Therapeutic applications of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have attracted considerable attention because of their immunomodulatory properties against immune-mediated, inflammatory diseases. Here, we demonstrated enhanced immunomodulatory properties of EVs secreted from endoplasmic reticulum (ER) stress inducer thapsigargin (TSG)-primed human Wharton’s jelly-derived MSCs (WJ-MSCs). EVs from TSG-primed WJ-MSCs (TSG-EV) showed increased yield and expression of immunomodulatory factors, such as transforming growth factor-β1 (TGFβ), cyclooxygenase-2 (COX2), and especially indoleamine 2,3-dioxygenase (IDO), compared to control EVs. TSG-EV showed a significantly enhanced immunosuppressive effect on human peripheral blood-derived T cell proliferation and Th1 and Th17 differentiation, whereas Treg and M2-type macrophage were enriched compared to a control EV-treated group. Furthermore, TSG-EV substantially mitigated mouse experimental colitis by reducing the inflammatory response and maintaining intestinal barrier integrity. A significant increase of Tregs and M2-type macrophages in colitic colons of a TSG-EV-treated mouse suggests an anti-inflammatory effect of TSG-EV in colitis model, possibly mediated by Treg and macrophage polarization. These data indicate that TSG treatment promoted immunomodulatory properties of EVs from WJ-MSCs, and TSG-EV may provide a new therapeutic approach for treatment of colitis. Previous reports demonstrated promising therapeutic effects of mesenchymal stem cells (play an important role in immune regulation by mediating the delivery of secreted MSCs) through immunosuppressive, angiomodulatory, and paracrine factor activities ,2,3. MSCTherapeutic effects of MSC EVs have been studied to target autoimmune uveitis, inflammatory lung diseases, graft-versus-host disease, and liver fibrosis ,14,15,16The newly synthesized proteins undergo folding and translocation through the endoplasmic reticulum (ER) membrane and ER plays an important role in coordinating signaling pathways to ensure cellular homeostasis. However, under stressful pathological and physiological conditions, when ER is unable to maintain homeostasis, the unfolded protein response (UPR) is activated and may have an impact on cellular function ,22,23. PHere, we investigated the immunomodulatory properties of EVs released from TSG-treated WJ-MSCs on activation and polarization of human T cells and macrophages. In addition, we examined the therapeutic efficacy of TSG-primed EVs in murine colitis and examined the possible biological mechanism underlying the therapeutic value of EVs.2. Upon reaching 80~90% confluence, cells were detached with 0.05% Trypsin-EDTA (Gibco) for further subcultures.For the isolation of Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs), Wharton’s jelly tissue was obtained from the umbilical cords after normal full-term delivery with the written informed consent of the mothers and the approved by the Asan Medical Center Institutional Review Board (protocol no. 2015–3030). The WJ-MSCs were isolated as previously described . In this5 for overnight and treated with Thapsigargin  . After 24 h incubation, 0.5 mg/mL MTT -2,5-diphenyltetrazolium bromide) solution (Sigma-Aldrich) were added in the medium and incubated for an additional 4 h. The crystals were dissolved with lysis buffer (0.04 M HCl in isopropanol) and absorbance was measured at 570 nm using a spectrophotometer .WJ-MSCs were cultured in 24-well cell plate at a density of 2 × 106) were cultured overnight in 100 mm culture dish, and then stimulated with 1 μM TSG (Sigma-Aldrich) for 24 h. The medium was then replaced with medium supplemented with 10% EV-depleted FBS. EV-depleted FBS was obtained by ultracentrifugation at 100,000× g for 18 h. After 72 h incubation, the culture medium was collected and centrifuged at 300× g for 10 min, 2500× g for 25 min, 10,000× g for 30 min at 4 °C. The supernatant was filtered through 0.22 µm filters and ultracentrifuged at 100,000× g for 2 h. Then, the EV pellet was washed by centrifugation in sterile PBS at 100,000× g for 2 h, suspended in 200 μL of PBS and stored at −80 °C until use. Although this protocol aims to isolate exosomes with microvesicle elimination step, we used the term “EV”, rather than “exosomes” to follow the nomenclature recommendation by the International Society for Extracellular Vesicles (ISEV) [WJ-MSC EVs were prepared by ultracentrifugation as previously described . BrieflyWJ-MSC lysates and EVs were lysed with RIPA buffer (Thermo Fisher) with protease inhibitor (Thermo Fisher). Equal amounts of proteins or EV were subjected to SDS-PAGE and analyzed with primary antibodies: rabbit polyclonal CD63 , rabbit polyclonal COX2 , mouse monoclonal IDO , rabbit polyclonal TGFβ1, rabbit monoclonal GRP78 , mouse monoclonal CHOP , mouse monoclonal GAPDH . The bands were detected using enhanced chemiluminescence (Thermo Fisher) and images were captured using LAS-3000 system .Total RNA was extracted from cells, EVs, or tissue samples using TRizol reagent , and cDNA was synthesized from total RNA using Superscript™ III reverse transcriptase (Invitrogen). Quantitative PCR was measured using the SYBR Green Master Mix  in MX300P thermal cycler . The expression levels of mRNA were normalized to that of GAPDH. The primer sequences for qRT-PCR are listed in 5/well) in growth medium with and without EVs (4 μg) derived from naive or TSG primed WJ-MSCs for 72 h. Annexin V/7AAD  staining was performed to determine cell apoptosis according to the manufacturer’s instructions and analyzed by flow cytometry.Mononuclear cells (MNCs) were isolated from hUCBs using Ficoll-Hypaque  gradient centrifugation as described previously , and resHuman peripheral bloods from healthy donors were provided from the Korean Red Cross Blood Services  under approval of the Institutional Review Board (IRB) of the Catholic University (IRB No.2019-2891-0003). Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over a Ficoll-Hypaque density gradient as described previously , and res5/well) were cultured in 96 well plate with or without EVs (4 μg) derived from naive or TSG primed WJ-MSCs, in the presence of anti-CD3/CD28 microbeads (Gibco) and recombinant human IL-2 . After 6 days of incubation, the cells were stained with human monoclonal antibodies against CD45-APC-H7 (clone 2D1), CD3-PE-Cy5 (clone HIT3α), CD4-FITC (clone RPA-T4), and CD8-BV421  and measured by flow cytometry. All cells were gated on 7AAD negative cells.The proliferation of T cells was quantified by CFSE dilution. Isolated PBMCs were labeled with 2 μM carboxyfluorescein diacetate succinimidyl ester . CFSE-labeled PBMCs  according to the manufacturer’s protocol. CD4+ T cells were stimulated with of anti-CD3/CD28 microbeads and IL-2 (20 ng/mL) and added with specific cytokine for the differentiation of specific Th cells; IFNγ (25 ng/mL) and IL-12 (25 ng/mL) for Th1; IL-6 (50 ng/mL) and TGFβ (25 ng/mL) for Th17; TGFβ (25 ng/mL) and Retinoic acid (10 nM) for Treg. Cytokine-treated CD4+ T cells (5 × 105/well) were cultured for 5 days by adding EVs derived from naive or TSG primed WJ-MSCs in a 24 well plate. For intracellular staining, cells were stimulated with a cell stimulation cocktail (Invitrogen) for 5 h before staining. The cells were stained with cell surface marker anti-CD3-PE-Cy5, anti-CD4-FITC and anti-CD25-APC  antibodies and then fixed/permeabilized and stained with fluorescence-labeled anti-IFNγ-APC (clone 4S.B3), anti-IL-17A-PE (clone SCPL1362), and anti-Foxp3-PE  antibodies. Staining cells were analyzed by flow cytometry.CD4+ monocytes were isolated from PBMCs lymphocytes using CD14 conjugated microbeads (Miltenyi Biotec). To generate macrophages, monocytes (5 × 105/well) were cultured for 6 days in RPMI 1640 medium (Gibco) containing 10% FBS with GM-CSF  or M-CSF (100 ng/mL) in a 24 well plate. For M1 polarization, GM-CSF derived macrophages were stimulated with IFNγ (20 ng/mL) plus LPS (1 μg/mL) in presence of EVs derived from naive or TSG primed WJ-MSC for 48 h. For M2 polarization, M-CSF derived macrophages were stimulated with IL-4 (20 ng/mL) plus IL-13 (20 ng/mL) in presence of EVs derived from naive or TSG primed WJ-MSCs for 48 h. After co-culture, the cells were stained with human monoclonal antibodies against CD14-APC-Cy7 (clone MφP9), CD80-APC-R700 (clone L3307.4), CD86-BV650 (clone 2331), CD206 (clone 19.2), and CD163- BV421 (clone GHI/61. BD Biosciences) and analyzed by flow cytometry.CD14w/v) dextran sulfate sodium  in the drinking water for day 7. Mice were randomly divided into the four groups of 10 mice per group: (1) Control (negative control), (2) DSS with concentrated-CM derived from GW4869 treated MSCs (positive control), (3) DSS with EVs derived from naive MSC, (4) DSS with EVs derived from TSG primed MSC. At days 1, 3, and 5, mice were intraperitoneally injected with 200 μg EV diluted in 200 μL PBS. Mice were euthanized at day 10. The severity of colitis was assessed daily by utilizing the disease activity index (DAI), including body weight loss (0–4), stool consistency (0–4), stool blood (0–4), coat roughness (0–4), rectal prolapse (0–3), hunched posture (0–3), bedding contamination (0–2), and not inquisitive/alert (0–2). All animal experimental procedures were approved by the Animal Research Ethics Committee of the Catholic University of Korea (IACUC no. 2019-0301-03).Acute colitis in 7-weeks-old C57BL/6 mice was induced by the administration of 3% , embedded in paraffin, and sectioned at 5 µm. For histological analysis, colon sections were stained with hematoxylin and eosin (H&E). Histopathology scores were determined in a blinded manner by the degree of inflammatory cell infiltration (0–4) and the degree of tissue injury (0–4).g. The isolated cells were stained with anti-mouse CD11b-BV480 (clone M1/70), anti-mouse F4/80-BV421 (clone T45-2342), anti-mouse CD206-Alexa Flour 647 (clone MR5D3) and anti-mouse arginase1-PE-Cy7  and analyzed by flow cytometry.Lamina propria cells were isolated from the intestine as previously described . The colg for 10 min. The supernatant