Synthetic mRNA provides a template for the synthesis of any given protein, protein fragment or peptide and lends itself to a broad range of pharmaceutical applications, including different modalities of cancer immunotherapy. With the ease of rapid, large scale Good Manufacturing Practice-grade mRNA production, mRNA is ideally poised not only for off-the shelf cancer vaccines but also for personalized neoantigen vaccination. The ability to stimulate pattern recognition receptors and thus an anti-viral type of innate immune response equips mRNA-based vaccines with inherent adjuvanticity. Nucleoside modification and elimination of double-stranded RNA can reduce the immunomodulatory activity of mRNA and increase and prolong protein production. In combination with nanoparticle-based formulations that increase transfection efficiency and facilitate lymphatic system targeting, nucleoside-modified mRNA enables efficient delivery of cytokines, costimulatory receptors, or therapeutic antibodies. Steady but transient production of the encoded bioactive molecule from the mRNA template can improve the pharmacokinetic, pharmacodynamic and safety properties as compared to the respective recombinant proteins. This may be harnessed for applications that benefit from a higher level of expression control, such as chimeric antigen receptor (CAR)-modified adoptive T-cell therapies. This review highlights the advancements in the field of mRNA-based cancer therapeutics, providing insights into key preclinical developments and the evolving clinical landscape. Over the last 30 years, extensive efforts have been made to increase intracellular stability, translational efficiency and uptake of mRNA. These optimizations were achieved by modification of its non-coding elements , contains a 5′ cap, UTRs embracing the coding region and a 3′ poly(A) tail, thus resembling naturally occurring processed mature mRNA molecules, and is generated by in vitro transcription (IVT) from a linear DNA template. Exogenous mRNA enters the cell either by directly passing through the cytoplasmic membrane , or by endocytosis followed by endosomal escape (if delivered as naked or formulated mRNAs). mRNA does not enter the nucleus, nor integrates into the genome. Translation occurs in the cytosol and the protein derived from the synthetic mRNA is not distinguishable from protein translated from endogenous mRNA. The protein undergoes post-translational modifications and is routed to subcellular compartments, such as the secretory pathway, the cell membrane, the nucleus, mitochondria or peroxisomes, via targeting sequences or transmembrane domains. Eventually, the protein is degraded and peptides are presented on major histocompatibility (MHC) complexes.In parallel to its translation to protein, exogenous mRNA exerts its activity as a natural ligand of endosomal toll-like receptors (TLRs) 3, 7 and 8, or of retinoic acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) in the cytoplasm, and triggers the release of type I interferon (IFN) and pro-inflammatory cytokines , provid1-methylpseudouridine, 2-thiouridine, 5-methylcytidine or N6-methyladenosine [In cancer immunotherapy, the most advanced application of mRNA is therapeutic vaccination, which leverages both the capability of mRNA to deliver genetic information and its innate immunostimulatory activity. The latter is particularly important for breaking immune tolerance when cancer-associated self-antigens are targeted. However, IFN-stimulated genes (ISGs), such as IFN-inducible double-stranded (ds)RNA-activated protein kinase (PKR), and 2′,5′-oligoadenylate synthetase (OAS) with subsequent RNase L expression, initiate a state of anti-viral defense, characterized by stalled mRNA translation and increased targeting of mRNA for degradation , such as differentiation antigens, overexpressed antigens, cancer/testis antigens, or truly tumor-specific antigens (TSAs) not subject to immune tolerance, such as viral and mutated neoantigens. Various formats can be used for antigen delivery, including viral vectors, DNA, peptides, mRNA, or dendritic cells (DCs) pulsed with any of these.mRNA has emerged as an attractive cancer vaccine format as it provides both antigen delivery and innate immune activation-mediated co-stimulation in a spatiotemporally aligned manner. mRNA vaccines encode the full or partial sequence of a TSA or TAA, and do not rely on prior identification of a patient’s human leukocyte antigen (HLA) haplotype or epitope prediction.The feasibility of mRNA-based cancer vaccination was first demonstrated about 25 years ago . Since tAs professional antigen presenting cells (APCs), DCs constantly engulf cellular material from their surroundings. To efficiently deliver tumor antigen to DCs, many early studies focused on ex vivo DC loading and re-infusion of the transfected cells to patients via the subcutaneous, intranodal (injected into a particular lymph node) or intravenous route. The generation of DC vaccines requires isolation of monocytes or hematopoietic progenitor cells from the blood (leukapheresis), ex vivo cultivation, differentiation and tumor-antigen loading and is labor-intensive, costly and complex .One of the approaches to pulse DCs with mRNA encoding tumor antigens is the use of bulk mRNA extracted from autologous tumors. Whole tumor mRNA-loaded DC vaccines demonstrated safety in phase I and I/II clinical trials in patients with renal cell carcinoma  , pediatrAnother approach is to resort to defined TAAs and generate synthetic mRNA for ex vivo DC transfection, as has been done with prostate-specific antigen (PSA) , carcinoIn a randomized phase II clinical trial (NCT01446731), a DC vaccine with mRNA encoding PSA, prostatic acid phosphatase (PAP), survivin and hTERT failed to significantly increase the disease-specific survival of docetaxel-treated, castration-resistant prostate cancer patients  . TAA-speIn 78 melanoma patients with resected regional lymph node metastases , vaccination with gp100- and tyrosinase-mRNA loaded DC vaccines as an adjuvant treatment showed a favorable safety profile and induced tumor-specific T-cell responses in 71% of the patients . The oveGenerally speaking, T-cell responses induced by mRNA-loaded DC vaccines have been rather weak, which may contribute to the low clinical efficacy. DC differentiation, maturation and antigen loading directly impact DC homing and T cell co-stimulation , and ar+ T-cell responses and led to a more potent CEA-specific CTL response in vitro [Cognate T-cell help is a critical factor that determines the extent of vaccine-induced T-cell responses. This requires the vaccine antigen to be presented on MHC class II molecules of the DC. mRNA transfected into DCs undergoes cytoplasmic translation, enters the endogenous processing compartment and is therefore presented on MHC class I rather than MHC class II molecules. Several strategies have been followed to enhance MHC class II antigen presentation by routing the antigen through lysosomal compartments, such as fusion of the vaccine antigen to lysosome-associated membrane proteins (LAMPs) , 66, or in vitro . CEA LAMin vitro , 69, 70,in vitro . Four onAs ssRNA and dsRNA trigger TLR activation, pulsing of DCs with mRNA provides a maturation signal irrespective of the biological activity of the encoded antigen. Several strategies aim to further improve the T-cell priming capacity of tumor-antigen loaded DC vaccines. These include co-transfection with immunostimulatory ligands and receptors for enhanced DC maturation and T-cell co-stimulation, which can be introduced by mRNA. In preclinical studies, DC co-transfection of cancer antigens with OX40 ligand (OX40L), 4-1BB ligand (4-1BBL), glucocorticoid-induced tumor necrosis factor receptor (GITR) ligand (GITRL), CD40 ligand (CD40L), constitutively active (ca)CD40 or inducible T cell co-stimulatory ligand (ICOSL) mRNA improved DC co-stimulation and resulted in enhanced T-cell priming –76. One + T cells were significantly enhanced by co-culture with IL-12 and MART-1 mRNA-transfected DCs compared to MART-1 mRNA-transfected DCs alone.mRNA-encoded cytokines are another class of molecules used to enhance DC maturation and T-cell priming capacity. In the context of autologous DC vaccines loaded with whole tumor mRNA preparations or with synthetic TAA-encoding mRNA, co-transfection with cytokine-encoding mRNA, such as GM-CSF, IL-12 and IL-15, was explored –84. Co-tThe direct injection of mRNA is an alternative to DC vaccines and circumvents the necessity of DC isolation, ex vivo cultivation and re-infusion. Injected mRNA is taken up by local cells, including APCs, and translocates to the cytoplasm for translation. Various mRNA vaccines are currently being evaluated, using different nanoparticle formulations, delivery routes and structural backbones, of which the most advanced candidates are summarized below.