suffering from irresectable tracheal stenosis often face limited treatment options associated with low quality of life. To date, an optimal tracheal replacement strategy does not exist. A tissue-engineered tracheal substitute promises to overcome limitations such as implant vascularization, functional mucociliary clearance and mechanical stability. In order to advance a tracheal mucosa model recently developed by our group, we examined different supporting cell types in fibrin-based tri-culture with primary human umbilical vein endothelial cells (HUVEC) and primary human respiratory epithelial cells (HRE). Bone marrow-derived mesenchymal stromal cells (BM-MSC), adipose-derived mesenchymal stromal cells (ASC) and human nasal fibroblasts (HNF) were compared regarding their ability to promote mucociliary differentiation and vascularization  Among many diseases requiring tracheal resection, airway stenosis caused by long-term intubation represents the most common one . Howeveret al. doubted the use of synthetic tracheal prostheses, as the respiratory tract does not provide a sterile environment and bacterial contamination prevents successful prosthesis integration , human nasal fibroblasts (HNF) and human nasal epithelial cells (HNEC) in fibrin gel  and cultured in Mesenpan medium (PAN-Biotech) supplemented with 2% FCS (Gibco) and 1% antibiotic/antimycotic solution  up to passage 4.ASC were isolated from abdominal fat tissue obtained from the Clinic for Plastic, Hand and Burns Surgery  and expanded in supplemented Mesenpan medium up to passage 3.HNF were isolated from nasal conchae received from the Clinic for Otorhinolaryngology  and cultured in Dulbecco`s Modified Eagle Medium  supplemented with 10% FCS and 1% ABM up to passage 3.Cell identities of HUVEC, BM-MSC, ASC and HNF were verified in previous studies using the same isolation protocols . Additio2. Gels were either molded in 24-well plates (VWR) for co-cultures with HUVEC and monocultures (350 µL) or in Transwell® Inserts  combined with ThinCert™Plates (Greiner Bio One) when used for ALI (206 µl). Polymerization of the gels was achieved by incubation at room temperature for 20 min followed by incubation at 37°C and 5% CO2 in a humidified atmosphere for another 20 min. In case of co-cultures with HRE and tri-cultures, HRE were then seeded on top of the gels with a final density of 80,000 cells/cm2. For the HRE monoculture, cells were seeded onto Transwell inserts coated with collagen IV from human placenta . Detailed media composition was described previously  in a fibrin gel or HRE were seeded on top of a gel containing one of the supporting cell types. Additionally, a co-culture of HUVEC and HRE as well as monocultures of HUVEC and HRE were prepared as controls. Fibrin gels were molded according to previously published protocols . Brieflyeviously . In shor2, samples were coated with a 10 nm gold/palladium layer . SEM was carried out in a high vacuum environment at 10 kV acceleration voltage with an environmental scanning electron microscope .Cell differentiation was analyzed by scanning electron microscopy (SEM), two-photon scanning electron microscopy (TPLSM) and histologic and immunohistochemical stainings. To evaluate ciliation of HRE after 4 weeks of ALI, SEM analysis was conducted by the facility for electron microscopy of the medical faculty of RWTH Aachen University. After fixation in 3% glutaraldehyde (Agar scientific), samples were rinsed with 0.1 M sodium phosphate buffer (Merck) followed by dehydration in an ascending ethanol series. Subsequent to critical point drying in liquid CO® 594 goat anti-mouse IgG . A selection of tri-culture samples was further stained sequentially using mouse anti-vimentin , Alexa Fluor® 488 goat anti-mouse IgG  and DAPI (Molecular Probes). The TPLSM was performed using a two-photon laser scanning microscope (Olympus FluoView 1000 MPE) with a ×25 water-objective NA 1.05  and a MaiTai Deep-See Titan-Sapphire-laser. FluoView FV 4.2 Software was used for the visualization while images were processed and evaluated with Imaris 9.6.0 software (Bitplane Inc. South Winsor). Vascularization was quantified by evaluating the average volume, surface area, length and number of branching points of vascular-like structures. Three images per gel were taken for the statistical result evaluation.Vascularization of the samples was analyzed using TPLSM after previously established protocols . After fpan-cytokeratin , mouse anti-mucin-5AC , rabbit anti-claudin-1 , mouse anti-acetylated tubulin  and mouse anti-CD31 . Fluorescently labelled secondary antibodies Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (both Invitrogen) were used for detection. All antibodies were diluted in PBS containing 1% w/v bovine serum albumin  and 0.1% w/v sodium azide (Sigma-Aldrich). Counterstaining of cell nuclei was carried out using DAPI. For observation, an inverted fluorescence microscope  was used.For histologic and immunohistochemical evaluation, samples were embedded in 2% agarose, fixed in Carnoy’s solution