was mixed with MPO assay buffer and MPO substrate, incubated at 25 °C for 120 min, then tetramethylbenzidine (TNB) probe was added. Absorbance was measured at 412 nm using a spectrophotometer (Biotek).Infiltration of neutrophils into the colon tissue was measured with the MPO Colorimetric Activity Assay Kit (Sigma). Briefly, the colon was homogenized in MPO assay buffer and supernatants were collected by centrifugation at 13,000× p-value < 0.05 were considered to indicate statistically significant.Statistical analysis was performed using GraphPad Prism v8.0.1  with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. All data were presented as the mean ± S.D. WJ-MSCs were isolated and cultured as previously described  and furtNext, we investigated the effect of TSG on EV release. EVs were isolated from the culture supernatant by standard ultracentrifugation methods. . We founEVs were isolated from the culture supernatant of naive (CTL-EV) or TSG-primed WJ-MSCs (TSG-EV) by standard ultracentrifugation methods. Transmission electron microscopic analysis showed that isolated EVs include round vesicles of 50–120 nm in diameter A. The EVAs pro- and anti-inflammatory cytokine secretion pattern determines the immunomodulatory properties of MSCs or MSC-derived EVs, we examined the expression of inflammatory genes. TSG-EV had significantly higher expression levels of IL-10, TGFβ, COX2, and IDO compared to CTL-EV but lower expression of pro-inflammatory cytokines including IFNγ, TNFα, and IL-1β D. Consis+ T, and CD8+ T cell proliferation compared to naive WJ-MSCs  and M2-type macrophage polarization ,33. Thuso CTL-EV . To inveo CTL-EV . The M1-ed group . These rWe investigated the potential protective effect of TSG-EV in a 3% DSS-induced colitis murine model A. As a vHistological examination showed that TSG-EV-treated mice maintained colon tissue integrity; significantly reduced structure destruction, crypt loss, and infiltration of inflammatory cells; and had lower histological score compared to CTL-EV-treated mice G,H. MoreNext, we investigated the immune response modulation of EV to attenuate DSS-induced colitis. Expression of pro-inflammatory cytokines, including IL-1β, IFN-γ, and TNFα was decreased in colon tissue of EV treated mice compared to DSS-induced colitis mice, whereas expression of the anti-inflammatory cytokines IL-10 and TGFβ were significantly increased in colon tissue of EV treated mice A. In parImmune cell response plays a crucial role in the pathogenesis of inflammatory bowel disease (IBD). In particular, the balance between Tregs and other T cells in the intestinal microenvironment plays an important role in alleviation of colitis . Therefo+CD11b+ cells (macrophages) in the lamina propria of colons compared to the negative control mice (+CD11b+ cells (Macrophages play an important role in the pathogenesis of IBD by mediating inflammatory responses through M1/M2 polarization in the colon . Thus, wrol mice E,F. Treab+ cells E,F, but b+ cells G. These During the last decade, our group and others have reported therapeutic properties of MSCs for immune-/inflammation-mediated diseases, including autoimmune uveitis, inflammatory lung diseases, graft-versus-host disease, and liver fibrosis ,39,40,41In light of previous studies that demonstrated that ER stress enhances the release of EVs ,26 and EPrevious studies have shown that Treg and/or macrophages play an important role in IBD pathogenesis by regulating the function of neutrophils, dendritic cells, and other inflammatory cells ,52. In lIn summary, our findings showed that TSG-mediated ER-stress induction was sufficient to increase the release of WJ-MSC-EVs containing up-regulated anti-inflammatory immunomodulators. Infusion of TSG-EV in experimental colitis significantly enhanced M2-type macrophage polarization and Treg induction in inflamed colons. Our data propose a novel therapeutic strategy for increase the yields of EVs from the MSCs by TSG priming and treatment of colitis based on use of EVs from TSG-primed MSCs.


Cyclic dinucleotides (CDNs) are promising vaccine adjuvants inducing balanced, potent humoral, and cellular immune responses. How aging influences CDN efficacy is unclear. We examined the vaccine efficacy of 3′,5′-cyclic diguanylic acid , the founding member of CDNs, in 1-year-old (middle-aged) and 2-year-old (aged) C57BL/6J mice. We found that 1- and 2-year-old C57BL/6J mice are defective in CDG-induced memory T helper (Th)1 and Th17 responses and high-affinity serum immunoglobulin (Ig)G, mucosal IgA production. Next, we generated two novel tumor necrosis factor (TNF) fusion proteins that target soluble TNF (solTNF) and transmembrane TNF (tmTNF) to monocyte-derived dendritic cells (moDCs) to enhance CDG vaccine efficacy in 1- and 2-year-old mice. The moDC-targeting TNF fusion proteins restored CDG-induced memory Th1, Th17, and high-affinity IgG, IgA responses in the 1- and 2-year-old mice. Together, the data suggested that aging negatively impacts CDG vaccine adjuvanticity. MoDC-targeting TNF fusion proteins enhanced CDG adjuvanticity in the aging mice. The current COVID-19 pandemic has highlighted the vulnerability of aging populations to emerging pathogens where people 45 and older account for ~97% of COVID-19 deaths –4. Immun® decreases with age . Almost all studies on CDN adjuvanticity were done in young mice (<3 months old). How aging influences CDN efficacy in vivo is not clear.Cyclic dinucleotides (CDNs) are a promising class of vaccine adjuvants inducing potent, balanced, and long-lasting humoral and cellular adjuvant responses for cancers and infectious diseases –15. CDNsin vivo . C57BL/6J mice were obtained, bred, maintained, and aged in the Animal Research Facility at the University of Florida. Both males and females were used to carry out the experiments.6CGG) (1.5 μg/mouse) alone or mixed with CDG (5 μg/mouse) and/or soluble TNF (solTNF)-Fc [immunoglobulin G2A (IgG2A)] (100 ng/mouse) and transmembrane TNF (tmTNF)-Fc (IgG2A) (100 ng/mouse) in total 40 μl endotoxin-free phosphate buffered saline (PBS). Sera were collected after 30 and 60 days of the last immunization. Bronchoalveolar lavage fluid (BALF) was collected on day 60.For intranasal immunization, mice were immunized with protein antigen H1N1-nucleoprotein (NP), pneumococcal surface protein A (PspA), 4-hydroxy-3-nitrophenylacetyl hapten conjugated with chicken gamma globulin (NPD221N/A223R-Fc (IgG2A) (lot # CB190829) fusion proteins were made by Creative® Biolabs. The fusion protein has N-Terminal Histidine tag. They were expressed in CHO cells and purified with affinity chromatography. The purity is >95% as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (Coomassie blue densitometry). Endotoxin level of the fusion proteins is less than 0.5 EU/mg fusion protein.The TNF-Fc (IgG2A) (lot # CB181108) and TNFStreptococcus pneumoniae Family 2, Clade 3 Pneumococcal Surface Protein A (PspA UAB099) with C-Terminal Histidine Tag, Recombinant from Escherichia coli, NR-33179.The following reagent was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH): 2BSA, NP23BSA, washed with 1 × PBS with Tween 20 (PBST), blocked with 2% bovine serum albumin (BSA) in 1 × PBS, and incubated at 37°C -13, IL-5, interferon (IFN)-γ, and IL-17a by ELISA.Lung and spleen cells were harvested and digested as before . The celLungs were harvested and processed as discussed above. The single-cell suspension was prepared and analyzed by BD™ LSR II and FACScan flow cytometry .hiCD11c+CD11b−CD64−, moDCs are MHCIIhiCD11c+CD11b+CD64+, and cDC2 are MHCIIhiCD11c+CD11b+CD64−  containing protease inhibitors at −80°C. Later, the lung was thawed on ice and homogenized with Minilys®  using Precellys lysing kit. Lung homogenates were transferred to a 1.5-ml tube and harvested at 14,000 g for 30 min at 4°C. The supernatant was collected and analyzed for TNF production by ELISA.Mice were intranasally immunized with either PBS or CDG (5 μg) and sacrificed after 5 h by COp < 0.05 was considered significant. * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. n.s. represents non-significant.GraphPad Prism v6.05 software was used for data preparation and statistical analysis. One-way or two-way ANOVA followed by Tukey's multiple comparison tests were employed. Data exclusion was justified when positive or negative control did not work. All experiments will be repeated at least three times. All repeats are biological replications that involve the same experimental procedures on different mice. Experiments comparing different genotypes, adjuvant responses are designed with individual treatments being assigned randomly. Where possible, treatments will be assigned blindly to the experimenter by another individual in the lab. When comparing samples from different groups, samples from each group will be analyzed in concert, thereby preventing any biases that may arise from analyzing individual treatments on different days.p < 0.0001; p < 0.001) than the adult mice (p < 0.0001) in aged mice than the adult mice.CDG induces balanced, potent, and long-lasting vaccine adjuvant responses in young animals , 20–22. ult mice . Similarex vivo recall assay showed that 60 days post-immunization, aged mice had dramatically (10-folds) reduced memory Th1 and Th17 responses in the lung (+ PD1+ CD4+ TFH cells than the adult mice (CDG immunization generates potent and long-lasting memory T helper (Th)1, Th2, Th17 response in adult mice , 20–22. the lung . The lunthe lung . CDG indult mice . Togethein vivo (+ and TNFR2− cDC2 subsets (+ cDC2 subset (R2D2) mediates the cellular immune responses of CDG adjuvant, while the TNFR2− cDC2 subset activates moDCs to mediate the humoral responses of CDG -κB pathway RelB. Again, CDG-induced pRelB was reduced in lung moDCs from the aged mice  and high-affinity (anti-NP2) antigen-specific antibodies influenced by aging. High-affinity antibody formation is a hallmark of germinal center formation and provides rapid neutralization of extracellular pathogens.Adult mice (~3 months old), not aged mice (~18 months old), exhibit potent CDG-induced