+ and CD8+ T-cell responses in ex vivo expanded PBMC were detected by intracellular IFNγ staining upon re-stimulation with tumor mRNA-transfected PBMC. Vaccine-induced humoral responses were analyzed by incubating allogenic melanoma cell lines with plasma samples taken before or during the therapy followed by flow cytometry analysis to detect cell-bound IgG antibodies. Upon vaccination, an increase in the amount of tumor-cell specific IgG antibodies was observed in the plasma of four out of the 13 patients. Objective responses were not apparent in this trial. In a phase I/II clinical study with 30 stage IV RCC patients, intradermal administration of naked mRNA encoding mucin 1 (MUC1), CEA, human epidermal growth factor receptor-2 (HER-2)/neu, telomerase, survivin and MAGE-A1 combined with GM-CSF was well tolerated and associated with stable disease in 15 patients and one partial response [Non-formulated or “naked” mRNA administered intradermally or into lymph nodes  has been shown to prime T-cell responses in mice –88 and hresponse . AntigenIn 2012, a phase I study of 29 advanced melanoma patients was initiated, which proved that intranodal injection of mRNA is both feasible and safe (NCT01684241) (unpublished results). Vaccination induced either de novo or expanded existing T-cell responses against the vaccine-encoded antigens New York esophageal squamous cell carcinoma-1 (NY-ESO-1) and tyrosinase in six out of 13 evaluable patients. New lesions occurred in only eight of the 29 patients during the course of the study. In another phase I study of 20 melanoma patients (NCT03394937), intranodal injection of tyrosinase, gp100, MAGE-A3, MAGE-C2, and melanoma antigen preferentially expressed in tumors (PRAME)-encoding mRNA together with TriMix mRNA was well tolerated. As observed by IFNy ELISpot and intracellular cytokine staining, T-cell responses against at least one TAA were induced in 35% of patients .+ and CD8+ T-cell responses against multiple vaccine neoepitopes in 13 evaluable melanoma patients. Objective responses were observed and the cumulative rate of recurrences was significantly reduced by vaccination, resulting in a sustained progression-free survival. The vaccine was well tolerated.Mutated neoantigens are promising vaccine targets as they are abundant in many tumor types and highly tumor-specific . NeoantNanoparticulate formulations protect mRNA from extracellular RNases and improve uptake by APCs in vivo. Formulations typically contain polymers, such as protamine, or cationic and ionizable lipids with or without polyethylene glycol (PEG) derivatives to enable complexing with mRNA via electrostatic interactions and condensing of the RNA molecules .+ and CD8+ T cells in two of four immunologically evaluable patients. One complete response occurred in seven patients with measurable disease. In addition, a vaccine encoding six protamine-formulated prostate cancer-specific antigens , prostate stem cell antigen (PSCA), 6-transmembrane epithelial antigen of prostate (STEAP), PAP and MUC1) was investigated in a phase I/II trial in patients with asymptomatic or minimally symptomatic metastatic castrate-resistant prostate cancer (NCT01817738) [n = 197) [One of the mRNA formulations in advanced clinical development is based on protamine, a polycationic peptide that complexes negatively charged mRNA for intradermal delivery , 96–100.1817738) –44. The n = 197) , the vacA major limitation of local administration is that only the injected or the draining lymph nodes are accessed and participate in antigen-specific T-cell priming. Other formulations are required for intravenous administration to deliver mRNA to secondary lymphoid organs body-wide, including the spleen as the largest agglomeration of immune cells. Formulation technologies developed for intravenous administration not only protect RNA integrity, but also promote its targeted expression in lymph node-resident DCs.mRNA lipoplexes (RNA-LPX) are based on cationic liposomes as broadly used for transfection reagents in vitro . Specifin = 25), vaccination alone mediated one metabolic complete response, three partial responses and seven cases of stable disease. In combination with anti-PD-1 antibodies , six partial responses and two cases of stable disease were observed. RNA-LPX vaccine-induced CD4+ and CD8+ T-cell responses were observed in the majority of patients reaching up to low double-digit percentages of total circulating CD8+ T cells. A randomized phase II study will investigate this melanoma TAA-specific RNA-LPX vaccine in combination with the antagonistic anti-PD-1 antibody cemiplimab (NCT04526899). The same platform encoding other tumor type specific TAAs is being evaluated in various phase I and I/II clinical trials in patients with triple negative breast cancer , ovarian cancer (NCT04163094) and prostate cancer (NCT04382898).Intravenously administered negatively charged to near-neutral RNA-LPX vaccines have been shown in mice to target APCs resident in lymphoid organs, including the spleen, lymph nodes and bone marrow, while inducing strong local (cell-specific activation) and systemic (proinflammatory cytokine abundance in the circulation) type I IFN-dominated immune modulation . In seve+ and PD-L1+ head and neck squamous cell carcinoma (HNSCC) (NCT04534205). Another example is the so-called individualized neoantigen-specific immunotherapy (iNeST) program, which treats each patient with their ‘on-demand’ manufactured individualized set of cancer mutations. This truly personalized treatment approach is being tested in various tumor types and treatment lines in the phase I and II studies NCT03289962, NCT03815058, and NCT04486378.Moreover, several clinical trials are testing this intravenous RNA-LPX technology in conjunction with TSAs, which are expected to be less compromised by central immune tolerance. One example is a human papilloma virus (HPV)16 E6 and E7 mRNA-encoding RNA-LPX vaccine, which is being evaluated in a randomized phase II trial in combination with pembrolizumab in patients with HPV16+ Lewis lung tumors. Safety and efficacy of this concept are currently being evaluated in a phase I/II clinical study in patients with solid cancers (NCT04503278).The same RNA-LPX vaccine platform was utilized to expand chimeric antigen receptor (CAR)-engineered T cells against claudin 6 (CLDN6), a carcinoembryonic cell surface antigen, in mice . Vaccina1-methylpseudouridine-modified mRNA with blunted innate immune modulatory activity to promote potent TH1, T follicular helper and germinal center B cell responses for the induction of neutralizing antibodies as part of the multi-effector immune response [Lipid nanoparticles (LNPs) are mRNA formulations based on a mixture of cholesterol, ionizable and helper lipids, and PEG-derivatives . Intramuresponse , 122.In the cancer therapeutic field, intramuscularly administered, LNP-formulated nucleoside-modified mRNA vaccines encoding 20 patient-specific mutated neoepitopes are currently being evaluated in combination with pembrolizumab in patients with solid cancer  and melanoma . Data from a recent phase I clinical trial in four gastrointestinal cancer patients (NCT03480152) suggest that vaccination with neoantigen-specific RNA-LNP is safe and induces mutation-specific T-cell responses  with no Besides the clinically explored mRNA formulations discussed above, a plethora of different in vivo DC-targeting mRNA nanoparticles are under development. A recent study analyzed over 1000 mRNA lipid formulations for transfection efficacy. A subset of these were tested for in vivo protein production, APC maturation in local lymph nodes, induction of T and B cell responses in naïve, and anti-tumor efficacy of Trp2, OVA and E7-encoding mRNA lipid formulations in B16, B16-OVA and TC-1 tumor-bearing mice . The forAnother immunotherapy approach in cancer is based on the redirection of T cells against tumor cells by stable integration of tumor-specific antigen receptors, either with T cell receptors (TCRs) which recognize MHC-presented epitopes from both intra- and extracellular antigens, or CARs which bind to tumor surface antigens and are MHC-independent. T cells are then expanded in cell culture systems. Patients are treated with a lymphodepleting chemotherapy regimen prior to adoptive cell transfer (ACT) of such engineered cell products to improve engraftment.In the ACT field, CD19-directed CAR-T cells are the most advanced. Several products have been market authorized for the treatment of B-cell leukemia and lymphoma , 126 bas1-methylpseudouridine modification have been shown to enhance CAR expression and result in more functional CAR-T cells [Electroporation of mRNA CARs is a practical and scalable method accomplishing lymphocyte transfection rates of more than 90% without affecting the viability of the cell product, and is used in the vast majority of studies –136. mRN-T cells , but havmRNA CAR-T cells must be administered repeatedly, in contrast to classical CAR-T cells which constitutively express the CAR. Consequently, mRNA CAR-T cells are produced in large quantities and cryopreserved in batches that can be thawed for each treatment cycle. GMP-compliant protocols for the large-scale manufacturing of mRNA CAR-T cells have been established , 154, anEarly studies with TCRs specific for NY-ESO-1, MART-1 or p53  and a CATwo phase I studies  were initiated based on the observation that repeated injection of mesothelin-directed  