and dehydrated in an ascending ethanol series followed by incubation in xylene and embedding in paraffin. Native human nasal concha tissue was fixed in 4% paraformaldehyde solution before dehydration and paraffin embedding as described above. Afterwards, 5 µm-thick deparaffinized sections were stained by periodic acid Schiff’s reaction (PAS reaction) to analyze epithelial cell differentiation using a bright field light microscope. Briefly, deparaffinized samples were first hydrolyzed in 1% w/v periodic acid solution before staining with Schiff’s reagent (both Merck). After a washing step in 35°C warm tap water, cell nuclei were counterstained with Mayer’s hematoxylin (Sigma-Aldrich). Immunohistochemical analysis was also performed on Carnoy-fixed sections after a previously described protocol . After dTo evaluate differences in signaling patterns between cultures, a LEGENDPlex™ bead-based immunoassay (BioLegend) for eight human cytokines including vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-6 (IL6), interleukin-8 (IL8), angiopoietin 1, angiopoietin 2, fibroblast growth factor basic (FGF-b), placenta growth factor (PIGF), interleukin-33 (IL33), thymic stromal lymphopoietin (TSLP) and transforming growth factor-beta1 (TGF-β1) was performed on media supernatant at the end of the ALI culture following the manufacturer’s instructions. Results were evaluated according to the manufacturers’ instructions.To compare the ciliation amount between different cultures, ten independent and in terms of mucociliary differentiation well educated observers classified SEM images blinded for the condition as previously described . The scop-value of p < 0.05 was considered as statistically significant.Statistical analysis was performed using GraphPad Prism software 9.2.0. For vascularization and ciliation quantification, statistics were performed by one-way analysis of variance (ANOVA) with Tukey’s post-hoc test. Standard deviation of ciliation scores was determined by Gaussian error propagation. Cytokine production results were analyzed by two-way ANOVA with Tukey’s post-hoc test. A in vitro, tri-cultures with HRE, HUVEC and one of three different supporting cell types BM-MSC, ASC or HNF were created in fibrin gel. Furthermore, vascularization and mucociliary differentiation were tested in co-culture models of HRE or HUVEC combined with one of each of the three supporting cell types. Tri-cultures as well as co-cultures with HRE were cultured under submerged conditions for 1 week before switching to ALI conditions for 4 weeks. Co-cultures with HUVEC were vascularized for 2 weeks. At the end of the culture period, mucociliary differentiation of HRE on top of the gels and formation of vascular-like structures in the gels were analyzed using histological and immunohistochemical methods as well as SEM and TPLSM. Differences in cell communication via cytokine secretion were evaluated using LEGENDPlex immunoassay.To engineer a vascularized and mucociliated respiratory mucosa based on MSC Cell morphology, presence of goblet cells and mucus production in co- and tri-cultures were analyzed using PAS reaction . All culTo confirm mucociliary differentiation and formation of vascular-like structures, immunohistochemical stainings of endothelial cell marker CD31 and of characteristic epithelial cell markers pan-cytokeratin, mucin5AC, claudin-1 and α-tubulin were performed . Pan-cytCilia formation was additionally analyzed by SEM and could be confirmed for all cultures . In compEstimation of the differences in ciliation amounts between different culture conditions by independent observers based on SEM images revealed significant differences between almost all groups : only thThe influence of different supporting cell types in co-cultures with HUVEC and in tri-cultures on formation of vascular-like structures was evaluated using TPLSM , followeStatistical analysis of the TPLSM images showed significantly increased structure length and area for tri-cultures with BM-MSC in comparison to co-cultures with ASC or HNF . In addiIn order to investigate differences in cytokine production between the cell types, media supernatants were analyzed for levels of eight cytokines involved in vascularization processes and inflammation by LEGENDPlex bead-based immunoassay . CytokinHUVEC-co-cultures with ASC or HNF in addition exhibited significantly higher levels of VEGF  when compared to all other groups . DiffereIn summary, using BM-MSC as supporting cell type in tri-cultures led to well differentiated respiratory epithelium and a high degree of vascularization. HNF-supported tri-cultures also showed mucociliary differentiation and formation of vascular-like structures although to a lesser degree than with the BM-MSC. HRE in tri-cultures containing ASC were mostly undifferentiated while differences in vascularization were marginal. Co- and tri-cultures containing BM-MSC or HNF contained a basal cell layer positive for pan-cytokeratin, ciliated cells, mucus-producing goblet cells, tight junctions and epithelial cytoskeleton