humoral and cellular responses . We askeSurprisingly, while CDG still induced antigen-specific Th2 responses in the lung of 1-year-old mice , CDG barClinicalTrials.gov NCT02675439). Here, we discovered that CDG adjuvanticity depends on age. To translate CDG research into the clinic, we need methods to improve CDG adjuvanticity in aging humans.The discovery that 1-year-old healthy mice already had waning CDG vaccine response was potentially significant in the translational research of CDNs. The median age for COVID-19 patients in the US is ~63 years old . The medMoDC activation is essential for CDG adjuvanticity . Aged miD221N/A223R-Fc (IgG2A) fusion proteins to target solTNF or tmTNF to moDCs , more than 1,000-fold higher than its next binding partner IgG2b-Fc  class IIlow/int macrophages were also targeted (in vivo (We generated TNF-Fc (IgG2A) and TNFto moDCs . The TNFot TNFR1 . MoDCs eot TNFR1 . FcγRI bIgG2b-Fc . We show4+ moDCs . The CD6targeted . Howeverfl/flCD11ccre mice. CDG does not generate lung TNF or vaccine responses in the IRF4fl/flCD11ccre  plus CDG generated high-affinity serum IgG antibody in the IRF4cre mice , suggestfl/flCD11ccre mice (fl/flCD11ccre mice (SolTNF-Fc (IgG2A) did not restore CDG-induced antibody responses in the IRF4cre mice . This wacre mice . Last, acre mice  as we prcre mice .6CGG to separate high- vs. low-affinity antibodies by immunization. We immunized 1-year-old C57BL/6J mice with NP6CGG alone, CDG/NP6CGG, or CDG/NP6CGG with solTNF-Fc or tmTNF-Fc twice at a 2-week interval. Lung memory T cell responses were determined by ex vivo recall assay 60 days post the last immunization. Remarkably, both solTNF-Fc and tmTNF-Fc restored CDG-induced lung memory Th1 and Th17 responses in the 1-year-old C57BL/6J mice  or solTNF-Fc (IgG2A) can enhance CDG adjuvanticity in 1-year-old middle-aged C57BL/6J mice. Again, we used the model antigen NP/6J mice . The moD/6J mice . Consist/6J mice . The 1-y/6J mice . We foun/6J mice .Last, we immunized (i.n.) 1-year-old mice with CDG-adjuvanted mucosal pneumococcal protein subunit vaccine CDG/PspA with or without TNF-Fc fusion proteins twice at 2 weeks' interval. Serum anti-PspA IgG was determined 120 days post the last immunization. Remarkably, 1-year-old mice immunized with CDG/PspA plus TNF-Fc fusion proteins had stronger serum anti-PspA IgG 4 months post-immunization than the 1-year-old mice immunized with only CDG/PspA . Togethede novo. We used a much lower dose of TNF-Fc (IgG2a) (100 ng) than previous TNF (2 μg) used as a mucosal adjuvant  and tmTNF-Fc (IgG2A) twice at a month's interval. Serum antigen-specific antibodies were determined in the serum on days 30 and 60 post last immunization. As expected, CDG did not induce durable and high-affinity IgG in the aged mice (p < 0.0001). The enhancement of antibody production was across the spectrum, including augmented production of IgG1, IgG2C by moDC-targeting TNF fusion proteins  2-year-old C57BL/6J mice with NPged mice . In contged mice   2-year-old C57BL/6J mice 60 days post the last immunization and determined antigen-specific IgA titer including high-affinity IgA. A significant elevation in the IgA titer was observed in aged mice immunized with CDG/NP6CGG adjuvanted with solTNF-Fc (IgG2A) and tmTNF-Fc (IgG2A) as compared to the mice group immunized with only CDG/NP6CGG (p < 0.0001) (6CGG elicited primarily low-affinity lung IgA titer while CDG/NP6CGG with moDC-targeting TNF fusion proteins generated high-affinity lung IgA (p < 0.0001) in the 2-year-old mice  . Furtherold mice .Together, the data indicated that the moDC-targeting TNF fusion proteins markedly enhanced CDG-induced durable and high-affinity antibody responses, both in the serum and in the lung, in the 2-year-old C57BL/6J mice.via enhancing neutrophil recruitment, are essential for clearing bacteria, e.g., Streptococcus pneumoniae infections. Next, we examined memory T cell responses in 2-year-old mice immunized with the moDC-targeting TNF fusion protein-adjuvanted CDG/NP6CGG vaccine 60 days post the last immunization by ex vivo recall assay. We examined memory T cell production in the lung (mucosa) and spleen (systemic) in the immunized mice.CDG-induced memory T cell response, primarily Th1 and Th17, is impaired in aged mice . Vaccine6CGG only (6CGG plus moDC-targeting TNF fusion proteins elicited a significantly high level of Th1 (p < 0.0001), Th17 (p < 0.01) responses in the lungs  or Th17 (IL-17a) was detected in the lungs of aged mice immunized with CDG/NPCGG only . SimilarCGG only . Remarkahe lungs . Lung mehe lungs . Similarold mice . Differeold mice . Howeveresponses . The datin vivo mode of action of CDG adjuvant in the adult mice  to design new biologics to circumvent the defective component in CDG mode of action and restore CDG adjuvanticity in the aged mice. Our new vaccine strategy focuses on moDC targeting and TNF induction because (i) CDG adjuvanticity is mediated by two DC subsets: cDC2 and moDCs  to identify the ult mice , 22;  and the magnitude of CDG/PspA antibody production in 1-year-old mice. CDN-adjuvanted protein subunit vaccines protected adult mice from respiratory infections such as influenza  have established an excellent safety file in humans, including the elderly. The human body makes TNF. Low-dose (ng) targeted delivered TNF biologics are likely safe in the elderly as well. Nevertheless, a future study is needed to establish a safety profile for the CDG/TNF biologics vaccines for the elderly.As a vaccine specific to the elderly, safety is paramount. Intranasally administered CDG (5 μg/mouse) does not cause acute toxicity in mice , 43, 44 Last, the moDC-targeting TNF fusion proteins applied here did not have adjuvant activity when administered in the absence of CDG. Furthermore, TNF when used as a mucosal adjuvant mainly induces Th2 responses . Here, mAll datasets presented in this study are included in the article/The animal study was reviewed and approved by Institutional Animal Care and Use Committee, University of Florida.SM and LJ conceived the research. LJ designed the experiments, wrote the manuscript, and supervised the research. HG, SM, DK, and LJ performed experiments and analyzed the data. HG drafted the manuscript. All authors contributed to the article and approved the submitted version.LJ and SM are co-Inventors on a patent (PCT/US19/53548) on the moDCs-targeting TNF fusion proteins. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


On September 16, 2020, this report was posted online as an MMWREarly Release.Pregnant women might be at increased risk for severe coronavirus disease 2019 (COVID-19),possibly related to changes in their immune system and respiratory physiologyInternational Classification of Diseases, Tenth Revision,Clinical Modification, (ICD-10-CM)VSD is a collaboration between CDC’s Immunization Safety Office and nine U.S.health care organizations serving more than 12 million persons each year.Hospitalizations with a patient diagnosis of COVID-19 were identified using COVID-19Pregnant women with COVID-19 diagnoses were classified into the following three groupsbased on the primary reason for admission: 1) treatment of COVID-19 without an obstetricreason ; 2) an obstetric reason, along withsymptoms consistent with COVID-19 ; and3) an obstetric reason, without COVID-19–compatible symptoms (or with a historyof resolved COVID-19), but with a positive test result for SARS-CoV-2 at the time ofadmission. Demographic and pregnancy characteristics among pregnant women admitted forCOVID-19 were compared with those of women admitted for obstetric reasons. Birthoutcomes in pregnant women with SARS-COV-2 infection were compared with background ratesamong all pregnant women in eight VSD sites during the study period. This activity wasreviewed by CDC and was conducted consistent with applicable federal law and CDCpolicy.2) was 36.2% overall andwas higher among the 43 women hospitalized for COVID-19 (44.2%) than among the 62hospitalized for obstetric reasons (30.6%). Similarly, prevalence of gestationaldiabetes was higher among women hospitalized for COVID-19 (25.6%) than among thosehospitalized for obstetric reasons (8.1%).During March 1–May 30, among 4,408 persons hospitalized with a COVID-19 diagnosisat VSD sites, 105 (2.4%) pregnant women were identified. SARS-CoV-2 real-time reversetranscription–polymerase chain reaction test results were positive for 104 women.One additional woman, who had a negative SARS-CoV-2 test result, was symptomatic and hadclose contacts with confirmed COVID-19; she received a clinical diagnosis of COVID-19.Among these 105 pregnant women, 43 (41.0%) were hospitalized for COVID-19 illness and 62(59.0%) were admitted for obstetric reasons . Among tAmong all 105 pregnant women hospitalized with COVID-19, 93 (88.6%) had a pregnancyoutcome before July 31 , includiAmong 105 hospitalized pregnant women with COVID-19 diagnoses in VSD during March1–May 30, 41% were hospitalized because of COVID-19 illness, and 59% wereadmitted for obstetric reasons. Approximately 80% of those admitted for obstetricreasons were asymptomatic with COVID-19. This percentage is similar to findings froma New York City study that reported universal screening of obstetrics patients onadmission and found that among 13.7% of women with SARS-CoV-2–positive testresults, 87.9% were asymptomatic , compared with thosein the general population  this should be implemented. Fourth, this study did not collectinformation on prenatal care, which is known to affect pregnancy outcomes. However,VSD’s surveillance population is primarily insured and has high rates ofstandard prenatal care  than among those admitted forpregnancy-related treatment or procedures  and found to haveCOVID-19. Intensive care was required for 30% (13 of 43) of pregnant womenadmitted for COVID-19, and one pregnant woman died from COVID-19.Antenatal counseling emphasizing preventive measures, including use of masks,frequent hand washing, and social distancing, might help prevent COVID-19among pregnant women, especially those with prepregnancy obesity andgestational diabetes.