mRNA CARIn AML, lineage markers used as target antigens are expressed by hematopoietic stem cells and myeloid progenitor cells , 166. TamRNA CAR-T cells targeting protein kinase Met (c-Met) are in clinical development for the treatment of breast cancer and melanoma  , and B-cmRNA CARs are also used for retargeting of γδ T cells , 169, NKmRNA is also being explored for delivery of full IgG mAbs or scaffolds, including engineered bispecific antibody fragments, and addresses pharmacokinetic  as well as manufacturing challenges  to accelerate clinical development.mAbs are an established treatment modality in cancer and are used for targeting of tumor cells and for the modulation of immune cell responses. Most of the approved recombinant mAbs are administered intravenously to achieve systemic exposure.. HER-2/neu-targeting mAb trastuzumab is approved for the treatment of HER-2/neu-overexpressing breast cancer and metastatic gastric or gastroesophageal junction adenocarcinoma [Established standard-of-care mAbs are obvious starting points to explore mAb-encoding mRNA in conjunction with liver-targeting formulations for high protein production in liver-resident cells and release of the protein into the circulation. Rituximab, targeting CD20, was the first mAb approved for cancer therapy and is used for the treatment of non-Hodgkin’s lymphoma and chronic lymphocytic leukemia (CLL) . HER-2/narcinoma . In micearcinoma . Trastuzarcinoma .1-methylpseudouridine-modified and dsRNA-purified bispecific mAb-encoding mRNA  was translated in the liver after intravenous administration and resulted in sustained plasma antibody concentration over several days in mice. In contrast, non-nucleotide modified and non-dsRNA purified mRNA induced only minimal plasma concentrations of bispecific mAbs, highlighting the importance of engineering mRNA for enhanced protein production. The favorable pharmacokinetic profile of modified and purified mRNA was associated with the induction of T-cell cytotoxicity and superior anti-tumor activity against human ovarian cancer xenografts as compared to recombinant mAb.Engineered bispecific mAbs, comprising an anti-CD3 scFv fused to a tumor antigen-specific scFv, are able to redirect T cells to tumor cells and promote tumor cell killing . The sma+ T cell infiltration into the tumor [In contrast to systemic exposure mediated through intravenous injection, local mRNA translation by direct injection into a specific tissue or by transfection of specific cell types ex vivo restricts the therapeutic activity of the translated protein to the target cells or their intermediate surroundings. Intratumoral injection of mouse tumors with an antagonistic anti-PD-L1 mAb-encoding self-amplifying (sa)RNA, which is derived from alpha- or flaviviruses and encodes both the protein of interest and a replicase for RNA amplification, resulted in superior anti-tumor efficacy compared to the intraperitoneally or intratumorally administered recombinant protein counterpart. Intratumoral saRNA administration mediated anti-PD-L1 mAb translation in transfected cells and promoted CD8he tumor .+ T cells (Tregs). The concept was subsequently explored in a small trial including two patients with metastatic melanoma (NCT01216436)  of recombinant anti-GITR mAb . The admNCT0121646  blinatumomab approved for the treatment of certain types of acute lymphoblastic leukemia (ALL). Secretion of the BiTE led to the release of inflammatory cytokines and efficient killing of tumor cells by transfected T cells in vitro . These omRNA-encoded immunomodulators investigated so far comprise cytokines as well as co-stimulatory ligands and receptors . Autonomous stimulation of the trimeric IL-15R in cis enhanced the cytotoxicity of the T cells in vitro and when administered to mice, these cells proliferated better [IL-15 is functionally related to IL-2, as both signal via IL-2Rβ and the common γ chain (IL-2Rβγ), and thereby stimulate T cells and NK cells . Whereas T cells . IL-15 h T cells . Signalid better .H1 immunity and exerts significant anti-tumor activity in mouse tumor models. In humans, IL-12 is associated with potentially lethal toxicity upon systemic administration [1-methylpseudouridine-modified mRNA. In mouse tumor models, a single intratumoral injection promoted tumor regression, which required IFNγ and CD8+ T cells [1-methylpseudouridine-modified scIL-12 RNA-LNPs administered intravenously to mice bearing hepatocellular carcinomas reduced liver tumor burden and prolonged survival, without apparent liver toxicity [IL-12 is a potent mediator of Tstration , 195. Onstration . Local e T cells . In a cotoxicity . Reporte1-methylpseudouridine-modified mRNAs encoding IL-36γ , IL-23 (a regulator of inflammation) and OX40L demonstrated regression of established tumors in three syngeneic tumor models [Various mRNAs can be easily mixed and formulated together as a single drug product, a technological property that has encouraged the development of combinatorial approaches. Intratumoral administration of LNP-formulated Nr models . Inflammr models . Dose exr models .In a combination approach using a single mRNA construct, IFNβ was fused with the ectodomain of the transforming growth factor (TGF) β receptor II to stimulate innate immunity while antagonizing the suppressive function of TGFβ . In vitrInteractions of stimulatory ligands and receptors provide inflammatory signals to the immune system that can be exploited for cancer immunotherapy. mRNA can be used to transiently equip cells with stimulatory receptors, enabling transient activation of strong inflammatory signals.cis. Co-transfection of cytokine mRNAs with caTLR4 mRNA, caCD40 mRNA, or both, enhanced IFNγ production by T cells, induced upregulation of T cell activation molecules , and improved the cytotoxicity of TILs against autologous melanoma cells in vitro in comparison to cells transfected with caTLR4 mRNA, caCD40 mRNA, or both [Modification of intracellular receptor domains to generate ca receptors is helpful to provide cells with autonomous stimuli. The transfection of T cells, isolated either from PBLs or tumor-infiltrating lymphocytes (TILs), with caTLR4 mRNA, caCD40 mRNA, or both, enhanced the production of IFNγ and TNFα, upregulated 4-1BB and CD25, and increased the cytolytic activity against autologous melanoma cells in vitro , 203. Bu or both , 205.The transient nature of mRNA translation is appealing for the direct in vivo delivery of stimulatory ligand and receptor-coding mRNA. The injection of naked TriMix mRNA  directly into the tumor resulted in systemic therapeutic anti-tumor activity in various mouse models and led to a phase I clinical trial in patients with early, resectable breast cancer lesions (NCT03788083) . In addiResorting to the combination of co-stimulatory ligands and receptors , as well as cytokines , different mixtures of formulated 5′-methylcytidine- and pseudouridine-modified mRNAs were injected directly into mouse tumors. Among all tested combinations, the triplet of OX40L, CD80 and CD86 mRNA induced the strongest anti-tumor immunity in subcutaneous A20 and CT26 tumor-bearing mice .The development and worldwide approval or authorization of mRNA vaccines against SARS-CoV-2 within less than 1 year upon occurrence of the COVID-19 pandemic showcased the enormous potential of mRNA technology. COVID-19 mRNA vaccines were rapidly developed, have a favorable safety profile, and outperform established technologies with efficacies around 95% of preventing COVID-19 , 114.This rapid response to the pandemic leveraged the rich source of scientific, clinical, manufacturing and regulatory lessons learned over decades of exploring mRNA technology for the design and clinical development of therapeutic cancer vaccines. In turn, the insights gained in the process of advancing mRNA for the first time through all stages of pharmaceutical drug development to a commercial product will now feed back into its application for cancer immunotherapy.The beauty of mRNA technology is the broad bandwidth of its versatility. By modifying building blocks, structural elements, and formulations of the synthetic mRNA, a variety of features including targeting to defined cells, duration of expression, and immunological effects can be adapted. This expands the design space for mRNA beyond therapeutic cancer vaccination. mRNA is now utilized for the most diverse immunotherapeutic approaches, including the equipping of immune cells with antigen receptors and the in vivo production of therapeutic proteins such as antibodies or immunomodulators. mRNA CAR-T cells have the potential to improve the safety profile of CAR-T cell therapy and allow concurrent modification of the lymphocytes by co-delivery of additional mRNAs. mRNA-based immunomodulators successfully entered clinical testing. Intratumoral injection of mRNA-encoded cytokines and co-stimulatory ligands demonstrated feasibility and induction of anti-tumor immune responses.mRNA is expected to become one of the major pillars of drug development. With an increasing number of concepts entering clinical trials, the first approval of an mRNA therapeutic against cancer is nearing.