comparable to native tissue. Although ciliation amount in HNF-tri-cultures was comparable to tri-cultures with BM-MSC, and HNF-tri-cultures were also found to be positive for epithelial cell marker expression, epithelial morphology observed in PAS reaction and immunohistochemistry more closely resembled the native state in BM-MSC-tri-cultures with regard to cell arrangement, cell marker localization and layer thickness. Moreover, vascularization in tri-cultures with BM-MSC associated with a significantly higher number of branching points compared to all other cultures. Although differences in area and length of the vascular-like structures were only found to be statistically significant for BM-MSC-tri-cultures in comparison to co-cultures with ASC or HNF, an overall trend towards higher values in BM-MSC-tri-cultures was observed.in vitro culture of respiratory epithelial cells has become a common method in airway disease research nowadays. ALI-cultures have proven to be a suitable platform for studying viral infection pathways, drug testing, as well as toxicological assays and are widely used to model common respiratory diseases (To date, strategies for tracheal tissue engineering hardly reach translation into the clinic as they face three major challenges: poor mechanical properties of used scaffolds, inadequate vascularization and insufficient epithelialization . Neverthdiseases . Howeverdiseases . Most stdiseases , whereasdiseases . To our diseases .in vitro differentiation of HRE and formation of vascular-like structures mediated by HUVEC is analyzed. Because HNF had already proven to promote mucociliary differentiation and vascularization in tri-cultures with HUVEC and HNEC (In this study, influence of different supporting cell types, BM-MSC, ASC and HNF, on and HNEC , they weWe found BM-MSC to be superior to ASC and HNF with regard to mucociliary differentiation and vascularization both in tri-cultures and in co-cultures. This was followed by tri-cultures with HNF while tri-cultures containing ASC did not promote mucociliary differentiation. These results stand in some contrast to the results obtained in our previous study where we established the tri-culture system based on HNF, HUVEC and HNEC. Morphology of HNF-tri-cultures with HNEC was found to be more similar to the BM-MSC-tri-cultures presented in this study. The different origin of the epithelial cells can explain those differences. There is evidence in the literature that gene expression differs between airway epithelial cells from nasal and bronchial origin and that these cell types cannot be expected to behave in the same way .Interestingly, tri-culture reduced ciliation in comparison to the corresponding co-culture. The presence of a third cell type in the tri-culture system resulting in higher consumption of nutrients and growth factors might be a possible reason. Increasing the media exchange frequency or extension of the ALI period could therefore be strategies to increase ciliation in tri-cultures. However, this was not tested in this study in order to keep all parameters constant. In contrast, this effect was not generally discovered for vascularization. Based on the mucociliary differentiation observed in the co-culture of HUVEC and HRE see , a negatet al., who compared differences in molecular signatures of BM-MSC and dermal fibroblasts, recommend the use of BM-MSC for developmental and differentiation investigations (in vitro differentiation of respiratory epithelial cells. Although co-cultures with ASC exhibited a low degree of cilia and mucus formation, the corresponding tri-cultures lacked signs of complete mucociliary differentiation. This correlated with the general reduction of mucociliary differentiation in tri-cultures when compared to co-cultures. However, epithelial cell layers in ASC-co-cultures were unusually thick and deficient in cell polarization and a basal cell layer, showing signs of epithelial hyperproliferation. These results go in line with observations made by Kobayashi et al., who found mucin5AC and β-tubulin expression of tracheal epithelial cells in vitro to be reduced in co-cultures with ASC compared to co-cultures with fibroblasts. Moreover, the authors could show an enhanced epithelial proliferation in ASC-co-cultures (Most studies dealing with airway epithelial co-cultures use fibroblasts as supporting cell type . Their aigations . Althougigations . Taken tcultures .in vitro (in vitro, with BM-MSC showing a widespread network of smaller vascular-like structures and more branching points in co-culture with HUVEC compared to the other cell types (With respect to vascularization, both MSC and fibroblasts are known to have pro-angiogenic properties . Severalin vitro . In a prll types . This mall types  indicatell types .in vitro. In general, primary cell differentiation is often connected to high donor-to-donor variability (In this study, we analyzed levels of cytokines IL6, IL8, angiopoietin 1, angiopoietin 2, EGF, VEGF, FGF-b and PIGF known to be involved in vascularization . Rather iability . Statistiability . In addiiability . As VEGFiability . The roliability . Among oiability . Interesiability . Our datin vitro platform for studies on airway diseases.Regarding growth factors IL6, IL8, angiopoietin 1, EGF, FGF-b and PIGF, differences between cultures were found to be non-significant. The observed trend towards higher PIGF levels in cultures containing HUVEC can be explained by the fetal origin of the cells. The protein belonging to the VEGF family was found to be involved in several physiological and pathological vascularization processes. Although its pro-angiogenic effects might be beneficial for vessel formation, increased PIGF levels are connected to pathological angiogenic processes . In ordein vitro disease model might replace animal experiments in research on respiratory diseases like asthma or COVID-19.Further research is needed to evaluate the perfusability of the lumen-forming vascular-like structures and the options to connect the vascular network to the patient’s blood vessels during implantation. Furthermore, creation of upscaled mucosal patches and the combination with a suitable cartilage-like scaffold would represent the next steps towards the development of an implantable tissue-engineered tracheal replacement. In addition, employment of autologous respiratory epithelial cells derived from diseased patients could provide valuable information for new therapeutic approaches. Such further development towards an in vitro tri-culture model. We were able to reproducibly demonstrate that BM-MSC promote both epithelial differentiation and vascularization most effectively, followed by HNF. In summary, analysis of growth factor content in media supernatant showed reduced levels of pro-inflammatory and angiogenic cytokines in tri-cultures and HRE-co-cultures in comparison to co-cultures with HUVEC. Thus, our BM-MSC-based model of the respiratory mucosa providing a pseudostratified ciliated epithelium including mucus-producing goblet cells and a vasculature network marks a major step on the way to a tissue-engineered tracheal implant.In this study, we compared the influence of BM-MSC, ASC and HNF on the mucociliary differentiation of HRE and on the formation of vascular-like structures mediated by HUVEC in a fibrin-based


Apolipoprotein C-III (ApoC-III) is a widely known player in triglyceride metabolism, and it has been recently recognized as a polyhedric factor which may regulate several pathways beyond lipid metabolism by influencing cardiovascular, metabolic, and neurological disease risk. This review summarizes the different functions of ApoC-III and underlines the recent findings related to its multifaceted pathophysiological role.The role of ApoC-III has been implicated in HDL metabolism and in the development of atherosclerosis, inflammation, and ER stress in endothelial cells. ApoC-III has been recently considered an important player in insulin resistance mechanisms, lipodystrophy, diabetic dyslipidemia, and postprandial hypertriglyceridemia (PPT). The emerging evidence of the involvement of ApoC-III in the in the pathogenesis of Alzheimer’s disease open the way to further study if modification of ApoC-III level slows disease progression. Furthermore, ApoC-III is clearly linked to cardiovascular disease (CVD) risk, and progression of coronary artery disease (CAD) as well as the calcification of aortic valve and recent clinical trials has pointed out the inhibition of ApoC-III as a promising approach to manage hypertriglyceridemia and prevent CVD.Several evidences highlight the role of ApoC-III not only in triglyceride metabolism but also in several cardio-metabolic pathways. Results from recent clinical trials underline that the inhibition of ApoC-III is a promising therapeutical strategy for the management of severe hypertriglyceridemia and in CVD prevention. Apolipoprotein C-III (ApoC-III) is a small apoprotein (79 amino acid residues) that mainly resides in triglyceride-rich lipoproteins (TRLs) , and their remnants), low density lipoproteins (LDL), and high density lipoproteins (HDL). Current knowledge on ApoC-III suggests a multifaceted influence in several pathophysiological processes including TRLs metabolism, atherosclerosis, inflammations, glucose metabolism, and cardiovascular and neurological diseases .APOC3 gene maps on chromosome 11q23 closely linked with APOA1 and APOA4 genes in a cluster on 17 kb APOA1/C3/A4/A5 gene cluster .ApoC-III inhibition by antisense oligonucleotides (ASO) represents so far a unique and effective strategy to lower ApoC-III levels . Monocloeactions , 85. FurSeveral evidences highlight the role of ApoC-III not only in triglyceride metabolism but also in several cardio-metabolic pathways. Clinical trials have underlined that ApoC-III inhibition is an effective strategy for the management of severe hypertriglyceridemia. In the future, novel therapies directed toward ApoC-III inactivation may offer promising therapeutical strategy to manage severe hypertriglyceridemia and CVD prevention.