We aerosolized severe acute respiratory syndrome coronavirus 2 and determined that its dynamic aerosol efficiency surpassed those of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome. Although we performed experiment only once across several laboratories, our findings suggest retained infectivity and virion integrity for up to 16 hours in respirable-sized aerosols. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a readily transmissible zoonotic pathogen and the etiologic agent of the coronavirus disease (COVID-19) pandemic (www.chtechusa.com), and Aerogen Solo (AS) (https://www.aerogen.com), to generate viral aerosols (Fs) as a unitless quotient of initial titer (PFU/L in liquid stock) to the resulting aerosol (PFU/L aerosol) providing a quantitative indicator for comparing airborne fitness , Collison 6-jet (C6)  or MERS-CoV (p = 0.01). Because SARS-CoV was aerosolized into a different chamber/volume than MERS-CoV and SARS-CoV-2, we cannot rule out chamber effects for the difference in Fs between SARS-CoV and SARS-CoV-2. Our comparison of nebulizers showed improved Fs for SARS-CoV-2 with the C6 (p = 0.006) and the AS (p = 0.01) over the C3 but no difference between the C6 and AS (p = 0.46).We determined lization . When weFurther studies with SARS-CoV-2 at Tulane University preliminarily assessed the long-term stability of airborne virus. We used a custom-built rotating (Goldberg) drum to provide an environment in which rotational drum speed overcomes the terminal settling velocity of the 2–3-μm particles, thereby providing a static aerosol suspension of known volume . We meas1/2 = 2.36E+05; τ = 3.40E+05). The curve .We graphed plaque assay and RT-qPCR results and applied nonlinear least-squares regression analysis single-order decay with no outlier detection, resulting in a poor curve fit, which typically results from a lack of replicate samples. We detected infectious SARS-CoV-2 at all time points during the aerosol suspension stability experiment . A minorhe curve , panel Bhe curve , panel Ahttps://www.flickr.com/photos/niaid/albums/72157712914621487). Airborne SARS-CoV-2 maintained the expected morphologies, size, and aspect ratios up to 16 h. Specifically, virions aged for 10 min (We also performed a qualitative assessment of virion integrity after longer-term aerosol suspension . Scanninlog10 (The comparison of short-term aerosol efficiencies of 3 coronaviruses showed SARS-CoV-2 approximates or exceeds the efficiency estimates of SARS-CoV and MERS-CoV. Some efficiency determinations for SARS-CoV-2 ranged to −5.5log10 , a full Collectively, these preliminary data suggest that SARS-CoV-2 is resilient in aerosol form and agree with conclusions reached in earlier studies of aerosol fitness (Additional methods and results in a study of aerosol efficiencies of coronaviruses.


C. elegans to identify members of the lipid bilayer stress response. AID-mediated degradation of the mediator MDT-15, a protein required for the upregulation of fatty acid desaturases, induced the activation of lipid bilayer stress-sensitive reporters. We screened through most C. elegans kinases and transcription factors by feeding RNAi. We discovered nine genes that suppressed the lipid bilayer stress response in C. elegans. These suppressor genes included drl-1/MAP3K3, gsk-3/GSK3, let-607/CREB3, ire-1/IRE1, and skn-1/NRF1,2,3. Our candidate suppressor genes suggest a network of transcription factors and the integration of multiple tissues for a centralized lipotoxicity response in the intestine. Thus, we demonstrated proof-of-concept for combining AID and RNAi as a new screening strategy and identified eight conserved genes that had not previously been implicated in the lipid bilayer stress response.Alteration of the lipid composition of biological membranes interferes with their function and can cause tissue damage by triggering apoptosis. Upon lipid bilayer stress, the endoplasmic reticulum mounts a stress response similar to the unfolded protein response. However, only a few genes are known to regulate lipid bilayer stress. We performed a suppressor screen that combined the auxin-inducible degradation (AID) system with conventional RNAi in  Biological membranes play an important role in protein folding, signaling, secretion, and the turnover of proteins. Changes in the lipid composition of a membrane alter its properties, and thus, interferes with its function and leads to lipid bilayer stress (LBS) . On a ceXBP1 mRNA to stabilize the transcript and allow translation of the spliced XBP1 transcription factor (The unfolded protein response (UPR) sensors IRE1, PERK1, and ATF6 are sensitive to changes in membrane fluidity . Activatn factor . PERK1 pn factor . During n factor . These tC. elegans, loss of fatty acid desaturases fat-6 and fat-7 or mdt-15, which is a mediator subunit required for fat-6/7 expression, leads to a higher ratio of saturated fatty acids in the membrane. This activates the ER stress reporter hsp-4::gfp via the IRE-1/XBP-1 axis  system has been recently introduced to mediate fast and reversible degradation of targeted proteins in MDT-15 by AID was used to induce LBS, which was visualized using the ER-stress reporters Patf-4(uORF)::gfp and Phsp-4::gfp. We screened RNAi libraries targeting kinases and transcription factors. Out of 868 genes, we identified one known and eight novel hits that robustly blocked LBS response upon MDT-15 degradation.Here, we identify suppressors of lipid bilayer stress response using a novel approach combining AID and RNAi-based forward genetic screening. Degradation of OP50Escherichia coli, as described ::GFP::unc-54(3′UTR)], LSD2096: ieSi57  II; yh44 [mdt-15::degron::EmGFP] III; [Patf-4(uORF)::GFP::unc-54(3′UTR)], LSD2102: ieSi57  II; yh44 [mdt-15::degron::EmGFP] III; zcIs4 [Phsp-4::GFP] V.IJ1729: SJ4005.The screening strain was generated by crossing IJ1729 males with LD1499. 48 F2s were singled out and their offspring were placed onto plates containing 100 μM auxin and the upregulation of the reporter was determined . In paraFor image acquisition, the animals were placed onto fresh 2% agar pads and anesthetized with 1 mM tetramisole, as previously described . Images GFP fluorescent levels were scored by inspecting transgenic animals with a fluorescent dissecting scope while still on the culturing/treatment condition plates  was dissolved in 10 mL DMSO to yield a 40 mM stock solution and stored at 4°. The stock was further diluted in M9 to 100 μM before use.A detailed step-by-step protocol can be found in the supplementary, and a schematic outline is shown in Briefly, 24-well plates were filled with Nematode Growth Medium (NGM) containing ampicillin (100 μg/ml), tetracycline (12.5 μg/ml), and 1mM Isopropyl β-d-1-thiogalactopyranoside (IPTG) seeded with 50 μl of freshly grown RNAi bacteria and dried in a sterile laminar flow cabinet. The following day, plates containing gravid LSD2096 adults were washed off and discarded, and the laid eggs were scratched off and collected. Approximately 30-40 eggs were pipetted into each well and incubated at 20°. After 48 hr, the wells were top coated with 50 μl of 100 μM auxin and dried in a sterile laminar flow cabinet at 20° overnight. The following day, the wells were screened for the suppression of the GFP signal. The kinase and transcription factor libraries were screened twice. The preliminary hits had to pass three additional runs on 6 cm plates successfully.RNAi and double RNAi were performed as described (RNAi bacteria cultures were grown overnight in Lysogeny Broth (LB) medium with carbenicillin (100 µg/mL) and tetracycline [12.5 µg/mL], diluted and grown again for 4-6 hr. Bacteria were then concentrated by centrifugation and induced with 1 mM IPTG and spread onto NGM plates containing 1 mM IPTG, tetracycline (12.5 µg/mL) and ampicillin (50 µg/mL). For the empty RNAi vector (EV), plasmid pL4440 was used as a control.bacterial RNAi cultures were grown separately, and after the concentration step, mixed in a 1:1 ratio and seeded onto culture plates, as described above for single RNAi.Animals, RNAi bacteria, and plates were prepared as above, without the addition of auxin. Heat-shock was carried out for 1 hr at 37°, incubated for 5 hr at 25°, and GFP expression was determined in the animals. Plates were top coated with 0.5 ml of 35 μg/ml tunicamycin , incubated for 6 hr at 25°, and GFP expression was again determined.https://doi.org/10.25387/g3.12783044.Strains used in this study are either available from CAENORHABDITIS GENETICS CENTER (CGC) or upon request. Supplementary materials are available at figshare: mdt-15 or fat-6/7, resulting in the upregulation of ER-stress reporter Phsp-4::gfp (hsp-4::gfp upon RNAi against mdt-15 and fat-6/7 (atf-4(uORF)::gfp, which was also