Although laboratory evidence suggests that antibody responses following COVID-19 vaccination provide better neutralization of some circulating variants than does natural infection (*Kentucky residents aged ≥18 years with SARS-CoV-2 infection confirmed by positive nucleic acid amplification test (NAAT) or antigen test resultsVaccination status was determined using data from the Kentucky Immunization Registry (KYIR). Case-patients and controls were matched to the KYIR database using first name, last name, and date of birth. Case-patients were considered fully vaccinated if a single dose of Janssen (Johnson & Johnson) or a second dose of an mRNA vaccine (Pfizer-BioNTech or Moderna) was received ≥14 days before the reinfection date. For controls, the same definition was applied, using the reinfection date of the matched case-patient. Partial vaccination was defined as receipt of ≥1 dose of vaccine, but either the vaccination series was not completed or the final dose was received <14 days before the case-patient’s reinfection date. Using conditional logistic regression, ORs and CIs were used to compare no vaccination and partial vaccination with full vaccination among case-patients and controls. SAS  was used for matching and statistical analyses. This activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.Overall, 246 case-patients met eligibility requirements and were successfully matched by age, sex, and date of initial infection with 492 controls. Among the population included in the analysis, 60.6% were female, and 204 (82.9%) case-patients were initially infected during October–December 2020 . Among cThis study found that among Kentucky residents who were previously infected with SARS-CoV-2 in 2020, those who were unvaccinated against COVID-19 had significantly higher likelihood of reinfection during May and June 2021. This finding supports the CDC recommendation that all eligible persons be offered COVID-19 vaccination, regardless of previous SARS-CoV-2 infection status.Reinfection with SARS-CoV-2 has been documented, but the scientific understanding of natural infection-derived immunity is still emerging , which limited statistical power. The lower odds of reinfection among the partially vaccinated group compared with the unvaccinated group is suggestive of a protective effect and consistent with findings from previous studies indicating higher titers after the first mRNA vaccine dose in persons who were previously infected (The findings in this report are subject to at least five limitations. First, reinfection was not confirmed through whole genome sequencing, which would be necessary to definitively prove that the reinfection was caused from a distinct virus relative to the first infection. Although in some cases the repeat positive test could be indicative of prolonged viral shedding or failure to clear the initial viral infection (These findings suggest that among persons with previous SARS-CoV-2 infection, full vaccination provides additional protection against reinfection. Among previously infected Kentucky residents, those who were not vaccinated were more than twice as likely to be reinfected compared with those with full vaccination. All eligible persons should be offered vaccination, including those with previous SARS-CoV-2 infection, to reduce their risk for future infection.Reinfection with human coronaviruses, including SARS-CoV-2, the virus that causes COVID-19, has been documented. Currently, limited evidence concerning the protection afforded by vaccination against reinfection with SARS-CoV-2 is available.Among Kentucky residents infected with SARS-CoV-2 in 2020, vaccination status of those reinfected during May–June 2021 was compared with that of residents who were not reinfected. In this case-control study, being unvaccinated was associated with 2.34 times the odds of reinfection compared with being fully vaccinated.To reduce their likelihood for future infection, all eligible persons should be offered COVID-19 vaccine, even those with previous SARS-CoV-2 infection.


Management of triple-negative breast cancer (TNBC) is still challenging because of its aggressive clinical behavior and limited targeted treatment options. Cisplatin represents a promising chemotherapeutic compound in neoadjuvant approaches and in the metastatic setting, but its use is limited by scarce bioavailability, severe systemic side effects and drug resistance. Novel site-directed aptamer-based nanotherapeutics have the potential to overcome obstacles of chemotherapy. In this study we investigated the tumor targeting and the anti-tumorigenic effectiveness of novel cisplatin-loaded and aptamer-decorated nanosystems in TNBC.in vivo and ex vivo analyses.Nanotechnological procedures were applied to entrap cisplatin at high efficacy into polymeric nanoparticles (PNPs) that were conjugated on their surface with the epidermal growth factor receptor (EGFR) selective and cell-internalizing CL4 aptamer to improve targeted therapy. Internalization into TNBC MDA-MB-231 and BT-549 cells of aptamer-decorated PNPs, loaded with BODIPY505-515, was monitored by confocal microscopy using EGFR-depleted cells as negative control. Tumor targeting and biodistribution was evaluated by fluorescence reflectance imaging upon intravenously injection of Cyanine7-labeled nanovectors in nude mice bearing subcutaneous MDA-MB-231 tumors. Cytotoxicity of cisplatin-loaded PNPs toward TNBC cells was evaluated by MTT assay and the antitumor effect was assessed by tumor growth experiments We demonstrate specific, high and rapid uptake into EGFR-positive TNBC cells of CL4-conjugated fluorescent PNPs which, when loaded with cisplatin, resulted considerably more cytotoxic than the free drug and nanovectors either unconjugated or conjugated with a scrambled aptamer. Importantly, animal studies showed that the CL4-equipped PNPs achieve significantly higher tumor targeting efficiency and enhanced therapeutic effects, without any signs of systemic toxicity, compared with free cisplatin and untargeted PNPs.Our study proposes novel and safe drug-loaded targeted nanosystems for EGFR-positive TNBC with excellent potential for the application in cancer diagnosis and therapy.The online version contains supplementary material available at 10.1186/s13046-021-02039-w. Breast cancer (BC) is the most commonly diagnosed type of cancer in women and is the main cause of cancer-related deaths. Based on the expression of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/ErbB2), BC can be classified into three main groups: hormone receptor positive, HER2-enriched and triple-negative (TNBC) . TNBC, wvia receptor mediated endocytosis , were randomized into groups of three animals per group. Cy7@PNPs, Cy7@PNPs-CL4, Cy7@PNPs-SCR (5 nmol of dye/100 µl of injection) were injected into the lateral tail vein of mice maintained under isoflurane anesthesia using a catheter to improve the likelihood of a successful injection. Fluorescence Reflectance Imaging (FRI) acquisition was performed on the 750 nm channel before and at specified time points, i.e., 30 min, 1, 3 and 24 h after intravenous injection using an FMT 4000 imaging system , as previously reported  and the body weight was also measured. At day 23, mice were euthanized.Mice bearing subcutaneous MDA-MB-231 xenografts  followed by Tukey’s multiple comparison test. Post hoc, a Bonferroni test for multiple comparisons was applied within groups, and a Fisher’s least significant difference test between groups. The mean normalized SI of ex vivo organs and tumors were compared between groups with the Kruskal-Wallis test, and the Dunn’s test for multiple comparisons.For the tumor targeting analyses, corrected SI were tested for normality with the Shapiro-Wilk’s test and then compared between groups with a two-way repeated measures analysis of variance (RM-ANOVA) mixed-effect model with the Geisser-Greenhouse correction for sphericity, looking for both within and between groups significant differences. in vitro cytotoxicity, anti-tumor effect both in vivo and ex vivo. The common base for all the samples was the PLGA-b-PEG copolymer, able to generate stable nanoparticles’ emulsions. Thanks both to a very versatile formulation procedure, called water-in-oil-in-water double-emulsion, and to the lipo and hydro blocks of the polymer, it is easy to encapsulate molecules whether inside the ‘oil’ moiety, as the lipophilic dye BODIPY for visualization purposes in vitro, or inside the inner ‘hydro’ portion, as