STEMI patients with COVID-19 also received significantly less invasive cardiac procedures  and were more likely to receive systemic thrombolytic therapy  when compared to STEMI patients without COVID-19. Our findings are the result of complications of SARS-CoV2 infection as well as alterations in healthcare delivery due to the burden of the COVID-19 pandemic.The COVID-19 pandemic has impacted healthcare delivery to patients with ST-segment elevation myocardial infarction (STEMI). The aim of our retrospective study was to determine the effect of COVID-19 on inpatient STEMI outcomes and to investigate changes in cardiac care delivery during 2020. We utilized the National Inpatient Sample database to examine inpatient mortality and cardiac procedures among STEMI patients with and without COVID-19. In our study, STEMI patients with COVID-19 had higher inpatient mortality (47.4% vs. 11.2%, aOR: 3.8, 95% CI: 3.2–4.6,  Acute ST-segment elevation myocardial infarction (STEMI) is a cardiac emergency that requires timely reperfusion . The corp < 0.001) when compared to STEMI patients without COVID-19 [There is limited data suggesting that outcomes for STEMI patients during the early pandemic were inferior to pre-pandemic standards of care; one large database study n = 82,640) by Saad et al., 2021, showed that STEMI patients with COVID-19 had significantly higher rates of in-hospital mortality  2020 NIS database, which is based on hospitalizations from 1 January 2020 to 31 December 2020 . NIS is (a)Patient characteristics: sex, age, race, insurance status, median income based on zip code, medical comorbidities, and Elixhauser comorbidity score.(b)Hospital characteristics: geographic division, teaching status, and size.Baseline characteristics related to patient demographics and comorbidities as well as hospital characteristics for all included patients were extracted from the NIS database, as detailed below:(a)Length of stay.(b)Cost of hospitalization.(c)Utilization of intubation and mechanical ventilation.(d)Utilization of vasopressors.(e)Incidence of cardiogenic shock.(f)Disposition at discharge.(g)Utilization of coronary angiograms.(h)Utilization of percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), or systemic thrombolytic therapy for coronary revascularization.(i)Utilization of percutaneous left ventricular assist device (pLVAD) or intra-aortic balloon pump (IABP).(j)Utilization of temporary transvenous pacing or permanent pacemaker.Study outcomes were also obtained from the NIS database. The primary outcome of our study was in-hospital mortality. Secondary outcomes were as follows:(a)Arrhythmias: atrial fibrillation, atrial flutter, ventricular tachycardia, ventricular fibrillation, sinus bradycardia, first degree atrioventricular nodal block, second degree atrioventricular nodal block, and third degree atrioventricular nodal block.(b)Pericarditis.In addition to our primary and secondary outcomes, we examined cardiovascular complications associated with STEMI. We used ICD-10 codes  was utilized for statistical analysis. The unweighted sample was 6.34 million observations, and the weighted sample was 31.71 million discharges for the year 2020. Patients who were hospitalized with STEMI were retrieved from the NIS database using ICD-10-CM codes and was divided into two cohorts based on COVID-19 status. The described patient characteristics, hospital characteristics, primary and secondary outcomes, and in-hospital cardiovascular complications were extracted for each patient.p-value of less than 0.05 was considered significant. This multivariate logistic regression model was built using covariates with unadjusted p-values of less than 0.2 on initial univariate regression. We arbitrarily chose a p-value of less than 0.2 for covariate inclusion to broaden the number of variables accounted for by our regression model and to decrease the probability of missing potential confounders that may impact the standard error for our exposure variable (COVID-19 infection). Ultimately, our multivariate analysis adjusted for sex, age, race, median household income, insurance status, hospital geographic division, Elixhauser comorbidity score, need for mechanical ventilation, and numerous other in-hospital complications as denoted (*) in p-value of 0.05 was considered significant. Finally, multivariate logistic regression was also utilized for adjusted analysis of secondary outcomes related to illness severity  and secondary therapeutic procedural outcomes.Chi-square analysis was used to compare categorical variables and linear regression was used to compare continuous variables among both study cohorts (STEMI with COVID-19 vs. STEMI without COVID-19). For our primary outcome, in-hospital mortality, we utilized a multivariate logistic regression model to adjust for potential confounders; a two-tailed p < 0.001, of patients were above 70 years of age), had a greater proportion of Hispanics  and African Americans , and were more likely to have household incomes below $50,000  when compared to STEMI