induced upon knockdown of mdt-15 or fat-6/7 RNAi (atf-4(uORF)::gfp reporter contains two upstream open reading frames (uORF) in the 5′ untranslated region ::gfp over Phsp-4::gfp for its stronger induction of GFP, allowing easier detection in 24- or 96-well plates. Crossing mdt-15(tm2182) mutant with Patf-4(uORF)::gfp led to heterogeneous GFP expression; therefore, it was difficult to use this strain for screening. Hence, we switched to an endogenously degron-tagged mdt-15 strain (C. elegans expressed Patf-4(uORF)::gfp only at basal levels at 20°. Incubation with 100 μM auxin for 24 hr increased GFP levels drastically and homogenously throughout the mdt-15::degron; TIR1; Patf-4(uORF)::gfp transgenic animals (atf-4(uORF)::gfp . Upon treatment with auxin, we also observed typical mdt-15 phenotypes, such as small body size, reduced brood size, and a pale appearance in mdt-15::degron; TIR1; Patf-4(uORF)::gfp animals, consistent with previous reports ::gfp animals were sensitive to bleaching, normally used to synchronize C. elegans populations. Either, our mdt-15::degron; TIR1; Patf-4(uORF)::gfp screening strain carried a background mutation or degron-tagged mdt-15 may be partially hypomorph. However, the untreated mdt-15::degron; TIR1; Patf-4(uORF)::gfp screening strain appeared superficially wild type without any induction of GFP. We did not outcross the strain but rather decided to avoid bleach synchronization and continued with our screen by collecting laid eggs off the bacterial lawn. Thus, we established the mdt-15::degron; TIR1; Patf-4(uORF)::gfp strain for screening for LBS suppressors.Lipid bilayer stress can be induced by knocking down  fat-6/7 . We test6/7 RNAi . The Patynthesis . For scr animals , but didC. elegans kinases ::gfp induction upon mdt-15 degradation . XPB-1 is known to upregulate hsp-4 mRNA during UPR . In addition, knockdown of xbp-1 resulted in the complete absence of any GFP expression even in the control Patf-4(uORF)::gfp strain, suggesting that the transcription factor XBP-1 may be necessary for general expression of atf-4 in C. elegans. Together with XBP-1s importance in transcribing hsp-4, it is difficult to argue whether xbp-1 is necessary for LBS, and further research is required to elucidate this. Thus, we ended up with nine candidate suppressors of LBS from our combined AID with RNAi screen  or sams-1(RNAi). We found that single knockdown of the candidate genes did not induce Patf-4(uORF)::gfp reporter, except for RNAi against ire-1 or let-607 which induced the Patf-4(uORF)::gfp reporter , suggesting that loss of either these latter two genes may cause ER stress. By contrast, knockdown of ire-1 or let-607 suppressed Patf-4(uORF)::gfp induction upon mdt-15 degradation with 0.1 mM auxin (fat-6/7 with ire-1 or let-607 and double RNAi of sams-1 with ire-1 or let-607 displayed strong suppression of the Patf-4(uORF)::gfp reporter . Similarly, double RNAi of candidate genes with either fat-6/7 or sams-1 exhibited full suppression of Patf-4(uORF)::gfp reporter, except for elt-2 knockdown . Knockdown of elt-2 did not suppress fat-6/7(RNAi) , but suppressed sams-1(RNAi) Patf-4(uORF)::gfp reporter . This suggests a differential genetic activation of LBS from a higher ratio of saturated fatty acids in the membranes compared to depleting the cell’s phosphatidylcholine levels. Thus, our screening approach identified nine high confidence candidates. Eight of these nine candidates have not previously been described to mediate LBS in C. elegans.To validate that the nine potential candidates were suppressors of LBS and were not only specific to mM auxin  or with mdt-15. Our screen revealed a known molecular player (IRE-1) and identified several new genes important to mount a proper LBS response. Thus, our results provide proof-of-concept and support the feasibility of combined AID-RNAi screening approaches. Here we discuss the function of the suppressor genes briefly and propose a hypothetical model for the LBS pathway in C. elegans . Curiously, mdt-15 and skn-1c but not skn-1a, co-regulate targets involved in detoxification, such as gst-4 . This suggests that some of the observed phenotypes in mdt-15 mutants or knockdowns are skn-1-dependent. The mammalian SKN-1c ortholog NRF2 has been shown to have protective functions during palmitate-induced lipotoxicity in mammalian cells  and is related to ATF6 and SREBP ::gfp and Phsp-4::gfp expression mainly in the intestine . Despite the striking similarities shared by rege-1 and drl-1, only drl-1 modulates LBS. Intriguingly, drl-1 knockdown itself causes ER stress at the L2 stage, and this mounts a protective effect throughout life ::gfp transgenic animals in the 24-wells without food failed to upregulate the reporter.ntestine . Therefogsk-3) has been described as the busiest of all kinases with over 100 targets known and was found to attenuate palmitate-induced apoptosis (gsk-3 inhibits skn-1 and stabilizes CREB3, two mode-of-actions contradicting the results of our screen (gsk-3 here does not act as previously reported; therefore, we hypothesize other modes of action. One alternative mechanism could be via autophagy. Activation of the lipid bilayer stress activates autophagy via the IRE-1/XBP-1 axis (Glycogen synthase kinase-3 (gtf-2f2, ntl-4, and rpl-14, which are involved in transcription, RNA processing, and translation, respectively. Interestingly, although RNAi against gtf-2f2, ntl-4, and rpl-14 inactivate general processes, the heat- or tunicamycin induced UPR remained functioning and was not affected. This favors the model that UPR and LBS are differentially regulated (The last three hits consisted of egulated .skn-1 and elt-2, together with the previously characterized ire-1, are transcription factors involved in immune responses. let-607 may be activated in parallel with the canonical UPR arms, and drl-1 and gsk-3 modulate the ER stress response in our suggested model upstream or downstream, respectively. The last category consists of genes involved in general processes of gene expression. Interestingly, all eight novel candidate genes are well-conserved, suggesting the potential implications of these genes in the mammalian lipid bilayer stress response.We demonstrated the feasibility of combining AID and RNAi-based genetic screens. We report the identification of eight novel regulators of the lipid bilayer stress response and grouped them into three categories . skn-1 a


A 78-year-old man presented with epigastralgia and left lower-leg pain 17 days post-laparoscopic surgery for gastric cancer. Immediately after admission, he suffered cardiopulmonary arrest, and cardiopulmonary resuscitation was initiated. Return of spontaneous circulation was obtained after 20 min. An electrocardiography revealed atrial fibrillation with an S1Q3T3 pattern. Veno-arterial extracorporeal membrane oxygenation (ECMO) was initiated due to hemodynamic instability. Coronary angiography revealed no stenosis. Pulmonary angiography (PAG) revealed a massive thrombus in the right main pulmonary artery (PA) . Because


B cell maturation antigen (BCMA)-targeted immunotherapy has shown unprecedented results in the treatment of relapsed or refractory (R/R) multiple myeloma (MM). However, disease progression remains an issue attributed to variable BCMA expression, BCMA downregulation, and heterogeneity of tumor antigens in MM. Therefore, additional treatment options with novel therapeutic targets are warranted. G protein-coupled receptor, class C group 5 member D (GPRC5D), an orphan receptor expressed on malignant plasma cells with limited expression in normal tissue, has emerged as a promising therapeutic target for R/R MM. GPRC5D-targeted chimeric antigen receptor (CAR)-T and CAR-NK cell therapy, as well as bispecific T cell engagers, offer remarkable anti-tumor activities. We summarized some latest reports on GPRC5D-targeted treatments for R/R MM from the 2022 ASH Annual Meeting (ASH 2022). The prognosis of patients with relapsed or refractory (R/R) multiple myeloma (MM) is generally poor, and new therapeutic methods are urgently demanded. G protein-coupled receptor family C group 5 member D (GPRC5D) is primarily expressed on myeloma cells, and normal tissue expression is limited to the hair follicle, making it a promising therapeutic target for patients with MM –4. We suIn patients with R/R MM, anti-GPRC5D chimeric antigen receptor (CAR)-T cell treatment exhibited encouraging clinical efficacy and a manageable safety profile –4. Dr. BBesides GPRC5D CAR-T cell monotherapy, BCMA and GPRC5D dual-target CAR-T cell therapies , concurrent administration of GPRC5D-targeted CAR-T cells and BCMA-targeted CAR-T cells (NCT05431608) in patients with R/R MM are being investigated in clinical settings.Talquetamab JNJ-64407564) is a first-in-class, off-the-shelf, bispecific T cell engager antibody that targets both GPRC5D and CD3 Table . In the 4407564 iRG6234 is another exciting novel GPRC5DxCD3 BiTE–10. CarlSeveral GPRC5D CAR-T cell studies reported higher ORR 86%-100%) compared with GPRC5DxCD3 BiTEs (60.4%-74%); CR rates of GPRC5D CAR-T were also higher –6, 9 (Ta%-100% coFT555 is a multiplexed-engineered GRPC5D CAR-NK cell derived from an induced pluripotent stem cells (iPSC) master cell line Table 11. ComparIn conclusion, the ASH 2022 Annual Meeting exhibited notable advances in the field of GPRC5D-targeted therapies in MM, as summarized in Tables