the Cis-Pt to assess the anti-tumor effect in and ex vivo. For the selective targeting of EGFR, the so differently loaded PNPs have been further conjugated onto the external surface with CL4 aptamer, or its scrambled sequence (SCR) with no affinity for EGFR . In this experimental condition , treatments with either free Cis-Pt or Cis-Pt@PNPs-SCR resulted in a slightly but significantly delayed tumor growth compared to Ctrl treatment  and Cis-Pt@PNPs-SCR (P = 0.0009)  in EGFR-positive MDA-MB-231-derived tumors, but not in EGFR-negative tumors; (iii) durable tumor retention (at least 24 h) and (iv) no accumulation in healthy organs, i.e., lung and heart.Cis-Pt is one of the most potent antitumor agents known, widely used against a variety of solid tumors, including lung, head and neck, ovarian, testicular, bladder and colorectal cancers. Even though it has been nearly 40 years since the FDA approved of its use for cancer treatment, there is still a huge effort to fully elucidate how Cis-Pt works in order to improve its antitumor activity and reduce the side effects associated with patient treatment . Cis-Pt One intriguing result was the low-intensity signal detected in the spleen. While the liver and kidneys’ prominent role as excretory organs can easily explain their high signal intensities, the absence of almost any signal into the spleen suggests that the PNPs used in our study can escape, at least in part, the reticuloendothelial system (RES). Indeed, it has been well described how phagocytic cells belonging to the RES, i.e. monocytes and macrophages housed primarily in the liver and spleen, rapidly take up nanoparticles when coated by serum proteins and depending on their size and surface characteristics –65. The in vitro experiments, Cis-Pt@PNPs-CL4, systemically injected in nude mice bearing MDA-MB-231-derived xenograft tumors at a suboptimal therapeutic dose of Cis-Pt payload (~ 0.6 mg/kg), abolished tumor growth and induced apoptosis with a superior effect respect to free Cis-Pt and Cis-Pt@PNPs-SCR. Interestingly, cisplatin dosage used in mice to inhibit tumor growth (repeated iv injection of 3–5 mg/kg) is usually associated to certain degree of toxicity causing severe emaciation or even death of some mice during the study [Remarkably, cisplatin encapsulated in aptamer-conjugated nanoparticles caused up to 28- and 5-fold higher cytotoxicity in comparison with free drug and untargeted nanovectors (either unconjugated or conjugated with a scrambled aptamer), respectively, indicating that the targeted delivery system provided improved chemotherapy. We previously demonstrated the ability of CL4 aptamer to bind to (and inhibit) EGFR that is overexpressed, even if at different level, on MDA-MB-231 and BT-549 cells, thus indicating that the receptor is displayed on the surface of both cell lines in a status competent for recognition by the aptamer . Accordihe study , 39, 67.Our group has previously developed nanovectors with the PDGFRβ aptamer conjugated on the PNP outer shell that have been shown to be extremely efficient in delivering a PI3K/mTOR inhibitor to GBM implanted intracranially in mice, across the blood-brain barrier . PDGFRβ In this study, we successfully constructed PLGA-based nanoparticles loaded with Cis-Pt and conjugated on the outer shell to an EGFR-aptamer as a highly specific targeting agent. The resulting nanovectors proved to be a promising nanoplatform for active tumor-targeted Cis-Pt delivery with the capability of high-yield drug payload, tumor-specific targeting, and improved therapeutic efficacy than untargeted nanovectors and free drug. This novel approach could represent a new strategy for the treatment of EGFR-positive tumors. Interestingly, it may be applied to overcome the intrinsic resistance of TNBCs to EGFR inhibitors by using this receptor as an anchor point for the nanoparticles, decorated with CL4 aptamer, specifically delivering the cytotoxic drug inside them. Thus, a signaling molecule critical for cancer cell survival and proliferation is converted into a cancer vulnerability.Additional file 1:


Sarbercovirus subgenus as SARS-CoV [In a global pandemic involving respiratory pathogens such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), intensified scientific research is required to delineate pathways involved in infectivity, transmissibility, and pathogenicity of the causative pathogen. SARS-CoV-2, the causative agent of Coronavirus Disease 2019 (COVID-19), is highly contagious and significantly threatens public health. This single-stranded positive RNA virus consisting of approximately 30 kb genome size virus is from the same Two-dimensional cell lines display a wide range of usability and are readily available in a pandemic for identifying potential pathways of infection and tissue tropism of human pathogens. SARS-CoV-2 cultured in Vero-E6 cells (monkey kidney cells) showed high sequence homology and morphology similarity compared to viruses inoculated in human airway epithelial cells ,4. In adAnimal models are important to delineate the transmissibility, immunological responses, and clinical manifestations of respiratory infections. However, physiological differences in the models and the lack of appropriate reagents for some animals may hinder scientific progress. For example, structural differences between mouse and human ACE2 resulted in reduced infectivity in mice because the mouse ACE2 does not support the binding of the original SARS-CoV-2 strain . This liOther animal models such as ferrets share similar lung physiologies to humans; hence, it is not surprising that they are naturally susceptible to SARS-CoV-2 and display mild–moderate clinical symptoms ,25. LikeOrganoids are miniature three-dimensional structures, derived either from stem cells that are able to differentiate into organ-specific cell types and self-organize through spatially restricted lineage commitment, or from primary cells through cell sorting to generate structures that recapitulate the functions and architecture of the pertinent tissue . OrganoiThe use of human stem cell–derived airway organoids has proven to be extremely useful in SARS-Cov-2 research, as they have led to observations that are difficult to delineate and validate using either 2D cell lines or animal models ,48,49. FWhile the airway organoid model systems hold great promise in respiratory infection research, there is still room for improvement for existing models. The biomimicry of available organoids is still far from achieving the complexity of its native tissue. Although current versions of airway organoids present multiple differentiated epithelial cell types within the lungs, they are not representative of the systemic response during an infection. Hence, successful incorporation of nonepithelial cells such as immune, stromal, and vascular cells, as well as noncellular components, is necessary to advance existing models. For instance, coculture of alveolar macrophages with human-derived lung cells and SARS-CoV-2 demonstrated elevated levels of proinflammatory cytokines that are likely to contribute to cytokine storm in severe COVID-19 patients ,54; hencCOVID-19 has fast-forwarded the applications of airway organoids in respiratory infection research including cellular and inflammatory responses and therapeutics screening . Airway