patients without COVID-19. While there was variability in the geographic distribution of patients in both cohorts, there were no significant differences in hospital teaching status, in that most STEMI patients regardless of COVID-19 status were seen at urban teaching hospitals. Additionally, STEMI patients with COVID-19 had a higher proportion of Medicare beneficiaries  and a lower proportion of private health insurance beneficiaries  when compared to STEMI patients without COVID-19.We identified 172,185 patients with STEMI between 1 January 2020 and 31 December 2020. Of these, 5786 patients had a diagnosis of COVID-19 (3.4%). STEMI patients with COVID-19 were significantly older , congestive heart failure , uncomplicated hypertension , and smoking  when compared to STEMI patients without COVID-19. Conversely, this group had a higher proportion of complicated hypertension , complicated diabetes mellitus , and chronic kidney disease . There were no significant differences in the rates of chronic pulmonary disease or obesity between the two groups. STEMI patients with COVID-19 had a lower prevalence of cardiac comorbidities, including coronary artery disease .Additionally, we noted that STEMI patients with COVID-19 required mechanical ventilation  and vasopressors  at higher rates despite a lower adjusted incidence of cardiogenic shock  when compared to STEMI patients without COVID-19. STEMI patients with COVID-19 also had an increased length of stay  and a higher cost of hospitalization  Of those patients who survived, fewer STEMI patients with COVID-19 were able to return home  and more required skilled nursing or long-term acute care  when compared to STEMI patients without COVID-19. After multivariate adjustment, we found that the in-hospital mortality of STEMI patients with COVID-19 was significantly higher than STEMI patients without COVID-19 , African Americans , and Native Americans  had increased in-hospital mortality when compared to Hispanic, African American, and Native American STEMI patients without COVID-19, respectively. Conversely, a significantly lower proportion of Caucasians in the STEMI with COVID-19 cohort had in-hospital mortality when compared to Caucasians in the STEMI without COVID-19 cohort . We also performed subgroup mortality analysis and found that among STEMI patients with COVID-19, Hispanics . Apart from this, STEMI patients with COVID-19 were significantly less likely to receive all types of invasive cardiac interventions including coronary angiography , percutaneous coronary interventions , coronary artery bypass grafting , intra-aortic balloon pumps , percutaneous left ventricular assist devices , and transvenous pacing  when compared to STEMI patients without COVID-19. p = 0.02), atrial fibrillation , and ventricular fibrillation . Notably, STEMI patients with COVID-19 were more likely to receive systemic thrombolytic therapy when compared to STEMI patients without COVID-19  and were instead more likely to receive systemic thrombolytic therapy.Moreover, there are clinical markers to indicate that STEMI patients with COVID-19 were more critically ill, including higher levels of vasopressor and mechanical ventilatory dependence despite similar levels of cardiogenic shock. The higher mortality observed among STEMI patients with COVID-19 was also despite lower levels of pre-existing cardiac comorbidities prior to the presenting STEMI. Thus, we believe that the etiology of the observed marked increase in mortality is likely multi-factorial—related both to the infectious and cardiovascular complications of COVID-19 infection as well as system-wide alterations in delivery of care resulting in significantly fewer cardiac procedures. While our findings are consistent with prior literature, to our knowledge, this study has the largest sample size and shows a dramatic difference in STEMI mortality and cardiac procedural utilization between patients with COVID-19 and patients without COVID-19.Myocardial injury from hemodynamic instability or hypoxemia;Inflammatory myocarditis;Stress cardiomyopathy;Microvascular dysfunction;Thrombosis with coronary artery plaque destabilization due to inflammatory hypercoagulability.The mortality of COVID-19 due to acute infectious complications such as respiratory failure and septic shock is well-characterized. However, there is a growing body of literature establishing the role of acute SARS-CoV2 infection in the development of acute cardiovascular complications, which include various forms of myocardial injury , congestive heart failure, and cardiac arrhythmia ,9. The pObservational studies suggest that COVID-19 increases the risk of acute myocardial infection (AMI); one analysis found that the risk of AMI in patients with COVID-19 is significantly elevated  . There iWith respect to the lower utilization of invasive cardiac procedures among STEMI patients with COVID-19 and its relation to the observed increase in mortality for this cohort, there are several possible explanations. Firstly, a major contributing factor is