Numerous international studies have focused on the outcomes of COVID-19 therapies in adults, however data are limited on the outcomes for high-risk paediatric patients treated with similar agents.Data was collected retrospectively for high-risk children treated for symptomatic COVID-19 at a tertiary paediatric hospital in Australia, over a 12-month period (September 2021- August 2022). Indications for COVID-19 therapies are outlined in [Figure 1]; with the guideline being implemented in September, 2021.Note: Casirivimab-imdevimab & sotrovimab were included in previous iterations of the COVID-19 algorithm. Due to increased resistance with newer strains of the dominant Omicron variant, these therapies were removed from the algorithm in April 2022.A total of 139 children were treated with COVID-19 therapies based on having a significant underlying comorbidity meeting criteria [Table 1].Symptomatic, non-hypoxic COVID-19; The majority  received treatment for symptomatic, non-hypoxic COVID-19 [Table 2]. Three drug-related adverse events were noted that resolved with treatment cessation; an infusion reaction with sotrovimab; taste disturbance with nirmatrelvir/ritonavir; and elevated liver enzymes with remdesivir [Table 3]. Six (2.9%) children re-presented within 90 days of therapy requiring hospitalisation [Table 4]; 4 (3.9%) with ongoing COVID-19 symptoms requiring monitoring and 2 (2%) with suspected secondary infections.Symptomatic, hypoxic COVID-19; Thirty-seven (26.6 %) children were treated for symptomatic, hypoxic COVID-19 [Table 2]. Of these, 31 (83.8%) received dexamethasone; 9 (24.3%) remdesivir; 5 (13.5%) baricitinib; 5 (13.5%) tocilizumab, 1 (2.7%) sotrovimab and 1 (2.7%) anakinra. One death occurred, attributed to complications secondary to a bone marrow transplant and was unrelated to COVID-19 directed therapy. There were no other adverse effects. Early cessation of therapies prior to course completion was indicative of symptom resolution and discharge from hospital. Overall clinical course for symptomatic, hypoxic COVID-19 patients is outlined in Table 5. Hospitalisation within 90 days of receiving COVID-19 therapies was required for 4 (10.8%) patients; 3 (8.1%) with ongoing COVID-19 symptoms requiring monitoring and 1 (2.7%) with a suspected secondary infection [Table 4].The use of directed COVID-19 therapies in high-risk paediatric patients were largely well-tolerated and side effects self-resolved upon treatment cessation.All Authors: No reported disclosures


It is believed that intestinal recruitment of monocytes from Crohn’s Disease (CD) patients who carry NOD2 risk alleles may repeatedly give rise to recruitment of pathogenic macrophages. We investigated an alternative possibility that NOD2 may rather inhibit their differentiation from intravasating monocytes.The monocyte fate decision was examined by using germ-free mice, mixed bone marrow chimeras and a culture system yielding macrophages and monocyte-derived dendritic cells (mo-DCs).Nod2-deficient mice, despite a similar abundance of monocytes. This decrease was independent of the changes in the gut microbiota and dysbiosis caused by Nod2 deficiency. Similarly, the pool of mo-DCs was poorly reconstituted in a Nod2-deficient mixed bone marrow (BM) chimera. The use of pharmacological inhibitors revealed that activation of NOD2 during monocyte-derived cell development, dominantly inhibits mTOR-mediated macrophage differentiation in a TNFα-dependent manner. These observations were supported by the identification of a TNFα-dependent response to muramyl dipeptide (MDP) that is specifically lost when CD14-expressing blood cells bear a frameshift mutation in NOD2.We observed a decrease in the frequency of mo-DCs in the colon of NOD2 negatively regulates a macrophage developmental program through a feed-forward loop that could be exploited for overcoming resistance to anti-TNF therapy in CD. While the intermediate monocytes CD14+CD16+ regulate angiogenesis and modulate effector T cell activity . The inflammatory microenvironment, including increasing levels of CCL2 and IL-8, promotes the recruitment of monocytes in the intestine of IBD patients , 14. WhiNOD2 gene confer an increased susceptibility to CD, likely due to the loss of NOD2 function. Furthermore, patients who carry NOD2 risk alleles are at greater risk of developing stricturing disease, that corresponds to a narrowing of the intestine due to continued inflammation  associated with tolerogenic DC, in a GM-CSF-dependent manner in mice  of BM cells from wild-type (WT) (CD45.1) and Nod2-deficient mice (CD45.2)  F4/80+ macrophages and MHC II+ CD226+ F4/80lo subsets that differ in function and origin. The MHC II+ CD226+ F4/80lo subset is Irf4-dependent and is continuously renewed by blood monocytes   and germ-free (GF) mice at steady-state using the markers CD11c, CD11b, CCR2, Ly6C, and MHCII (+ monocytes (Nod2-deficient mice as what was observed in the GF condition (+CD11c+Ly6C+CCR2+MHCII+)   proporti propria , we reas+MHCII+ fraction , a well-established cocktail of conditioning soluble factors , mTORC1, and mTORC2, signaling pathways that are both involved in the generation and activity of tissue-resident peritoneal macrophages in vivo , 63, andn vitro . As we hn vitro . Interesn vitro . The PI3n vitro . As a ren vitro . These rIRF4 expression  were mined for candidate genes encoding for enzymes involved in glycolysis. We observed an MDP-induced upregulation of several glycolytic genes such as the enzyme Gapdh that regulates the conversion of D-glyceraldehyde 3-phosphate into 1,3-bisphosphoglycerate , revealed a trend to a lower glycolytic reserve in the absence of NOD2. These results suggest that NOD2 may promote a lower pH, by sustaining a higher glycolytic demand, which is required for mo-DCs differentiation  were incubated sequentially with MDP and then LPS or LPS alone. As expected, the cells pre-treated with MDP enhanced their subsequent cytokine response upon LPS treatment  was significantly induced among the 156 commonly differentially upregulated genes that are induced by MDP in CD14-expressing cells from either control or CD patients (NR4A3 is involved in the proper differentiation of Mo-DCs (IL12B and miR-155 in mutated patients as compared to the patients who did not bear this mutation (n=2) (via NF-kB (5/200)  . Given tf Mo-DCs . Further446 . Givon (n=2) . Pathway2.53E-7) . CD14-ex2.53E-7) . Intereshi monocytes in the colon, suggesting that systemic MDP does not affect the number of recruited colonic monocytic cells at baseline, but may rather trigger early changes in epigenetic regulation of mo-DCs development , cytokine production can be detected after 3h of culture of human mo-Macs or mo-DCs   . One may0.00206) . Inhibit0.00206) . Additio0.00206) , and blo0.00206) . In CD p0.00206) . Analysi0.00206) . Equallyf mo-DCs , is lessThus, by using BM chimera and fecal transplantation models, our study highlights the role of NOD2 and the microbiota in the reconstitution of monocyte-derived cells in the colon of mice at steady state while not affecting mo-Macs during colitis. NOD2 promotes mo-DCs development by interfering with mouse BM and human monocyte differentiation in macrophages. In addition, we observed that NOD2 impacts mo-DCs/mo-Macs differentiation through activation of the mTORC2 pathway and a metabolic switch in NOD2 transfected THP1 involving TNF-α. The involvement of TNF-α signaling pathway has been confirmed in transcriptomic data of CD patients. Here, we provided a better understanding of macrophage-differentiation inhibition by NOD2-mediated signaling, which may offer new therapeutic strategies aiming at limiting the detrimental effects of pathogenic macrophages in gut pathologies such as CD.