Transforming growth factor-beta (TGFB) is a ubiquitously expressed cytokine involved in numerous functions in both normal and cancer cells. Here, we review, for the first time, the evidence of how B-cell lymphoma cells respond to TGFB and utilise TGFB to modulate surrounding cells in the microenvironment. We highlight recent data supporting the bi-directional signalling between B-cell lymphoma cells and their microenvironment. Targeting TGFB signalling in B-cell lymphoma may provide a future therapeutic strategy but further research is required to understand how lymphoma cells interact in different microenvironmental contexts.Transforming growth factor-beta (TGFB) is a critical regulator of normal haematopoiesis. Dysregulation of the TGFB pathway is associated with numerous haematological malignancies including myelofibrosis, acute myeloid leukaemia, and lymphoid disorders. TGFB has classically been seen as a negative regulator of proliferation in haematopoiesis whilst stimulating differentiation and apoptosis, as required to maintain homeostasis. Tumours frequently develop intrinsic resistant mechanisms to homeostatic TGFB signalling to antagonise its tumour-suppressive functions. Furthermore, elevated levels of TGFB enhance pathogenesis through modulation of the immune system and tumour microenvironment. Here, we review recent advances in the understanding of TGFB signalling in B-cell malignancies with a focus on the tumour microenvironment. Malignant B-cells harbour subtype-specific alterations in TGFB signalling elements including downregulation of surface receptors, modulation of SMAD signalling proteins, as well as genetic and epigenetic aberrations. Microenvironmental TGFB generates a protumoural niche reprogramming stromal, natural killer (NK), and T-cells. Increasingly, evidence points to complex bi-directional cross-talk between cells of the microenvironment and malignant B-cells. A greater understanding of intercellular communication and the context-specific nature of TGFB signalling may provide further insight into disease pathogenesis and future therapeutic strategies. In recent years, the treatment landscape of B-cell non-Hodgkin lymphoma (B-NHL) has expanded to include targeted therapies antagonising the B-cell receptor pathway or antiapoptotic signalling. Despite impressive responses in some subtypes of B-cell lymphoma, a large fraction of patients inevitably relapses. The tumour microenvironment provides protective niches to malignant cells, shielding them from immune clearance and therapy . CharactThe TGFB superfamily of cytokines is an evolutionally conserved family of proteins found in all multicellular organisms. The superfamily is composed of a diverse number of ligands and includes TGFB, bone morphogenetic proteins (BMPs), inhibins, activins, and growth differentiation factors (GDFs). TGFB signalling is implicated in multiple cellular processes including growth, migration, extracellular matrix remodelling, invasion, epithelial–mesenchymal transition, and immune suppression. Although dynamically regulated under normal homeostasis, TGFB is frequently overexpressed in diseased states including malignant, inflammatory, and fibrotic pathologies.Three isoforms—namely, TGFB1, TGFB2, and TGFB3—are synthesised as prohormones in mammalian cells. Their respective genes encode a large N-terminal portion termed the latency-associated peptide (LAP), a signal peptide, and a short C-terminal segment that corresponds to the mature active cytokine ,5 Figur. The sigTGFB activation requires the release of LLC from the ECM, followed by proteolysis of LAP, leading to the release of active TGFB . ConsequTGFB ligands bind to the TGFB receptor complex composed of a tetrameric structure made up of two type-1 TGFB receptors (TGFBR1) and two type-2 TGFB receptors (TGFBR2). Less studied co-receptors—TGFBR3 (betaglycan) and endoglin—modulate the response of the receptor complex to TGFB ligands ,18. EngaFollowing TGFB ligand-receptor binding several mechanisms can modulate the strength of downstream signalling including inhibitory SMADS (i-SMADs)—namely, SMAD6 and SMAD7. SMAD7 can bind to TGFBR1 and inhibit R-SMAD binding . In addiRegulation of haematopoiesis relies on the balance of proliferation, differentiation, and apoptotic signals of haematopoietic progenitor cells. TGFB plays an important role but demonstrates cell and context-specific effects. Assays of CD34+ human stem cells treated with exogenous TGFB1 in vitro lead to growth arrest and inhibition of colony formation ,33. TGFBThe bone marrow niche is composed of a diverse set of cells that both secrete and modulate the TGFB milieu. Large quantities of latent TGFB are deposited in the bone matrix; however, relatively few BM cells exhibit significant phospho-SMAD2/3 (pSMAD2/3) expression in health suggesting tight spatial regulation . Non-myeBone-marrow-derived stromal cells (BMSCs) are also important players in the BM niche. Beyond modulating haematopoiesis, they can play fundamental roles in lymphomagenesis and are capable of enhancing malignant B-cell survival, migration, as well as protecting against spontaneous and drug-induced apoptosis ,43,44,45The effect of TGFB signalling on modulation of chemokine and cytokine secretion by BMSCs highlights a role for TGFB in the immune response. Indeed, lessons from murine models and solid malignancy highlight TGFB as a regulator of activation, recruitment, and function of multiple cells of the innate and adaptive immune system. Generally, TGFB has an immunosuppressive effect on the innate system. TGFB signalling supports M2 macrophage polarisation associated with the promotion of angiogenesis and tumourigenesis ,60. SimiAccumulating evidence highlights TGFB as an important regulator of B-cells including development, activation, and differentiation. Some of these effects are direct actions of TGFB but some aspects of B-cell response are indirect through intercellular communication.Beyond regulating the HSC pool, TGFB modulates subsequent B-cell development and activation. TGFB1 inhibits kappa-light-chain expression in murine pre-B-cell clones exerting an inhibitory selective effect on the acquisition of surface gamma chains ,68. FurtTGFB clearly plays a role in regulating B-cell proliferation and survival. In vitro TGFB1 triggers apoptosis in normal B-cells and some transformed B-cell lines, leading to induction of proapoptotic and downregulation of antiapoptotic proteins ,75,76,77Most studies of TGFB dysfunction in haematological malignancies have focussed on myeloid malignancies, given a higher frequency of specific oncoproteins and mutations in TGFB signalling components . NevertResistance to apoptotic and antiproliferative effects of TGFB in CLL has been attributed to the loss of surface TGFB receptors ,91,92. NAlthough the literature is limited, genetic and epigenetic changes have been identified related to TGFB-receptor regulation. Mutations within the putative signal sequence of the TGFBR1 gene have been identified in a small cohort of CLL patients and found to correlate with resistance to growth inhibitory effects of TGFB . SimilarAlthough earlier research focussed on receptor-mediated resistant mechanisms, a more recent study has revealed distinct intracellular signalling alterations in follicular lymphoma and DLBCL. SMAD1 appears increasingly important in mediating TGFB signalling in malignant B-cells. Gene expression profiling highlights SMAD1 as overexpressed in FL and EBV negative BL cells ,104. IndIn contrast to FL, aberrant loss of SMAD1 is common in DLBCL and found in >85% of DLBCL patients, likely reflecting differing fundamental biology . MechaniBeyond perturbed SMAD1 signalling, DLBCL cells can also mediate TGFB signalling through SMAD5 phosphorylation, which is again more commonly associated with BMP signalling . This suThe B-cell malignant microenvironment is made up of multiple different cell types whereby complex reciprocal signalling can occur through both tumoural and microenvironmental TGFB . We focuThe role of microenvironmental TGFB has been explored most in FL and has been linked to both stromal and T-cell reprogramming. Both FL cells and intratumoural T-cells secrete TGFB, which promotes regulatory CD4+ T-cell expansion and suppresses effector helper CD4+ cell function . In addiMore recent reports highlight complex cross-talk between malignant B-cells and stromal cells. FL B-cells produce extracellular vesicles (EVs) that are internalised by BMSCs, drive stromal polarisation, and enhance FL B-cell survival and quiescence . ExaminiThe role TGFB plays in the microenvironment of DLBCL is less clear. Early sequencing studies identified enrichment of TGFB1 signalling elements within prognostically favourable ‘stromal 1’ signature . A more Finally, microenvironmental TGFB orchestrates aspects of innate immune function. B-cell acute lymphoblastic leukaemia (ALL)-derived TGFB1 mediates natural killer cell dysfunction, demonstrating impaired interferon-gamma secretion and cytotoxicity, allowing ALL to evade immune clearance . ConversOverall microenvironmental TGFB predominantly leads to immune cell suppression and stromal reprogramming, leading to a protumoural niche. Moreover, there is increasing evidence of bi-directional cross-talk, whereby cells of the microenvironment promote senescence and survival in malignant B-cells. Although these aberrations should be considered on a disease-subtype-specific basis, it is likely that some of these changes promote tumour growth and seed relapse, offering a potential new route to therapy.A challenge to therapeutic translation of strategies targeting the TGFB axis is the heterogeneous