the strain of the COVID-19 pandemic on systems of healthcare delivery . ThroughWe also found both economic and racial disparities in mortality. Hispanics, African Americans, and those with lower household income had increased mortality when hospitalized with STEMI and COVID-19, which is consistent with prior literature . As expeOur study has several limitations. The first is possible detection bias, particularly as it relates to COVID-19 infection. The ICD-10 diagnosis code for COVID-19 was not developed until late 2020, which allows for the possibility that certain COVID-19 diagnoses were not captured in our dataset. Consequently, a subset of patients with STEMI may have had COVID-19, but were included in the non-COVID-19 cohort due to under diagnosis. Likewise, COVID-19 testing remained in short supply and the NIS database does not provide lab test results to confirm COVID-19 status. This principle applies to a lesser extent to inaccurately documented or undocumented comorbid diagnoses. The second limitation is that selection bias and confounding remain a possibility in this retrospective study. Our statistical analysis accounted for numerous demographic and clinical factors via multivariable logistic regression—however unaccounted variables may underlie our findings. This dataset did not include certain clinical, biochemical, or radiographic markers of COVID-19 severity which may conceivably have contributed more heavily to the observed mortality rather than the STEMI diagnosis.Based on this study, we recommend further examination of the cardiac care delivery process during the early pandemic. Future investigations should evaluate process measures that explore time-to-intervention, procedural complications, and barriers to definitive therapies such as PCI and CABG.We conclude that hospitalized STEMI patients with concomitant COVID-19 infection were nearly four times as likely to die in the hospital in comparison to STEMI patients without COVID-19 during 2020, in the early stages of the COVID-19 pandemic. This finding is likely driven by both clinical complications of COVID-19 infection as well as system-wide alterations in delivery of cardiac care due to the healthcare burden of the COVID-19 pandemic. Thus, in addition to focusing on risk mitigation strategies for preventing infection during future pandemics, it is equally important to develop strategies for maintaining other critical clinical processes to allow for the delivery of healthcare to patients with life-threatening conditions such as STEMI in order to reduce unnecessary morbidity and mortality.


Antigen testing for SARS-CoV-2 has become an increasingly prominent screening tool in the ongoing COVID-19 pandemic and can be performed multiple times a week. However, the optimal weekly frequency of antigen testing is unclear; the Centers for Disease Control and Prevention recommends 1–3 times a week, while some experts support testing 2–3 times a week. In our own laboratory, all staff (n = 161) underwent twice- and thrice-weekly antigen tests during different periods from August 2021 to the present as part of routine COVID-19 surveillance of healthcare workers. No cases of COVID-19 were detected with either regimen. While more frequent SARS-CoV-2 antigen testing may allow antigen testing to be an important surrogate for RT-PCR testing, performing SARS-CoV-2 antigen tests twice or thrice a week shows no inferiority to each other in screening for COVID-19. Antigen testing for SARS-CoV-2 has become an increasingly prominent screening tool in the ongoing COVID-19 pandemic. Indeed, when used to screen patients in an emergency department in a high prevalence setting, rapid antigen testing (LumiraDx SARS-CoV-2 Antigen test) had a sensitivity of 68.7% when compared to RT-PCR testing; sensitivities increased to 81% when confined to symptomatic patients only , which hAlthough RT-PCR testing remains the gold standard for diagnosing SARS-CoV-2 infection, it is expensive to perform, and requires a significantly longer time to report results. The advantage of antigen testing is that it is cheaper and easier to perform than RT-PCR testing and can provide results in as little as 15–20 min. Thus, antigen tests have gained widespread use in screening programs across the world. However, some screening programs only report findings based on the use of a single antigen test only. For example, one recent report  performeBoth the SD Biosensor Standard Q  and AbboOur laboratory has 161 staff members. As part of routine rostered testing, all staff has been tested with twice-weekly rapid antigen testing. In August 2021, one of our staff members (immunized with 2 doses of Pfizer BNT162b2 mRNA vaccine) developed COVID-19 symptoms and tested positive for SARS-CoV-2 on RT-PCR. Subsequently, all staff members underwent immediate RT-PCR testing every three days , and rapid antigen testing  every day for the next two weeks as previously reported . As no nIn COVID-19 screening programs that use antigen testing, the optimal time interval between tests is an