-/-), have free access to a standard laboratory chow diet in a temperature-controlled SPF environment and a half-daylight cycle exposure. C57BL/6J WT GF, Nod2-/- GF mice were bred at TAAM-CNRS and were transferred into autoclaved sterile micro-isolator cages. C57BL/6J WT mice were purchased from Janvier Laboratories, France. Ly5.1 WT mice (CD45.1) were purchased from Charles Rivers Laboratories, France. –/–Nod2 mice were provided by R.A. Flavell .All animal studies were approved by the local investigational review board of the Institut Pasteur of Lille (N°28010-2016012820187595). Animal experiments were performed in an accredited establishment (N° B59-108) according to governmental guidelines N°86/609/CEE. Age-matched and gender-matched C57BL/6J WT, Nod2-deficient mice (Nod2A single cycle of acute colitis was induced by giving mice 2% (wt/vol) DSS (TdB Consultancy) for a period of 4 to 5 days followed by normal drinking water for the indicated period of time with a threshold of the maximal lost weight of 20% of the initial weight. DSS was dissolved in drinking water and changed every 3 days. Signs of morbidity, including body weight, stool consistency, occult blood, or the presence of macroscopic rectal bleeding, were checked daily. At specific time points throughout the course of the challenge, mice were autopsied to assess the severity of the disease by measurement of colon lengths and cell composition by flow cytometry.6 fresh BM cells. Nod2-deficient animals were irradiated and reconstituted in a 1:1 ratio with bone marrow cells from WT (CD45.1) and Nod2-deficient mice (CD45.2). Blood was collected in heparin-containing tubes 7–8 weeks after BM transplantation and reconstitution efficiency was checked by flow cytometry . Twenty-four hours post-irradiation, mice received intravenously 2 × 10ytometry . CellulaFecal microbiota from WT mice was transplanted by gavage in GF mice that are deficient or not for Nod2 . The micLamina Propria Mononuclear Cells (LPMC) were prepared from murine intestines by enzymatic digestion as previously described . BrieflyBone marrow cells were flushed out of the mouse bones with complete RPMI 1640 (Gibco). A single-cell suspension was then prepared by repeated pipetting. Bone marrow-derived dendritic cells (BMDCs) were generated for 7 days in respectively RPMI-1640 medium (Gibco), supplemented with glutamine, penicillin, streptomycin, 2-mercaptoethanol , and 10% heat-inactivated fetal calf serum . The medium was supplemented with 20 ng/ml GM-CSF of J558 cells (GM-CSF-producing cells). Half of the medium was removed at day 3 and new medium supplemented with GM-CSF supernatant  was added (+ microbeads (Miltenyi Biotec) according to the manufacturer’s recommendations. Cells were cultured for 6 days with rhGM-CSF  and rhIL-4 . When mentioned, muramyl dipeptide (MDP)  was added from the beginning of a 5-day culture with GM-CSF and IL-4 (day 0). The mTOR activator MYH1485 , wortmannin  or rapamycin  were added at the start of the culture (day 0). Adalimumab (Humira M02‐497) was a gift from Abbott .Human Peripheral Blood Mononuclear Cells were prepared from buffy coats (Etablissement Francais du Sang (EFS), Lille, France) using Ficoll Paque . The use of human samples was approved by the French Ministry of Education and Research under the agreement DC 2013-2575. According to French Public Health Law , Institutional Review Board and written consent approval are not required for human non-interventional studies. Monocytes were positively isolated using CD14Cytokine levels were determined by ELISA kits (DuoSet), according to protocols provided by R&D Systems.Protein extraction was performed using RIPA buffer in the presence of a complete Mini EDTA-free protease inhibitor (Roche) and PhosSTOPTM phosphatase inhibitor (Roche). Protein separation was performed by SDS-page using Bolt 4 to 12% Bis-Tris protein gels (Invitrogen). Transferences were done in an iBlot 2 gel transfer device using iBlot 2 transfer nitrocellulose stacks (Invitrogen). Membranes were blotted against phospho-AKT (Ser473) , phospho-RAPTOR (Ser792) , phospho-p70 S6 Kinase (Thr389) , β-ACTIN  and their correspondent HRP-conjugated secondary antibodies. The revelation was performed using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and images were acquired using an ImageQuant LAS 4000 .Single-cell suspensions were stained and analyzed using a FACS LSR Fortessa™ system (BD Biosciences). Dead cells were excluded with the LIVE/DEAD Fixable Violet Dead Cell staining kit (Life technologies). The cells were then incubated for 10 minutes with purified rat anti-mouse CD16/CD32 (only for mouse cells) and normal mouse serum (Interchim) before being stained with various monoclonal antibodies for 20 minutes in the dark on ice. For mouse cells, lineage-positive cells were excluded using the PerCP5.5-conjugated anti-CD3 (17A2), anti-NK1.1 (PK136), anti-CD19 (6D5), anti-Ly6G (1A8) (Biolegend). PerCP-conjugated anti-CCR3 (83103) added to the lineage staining to exclude eosinophils was from R&D. Alexa Fluor 700-conjugated anti-Ly6C (AL21) was from BD Pharmingen. PECF594-conjugated anti-CD11c (HL3) was from BD Horizon. Allophycocyanin-Cy7-conjugated anti-CD11b (M1/70), Brilliant violet 510-conjugated anti-MHC Class II (I-A/I-E) (M5/114.15.2), Brilliant violet 650-conjugated anti-CD45.2 (104), Brilliant violet 711-conjugated anti-CD45.1 (A20), PE-conjugated anti-CD135 (A2F10), APC-conjugated anti-CD115 (AFS98), PE-conjugated anti-CD226 (10E5), FITC-conjugated anti-CD102 (3C4) were all from Biolegend. PE-conjugated anti-CCR2 (475301) was from R&D systems. The data were analyzed with Flowjo software V10.1 (TreeStar). For human cells, a similar procedure was used with anti-HLA-DR FITC , anti-CD1a APC , anti-CD16 PE-Cy7 , anti-CD14 PE .5 cells/mL to 8x105 cells/mL and routinely verified negatively for mycoplasma contamination by PCR analysis. THP1 stimulation was performed in 96-well flat bottom plates at 1x105 cells per well in a final volume of 200 μl. Cells were stimulated with two sequential treatments of 24 hours each. For the first 24 hours of treatment, cells were cultured in RPMI complete medium or RPMI medium with MDP at 100μg/ml. The second 24-hour treatment consisted of LPS (Invivogen) at 50ng/ml or RPMI medium. In selected experiments, the first treatment was MHY1485 (Sigma) at 2μM, rapamycin (Sigma) at 100nM, or wortmannin (Sigma) at 1μM. In some conditions, THP-1 macrophages were generated by adding PMA (5ng/ml) for 48h, followed by at least 2 days without PMA (The THP1 monocytic cell line was cultured in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), L-glutamine (Thermo Fisher), Penicillin and Streptomycin (Thermo Fisher), MEM non-essential amino acids (Thermo Fisher), sodium pyruvate (Thermo Fisher), HEPES (Thermo Fisher) and 0.05mM of 2-mercaptoethanol (Thermo Fisher). Cells were kept in the culture at cell concentrations ranging from 2x10hout PMA , 122. CuBM cells were flushed out of the mouse bones with complete RPMI 1640 (Gibco). A single-cell suspension was then prepared by repeated pipetting. BM-derived dendritic cells (BMDCs) were generated for 7 days in respectively RPMI-1640 medium (Gibco), supplemented with glutamine, penicillin, streptomycin, 2-mercaptoethanol ], and 10% heat-inactivated fetal calf serum . The medium was supplemented with 20 ng/ml GM-CSF of J558 cells (GM-CSF-producing cells). Half of the medium was removed on day 3 and a new medium supplemented with GM-CSF supernatant  was added  using the Seahorse Glycolysis Stress Test Kit by using a Seahorse bioanalyser.RNAs were extracted using the RNEasy mini kit (Qiagen). According to the manufacturer’s instructions, isolated RNA was reverse-transcribed with the cDNA synthesis kit (Agilent Technologies). The resulting cDNA  was amplified using the SYBR Green real-time PCR kit and detected on a Stratagene Mx3005 P (Agilent Technologies). qPCR was conducted using forward and reverse primers (sequences available upon request). The relative abundance of gene expression was assessed using the 2−ΔΔCt method. Actb was used as an internal reference gene in order to normalize the transcript levels.Data were analyzed using Prism6.0 . Statistical significance was assessed by non-parametric Mann-Whitney test or two-way ANOVA for multiple comparisons. Values represent the mean of normalized data ± SEM. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69446: GEO database (accession number GSE69446).Publicly available datasets were analyzed in this study. This data can be found here: All animal studies were approved by the local investigational review board of the Institut Pasteur of Lille (N°28010-2016012820187595).Conceptualization: CC, DAS, ES, MC, and LP. Methodology: CC, DAS, KR, OB, WL, MD, NW, ES, JK, MC, and LP. Formal analysis: CC, DAS, KR, OB, WL, NW, ES, JK, MC, and LP. Investigation: CC, DAS, KR, OB, WL, MD, NW, JK, MC, and LP. Writing – original draft: MC and LP. Writing – review and editing: all authors. Visualization: CC, DAS, MC, and LP. Supervision: LP and MC. Funding acquisition: MC and LP. All authors contributed to the article and approved the submitted version.