mechanisms of dysregulation. One approach would be to reactivate sensitivity to the proapoptotic effects of TGFB. The epigenomic evolution of DLBCL is associated with the methylation of TGFB signalling elements, offering the opportunity to utilise demethylating agents. Notably, pre- and post-treatment biopsies from a small cohort of patients treated with azacytidine, followed by standard chemoimmunotherapy in high-risk, newly diagnosed DLBCL, confirm SMAD1 demethylation and associated chemosensitisation . NeverthAn alternative approach to modifying intrinsic TGFB dysregulation is to inhibit prosurvival microenvironment signalling. Targeting these interactions is advantageous as microenvironmental cells are not under selective pressure. For example, microenvironmental disruption of the CLL niche potentiates the antitumour activity of Ibrutinib, and targeting stromal PKC-β in B-cell lymphomas enhances toxicity to common therapies ,125. TarFinally, the TGFB axis should be considered in the context of the expanding number of cellular-based therapies for B-cell lymphomas. Early research using EBV-specific cytotoxic T-cell lymphocytes used for the treatment of EBV-positive Hodgkin disease show that modification through the expression of a dominant-negative TGFBR2 resisted the immunosuppressive effects of TGFB and enhanced cytotoxicity ,133. A sMultiple mechanisms of intrinsic TGFB signalling dysfunction have been identified in B-cell malignancies with distinct alterations dependent on histological subtype. Resistance to tumour-suppressive mechanisms of TGFB may confer a selective advantage for clonal expansion through direct, but also indirect, effects on other cells in the microenvironment. Inhibiting reciprocal TGFB signalling between B-cells and microenvironmental cells offers a potential therapeutic window to target protumoural niches. Given the cell-context nature of TGFB signalling, future meticulous functional characterisation of the B-cell tumour microenvironment may provide insights into disease pathogenesis and guide therapeutic strategies.


Background. Some patients previously presenting with COVID-19 have been reported to develop persistent COVID-19 symptoms. While this information has been adequately recognised and extensively published with respect to non-critically ill patients, less is known about the incidence and factors associated with the characteristics of persistent COVID-19. On the other hand, these patients very often have intensive care unit-acquired pneumonia (ICUAP). A second infectious hit after COVID increases the length of ICU stay and mechanical ventilation and could have an influence on poor health post-COVID 19 syndrome in ICU-discharged patients. Methods: This prospective, multicentre, and observational study was carrid out across 40 selected ICUs in Spain. Consecutive patients with COVID-19 requiring ICU admission were recruited and evaluated three months after hospital discharge. Results: A total of 1255 ICU patients were scheduled to be followed up at 3 months; however, the final cohort comprised 991 (78.9%) patients. A total of 315 patients developed ICUAP (97% of them had ventilated ICUAP). Patients requiring invasive mechanical ventilation had more persistent post-COVID-19 symptoms than those who did not require mechanical ventilation. Female sex, duration of ICU stay, development of ICUAP, and ARDS were independent factors for persistent poor health post-COVID-19. Conclusions: Persistent post-COVID-19 symptoms occurred in more than two-thirds of patients. Female sex, duration of ICU stay, development of ICUAP, and ARDS all comprised independent factors for persistent poor health post-COVID-19. Prevention of ICUAP could have beneficial effects in poor health post-COVID-19. Clinical presentation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection ranges from mild to severe . DiseaseCurrently, no consensus definition for the symptoms of poor health post-COVID-19 exist; however, the most common symptoms include fatigue, shortness of breath, weakness, and asthenia . Some puIn the present manuscript, we analysed poor health post-COVID-19 in the initial cases of hospitalised patients with COVID-19 at 3-month follow-up after hospital discharge. We hypothesised that critically ill patients would present with high, persistent post-COVID-19 symptoms and significant abnormalities in both lung function tests and radiology. Herein, we summarise the poor health post-COVID-19, functional respiratory parameters, and radiological features of patients discharged from the ICU at 3-month follow-up. The main objective of this study was to determine the factors of the acute period associated with poor health post-COVID-19 in ICU survivors at 3-month follow-up following hospital discharge and identify the factors associated with poor recovery. We also aim to analyse lung function and radiologic abnormalities in critically ill patients after hospital discharge.We carried out a prospective, multicentre and observational cohort study at 40 selected ICUs in Spain with critically ill patients initially admitted from 16 February 2020 until 1 January 2021. We consecutively recruited patients with COVID-19 requiring ICU admission and performed a follow-up at three months after hospital discharge. This study is a pre-planned analysis of the ongoing multicentre study called CIBERESUCICOVID . We then asked staff from each centre to prospectively obtain data for ICU-admitted patients aged 18 years or older with positive polymerase chain reactions (PCR) for SARS-CoV-2. Re-admitted patients and previously tracheostomised patients were not included. The study received approval by the institution’s Internal Review Board . We obtained informed consent for most patients by using emergency consent mechanisms in accordance with ethics approval guidelines for the study. Further participating centres either received ethics approval from their institutions or had waived ethics approval. Finally, we de-identified all clinical data to allow for the waiver of informed consent.3 or leucopoenia < 4000/mm3; or purulent respiratory secretion [Recorded data included demographic characteristics, comorbidities, time course of illness, treatments administered, laboratory and microbiologic data, radiologic findings on chest X-rays, CT scans, ventilatory parameters in patients with invasive mechanical ventilation, complications during ICU stay, and outcomes. We determined disease severity and assessed organ failure using the Sequential Organ Failure Assessment (SOFA) score, calculating both within the first day of ICU admission . Ventilaecretion ,17. We cecretion .Poor health post-COVID-19 was defined by a voluntary report of any of the following symptoms after COVID infection: dyspnoea, weakness, asthenia, myalgia, cough, numbness, headache, anosmia and ageusia. We provided electronic case report forms using a secure website. For all patients, we recorded demographic characteristics, duration of ICU and hospital stays, the McCabe classification of comorbidities and likelihood of survival , or <1 year ), the SOFA score to predict hospital mortality, and outcome . We calculated static compliance of the respiratory system as tidal volume/. Chest CT scans and CT pulmonary angiograms were obtained when clinically indicated and technically feasible. All the radiological interpretation was performed by an independent certified radiologist in all cases, as per current clinical practice in the participating hospitals. The Modified Medical Research Council (mMRC) dyspnoea scale grades the impact of dyspnoea on daily activities throughout the prior seven days and thereby quantifies the disability or physical limitations associated with dyspnoea [The primary outcome of our study was to determine incidence and the factors associated with poor health post-COVID-19 in critically ill patients per clinical presentation at three months of hospital discharge. The secondary outcome included determining associations between poor health post-COVID-19 symptoms and abnormalities in lung function tests, radiologic characteristics, and laboratory parameters.p values were two-tailed. We considered differences as significant if p was less than 0.05. We reported categorical variables as numbers and frequencies (%), normally distributed continuous variables as means (standard deviation (SD)) and skewed continuous variables as medians ((interquartile range (IQR)). We performed both χ2 tests or Fisher’s exact tests to compare