open question. Studies on viral kinetics seem to offer some insight into this conundrum. In a recent study  where voThis finding has been confirmed in another study  where RTThe most appropriate use for frequent rapid antigen testing would be in the detection of early COVID-19 infection when patients have the highest viral loads (lower Ct counts) or are just becoming symptomatic. Indeed, twice-weekly antigen testing has excellent sensitivity (96.2%) when compared with RT-PCR testing in early disease (days 0 to 3 of symptom onset) . A meta-The most optimal method of screening for COVID-19 would be to use both antigen and RT-PCR testing in an orthogonal fashion, especially in settings of low disease prevalence to minimize false-positive antigen results. All “positive” antigen tests would be retested with RT-PCR and only those positive on both tests would considered true positives. Low disease prevalence would greatly reduce the positive predictive value of any individual test, leading to an increase in false-positive results. Orthogonal testing can greatly reduce the false positive rate . This isThe strength of our study is that we compared twice- vs. thrice-weekly COVID-19 screening using LFIAs in a disease-free population, which supplements studies that compared twice vs. thrice weekly testing in infectious cases , or repoOne limitation of our study is that two different rapid antigen testing kits were used by our staff over the time period, due to logistical supply constraints at our institution. Despite the varying positive predictive values of both tests differing at various levels of disease prevalence see , we did More frequent SARS-CoV-2 antigen testing can allow antigen testing to be an important surrogate for RT-PCR testing. However, performing antigen testing twice or thrice a week did not detect any new cases; thus, two tests per week may suffice.


Glioma remains the toughest brain tumor among all primary central nervous system (CNS) tumors. The complexity of its pathogenesis makes it difficult to achieve radical cure, especially in the case of glioblastoma multiforme , the most aggressive subtype of glioma . RecentlWu. et al. screened necroptosis (NCPS)-related genes  to construct a prognostic model for GBM, paving the way for the use of new targets for the diagnosis and treatment of glioma. Fang et al. proved that the transient receptor potential (TRP) family genes are promising immunotherapeutic targets and potential clinical biomarkers for glioma. Besides genetic mutations, increasing evidence shows that the tumor microenvironment is also important for glioma development and resistance to therapy [Alice Giotta Lucifero et al. found that the phosphatase and tensin homolog (PTEN)-related immune landscape mainly consists of Treg and M2 macrophages, which repress the antitumor immune activation and are responsible for triggering glioma cell growth and invasion. Li et al. demonstrated that the tyrosine phosphatase receptor type N (PTPRN) could be an independent prognostic factor and correlates with tumor immune infiltration in low-grade glioma.In this Special Issue, we collected 10 articles discussing the genomic alterations of glioma, its immune microenvironment, and multi-therapy for glioma.  therapy . Within Yu et al. suggested that irreversible electroporation possibly mediates glioma apoptosis via the upregulation of transcription factor AP-1 and Bim (Bcl2l11) expression. Programmed death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) play critical roles in tumor immune escape, and several immunotherapies, including PD-1/PD-L1 checkpoint inhibitors and chimeric antigen receptor-T cells (CAR-T), have already been applied in glioma therapy. Yu et al. explore the PD-1/PD-L1 protein expression in recurrent glioma and its paired primary tumor and reveal a tendency of increased PD-1/PD-L1 in recurrent glioma. However, immunotherapies proved therapy failures, which strongly indicates that beyond the T cell-based adaptive immunity, innate immunity might also be the key to regulating anti-tumor immunity in the glioma microenvironment. Qi et al. provided a cellular response to the interleukin-4 (IL-4)-related gene signature as an excellent immune biomarker of gliomas, which may be beneficial to develop novel immunotherapies for glioma.In the past decade, newly emerging therapeutic strategies such as tumor-treating fields (TTF) have been introduced in GBM patients . Yu et aCintia Carla da Hora et al. proposed patient-derived xenografts (PDX) or patient-derived GSC models in glioma research, which provide the possibility of studying glioma growth, treatment response, and survival outcome. In addition, Hannes Becker et al. conducted multilayered profiling of a platelet-derived growth factor B (PDGFB)-driven glioma mouse model and discovered radiological, histological, and metabolic features that are comparable to human high-grade glioma.Rodent models of glioma are still indispensable for understanding the basic principles of glioma development and tumor invasion . Rodent