Whole-body 18F]FDG has limited sensitivity in the staging of MZL [68Ga]Ga-PentixaFor ([68Ga]Ga-CPCR4.2) was performed, given that CXCR4 is overexpressed by most B- and T-cell neoplasms [18F]FDG, SUVmax = 4.77; [68Ga]Ga-CPCR4.2, SUVmax = 7.18) revealed no signs of lymphoma infiltration but characteristic epithelioid cell granulomas with pronounced CXCR4-expression in the surrounding rim of activated lymphocytes (C), consistent with the diagnosis of sarcoidosis, a multisystem inflammatory disorder of enormous heterogeneity in clinical presentation [As 8FFDG haseoplasms . ChemokiSince CXCR4 is abundantly involved in immune cell activation and several inflammatory processes, and especially expressed by macrophages and T-lymphocytes , intense


The COVID-19 pandemic has posed a significant threat to society in recent times, endangering human health, life, and economic well-being. The disease quickly spreads due to the highly infectious SARS-CoV-2 virus, which has undergone numerous mutations. Despite intense research efforts by the scientific community since its emergence in 2019, no effective therapeutics have been discovered yet. While some repurposed drugs have been used to control the global outbreak and save lives, none have proven universally effective, particularly for severely infected patients. Although the spread of the disease is generally under control, anti-SARS-CoV-2 agents are still needed to combat current and future infections. This study reviews some of the most promising repurposed drugs containing indolyl heterocycle, which is an essential scaffold of many alkaloids with diverse bio-properties in various biological fields. The study also discusses natural and synthetic indole-containing compounds with anti-SARS-CoV-2 properties and computer-aided drug design (in silico studies) for optimizing anti-SARS-CoV-2 hits/leads. Plasmodium falciparum and has subsequently progressed to pre-clinical trials as a potential antimalarial drug  followed by cyclization with concentrated sulfuric acid (H2SO4) at 90 °C and alkylation (13 (C1–C29) revealed 3C-like protease (3CLpro) [or main protease (Mpro)] SARS-CoV-2 inhibitory properties relative to Tideglusib (positive control) [A series of isatin derivatives kylation . Some ofcontrol)   were synthesized through the reaction of indole derivatives with Bunte salt ethyl acetate-2-sodium thiosulfate in iodine/DMSO at 60 °C followed by hydrolysis  and coupling with the appropriate amine thio]acetamides te amine . Some ofte amine . Considete amine  and thosE1–E5) with possible RdRp SARS-CoV-2 inhibitory properties have been explored. The efficacy of the most promising agents was comparable to that of Remdesivir (EC50 = 1.05 μM) [A set of acetamide-containing indoles (1.05 μM)   were synthesized via reaction of the appropriate indolecarboxylic acid with 3-chloropyridin-5-ols or 5-amino-3-chloropyridines using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and dimethylaminopyridine (DMAP) in methylene chloride (CH2Cl2) (pro inhibitory properties were observed for some of the synthesized agents [A variety of indole-chloropyridine conjugates (CH2Cl2) . Anti-SAd agents  . Anti-SARS-CoV-2 activity was observed for the compound  [Indomethacin-chloropyridine conjugate 16  is also 5.32 μM) .o-phenylenediamine under green conditions (in water at 75 °C) in a 2:1 molar ratio afforded the corresponding diindole-substituted benzimidazole 17  from the corresponding 3-hydroxy analogs  in the Vero E6 assay [Anti-SARS-CoV-2 3-alkenyl-2-oxindoles  analogs . Some of analogs  relativeE6 assay .Spirocyclic compounds are organic compounds with a rigid, 3D-geometrical structure. In 1911, A. Pictet and T. Spengler reported the first spiro-analog intermediate. Spiroindole-containing compounds are important due to the versatile biological properties established by diverse natural and synthetic analogs originating from the C-3 indolyl ring with many heterocycles affording various motifs.20 were synthesized through the cycloaddition of azomethine ylide (obtained from sarcosine and isatins) with 3,5-diylidene-4-piperidones (I1–I15) in the Vero E6 assay relative to the standards Hydroxychloroquine, Chloroquine, and Favipiravir [Spiroindoles eridones . Promisiipiravir   were recently reported with potential Mpro inhibitory properties, synthesized through azomethine (generated from the reaction of isatin and sarcosine) reaction with the appropriate 3,5-bis((E)-ylidene)-1-phosphonate-4-piperidone [Anti-SARS-CoV-2 spiroindole-containing compounds bearing a phosphonate group peridone  (Scheme It is notable that the spiroindoles with a sulfonyl group  are more22) with potential SARS-CoV-2 protease (Mpro “main protease” and PLpro “papain-like protease”) inhibitory properties  controls many essential viral processes including maturation, replication, and transcription. This makes it a potential target for optimizing therapeutics against COVID-19 [SARS-CoV-2 main protease 3 as a catalyst in refluxing ethanol [K1–K6) showed inhibitory properties for SARS-COV-2 Mpro (PDB: 6LU7) and spike glycoprotein (PDB: 7NX7) utilizing Auto Dock-Vina software (v. 1.1.2). For comparison, Remdesivir binding energy = −7.7, −6.5 kcal/mol is against main protease 6LU7 and spike glycoprotein 7NX7, respectively [In this section, representative examples of computationally predicted M ethanol   utilizing AutoDock and AutoDock vina software. They reveal considerable binding affinity in the protein active site  [Many mushroom metabolites have potential biological activities. Psilacetin, psilocin, and psilocybine, which are psilocybin-mushroom components, have been subjected to Mctively)   have also been explored against Mpro (PDB: 6YB7), spike protein (PDB: 6LZG), and RNA-dependent RNA polymerase (PDB: 6M71) in silico by the blind docking technique utilizing AutoDock Vina v.1.1.2. Some of the results suggested promising inhibitory properties that may help narrow the search for anti-SARS-CoV-2 candidates [A series of indolyl chalcones . The 5-bromo-substituted analog of bis-Schiff base 24 formed from the condensation of 2-(1-aminobenzyl)benzimidazole and the corresponding bis-isatin in ethanol containing a few drops of AcOH at room temperature (The Schiff bases formed from the condensation of isatin and 2-(1-aminobenzyl)benzimidazole revealed in silico possible Mperature  is the mperature .M1–M31) with in silico Mpro SARS-CoV-2 inhibitory properties were mentioned  [A set of 2-oxindole derivatives   . The most promising agents (N1–N5) were identified based on the observed binding affinities (O1–O3) with potential inhibitory properties against Mpro SARS-CoV-2 [A set of isatin-based protease inhibitors was collected from previous publications followed by in silico high throughput screening in the active pocket of Mfinities . SearchiRS-CoV-2   in addition to the in vitro activity (Vero E6 assay) of indole-containing compounds (P1 and P2) [Hattori et al. have also reported the in silico M and P2)   utilizing MOE (Molecular Operating Environment v.2019 software). It reveals hydrogen bonding interaction with CYS145 and GLU166 in addition to a π-interaction with HIS41 . Compound ID: ZINC95567760, which contains a fused indolyl heterocycle, also reveals a promising docking interaction in the PDB: 6LU7. Hydrogen bonding interaction with CYS145, in addition to π-interactions with GLU166 and GLY143, support these assumptions [A computational study considered food chemicals and components named as dark chemical matters could predict some effective anti-SARS-CoV-2 hits. Compound ID: ZINC4217536 (ZINC database) was mentioned as a promising antiviral active agent due to docking observations in Mumptions   for the identification of promising RdRp SARS-CoV-2 inhibitor agents ; the most promising compounds discovered are exhibited in A computational study has also explored isatin analogs (López-López et al.  have poiCOVID-19 has proven to be one of the most serious crises facing human health in recorded history. The scientific community has been tirelessly working to optimize effective therapeutics. While vaccination has been successful in controlling the pandemic, research into the effective treatment of current and future mutants remains crucial. One area of focus has been on the indole scaffold, which includes many alkaloid categories and has shown promise in the fight against COVID-19. Repurposed indole-containing drugs, as well as various natural and synthetic indole analogs, have been investigated for anti-SARS-CoV-2 efficacy. In silico studies were utilized to generate new hits and optimize leads against SARS-CoV-2. 3-Alkenyl-2-oxindoles and spiroindoles are potentially valuable anti-SARS-CoV-2 agents that can be synthesized in a regio-selective approach. From the cited reports, it can be concluded that indole-containing compounds are important lead molecules and can be further optimized for the development of potential agents against SARS-CoV-2.