qualitative variables and Student’s t tests and ANOVAs or Mann–Whitney U and non-parametric Kruskal–Wallis tests to compare normally distributed or skewed continuous variables, whenever appropriate. We undertook univariate analyses of predictors of post-COVID syndrome, using the χ2 test and Student’s t test. To explore the factors associated with post-COVID syndrome, a generalized linear model was used, defined by a binomial probability distribution and a log link function. The following variables were included in the multivariable model based on clinical relevance only: sex, SOFA, cirrhosis, non-invasive mechanical ventilation, invasive mechanical ventilation, ICU length of stay, corticosteroids, ICUAP, and ARDS. Relative risks (RRs) and their 95% confidence intervals were calculated. We performed a mixed-effects model [The study sample size was not formally calculated but instead based on the nature of the study and pre-planned dates. We used SPSS version 20) for data analysis. All  for dataWe monitored patients after hospital discharge for a median of 77 (57–99) days. A total of 1255 patients were scheduled to be followed up for three months. However, we could not perform a 3-month follow-up of 264 patients. Patients did not show up to their appointments despite multiple attempts. The final cohort comprised 991 (78.9%) patients. A total of 731 (73.8%) patients developed persistent post-COVID-19 symptoms. A flow chart with the percentage of patients with persistent post-COVID-19 symptoms is displayed in p = 0.05). After discharge, 33 (3.2%) patients presented with infectious complications, 172 (15.1%) attended the emergency department, and 63 (5%) were readmitted to hospital. No significant differences were observed in patients with or without persistent post-COVID-19 symptoms with respect to follow-up clinic visits . Patients with persistent post-COVID-19 symptoms required additional respiratory therapy after hospital discharge  and showed a trend of its presence in patients with persistent post-COVID-19 symptoms  or chest CT scans . Persistently abnormal chest X-rays were observed in 21.4% of patients. Patients with persistent post-COVID-19 symptoms more frequently had abnormal X-rays and chest CT scans showing diffuse interstitial lung patterns than those without such symptoms. Patients who required invasive mechanical ventilation more often presented interstitial lung disease patterns in chest CT scans. Further details are shown in We performed a radiologic work-up in 471 (47.5%) patients. A similar number of patients with and without persistent post-COVID-19 symptoms underwent either chest radiography  were performed more often in patients with persistent post-COVID-19 symptoms, while there were no significant differences in performing DLCO between patients with or without persistent post-COVID-19 symptoms . A total of 665 (67.4%) patients underwent invasive mechanical ventilation. Patients with invasive mechanical ventilation had a higher percentage of persistent post-COVID-19 symptoms than those without mechanical ventilation . We performed a pulmonary function test (PFT) in 535 (53.9%) patients and diffusing capacity of the lungs for carbon monoxide (DLCO) in 464 (46.8%). PFTs . Among the 258 patients with confirmed pathogen, 220 were ventilated. 44 (20%) had early VAP and 176 (80%) had late VAP. The most pathogens isolated were Gram negatives, but S. aureus was present in 46 (16%) cases. 40 patients had polymicrobial pneumonia.The present study analysed a multicentre cohort of patients admitted to the ICU with COVID-19. Few data are currently available on the follow-up of survivor patients with COVID-19 after discharge. Interestingly, we found that more than 70% of patients discharged from the ICU had persistent post-COVID-19 symptoms after three months had passed since hospital discharge; however, the hospital readmission rate within this period remained low, and only 15% needed to visit the emergency department. There was also a poor correlation between abnormal radiologic findings and persistent post-COVID-19 symptoms in critically ill patients after three months had passed since hospital discharge. Factors associated with persistent post-COVID-19 symptoms included female sex, duration of ICU stay, development of ICUAP and ARDS.With respect to the burden of follow-up on the healthcare system, X-rays and CT scans were performed in 4 of 10 ICU-admitted patients at three months after hospital discharge. As expected, normal X-rays were frequent in patients with good clinical resolution; interstitial patterns were more often seen in chest CT scans of patients with persistent post-COVID-19 symptoms. Recently, patients with previously undiagnosed fibrotic lung abnormalities have been reported to face the possibility of ARDS onset . In a coThe symptoms most frequently observed in patients with post-COVID-19 included dyspnoea, asthenia, and weakness. After analysis of the therapy provided, we found that oxygen therapy was provided significantly more often to patients with persistent post-COVID-19 symptoms. In a previous study in China, patients monitored for three months after hospital discharge presented with considerable radiologic and physiologic abnormalities . In anotA strength of our research includes the prospective follow-up of a detailed list of lung function parameters. Soriano et al.  recentlyAdditionally, we aimed to determine an association between ventilatory parameters in patients with invasive mechanical ventilation and persistent post-COVID-19 symptoms at the 3-month follow-up after hospital discharge. Our assessment of different respiratory parameters such as PaO2/FiO2 ratio and pCO2 showed a significant correlation across two variables: compliance at the time of intubation and PCO2 at day 3. Both parameters clearly reflect the damage caused to the respiratory system by a COVID-19 infection. It is interesting that compliance, albeit not the PaO2/FiO2 ratio, was a predictor of persistent post-COVID-19 symptoms. A recent study found that median time to intubation was twice as long in the very-low compliance group than in the low-normal compliance group . ReporteA factor associated with poor recovery was female sex. This is an intriguing finding, given that male patients are more widely reported to be admitted to the ICU ,37 for CLastly, the development of a ICUAP during the ICU stay was found to be an independent factor associated with poor recovery at 3-month follow-up. This is an especially important finding given the high incidence of nosocomial pneumonia in critically ill patients, especially in those that needed invasive mechanical ventilation. This finding stresses the importance of the prevention ICUAP in COVID-19 critically ill patients. The hypothesis behind this finding is that a second hit (ICUAP) after COVID-19 increased lung damage and consequently increased the risk of respiratory symptoms persistence at follow-up. Some multicentre, European manuscripts suggest that ventilator-associated lower respiratory tract infections (VA-LRTI) were more frequent in patients with COVID-19 than in patients admitted to the ICU with another virus (influenza) or in patients without viral infections . To the Our study has several limitations. Samples obtained from the centres may not be representative, given that the hospital units selected all had the research resources necessary to participate. We included an acceptable number of variables for follow-up analysis; however, certain functional tests were not recorded, including the six-min walk test (6MWT), an excellent tool for assessing sub-maximal exercise aerobic capacity and endurance. We preferred to determine PFT and imaging, as detailed extensively in this manuscript, and felt that the 6MWT might not be reproducible as a measure for oxygen desaturation. Another potential limitation was related to symptom collection. The objective of the study was to obtain a voluntary report of symptoms after the onset of COVID and some patients might have had these symptoms before contracting COVID for various reasons. Moreover, the database did not capture the frequency of headaches and/or the location of numbness. A strength of this manuscript is the availability of many data points from the acute period and including data of day 1 and day 3 to determine factors associated with long-COVID 19 syndrome.In conclusion, we evaluated many critically ill patients with COVID-19 after three months had passed following hospital discharge. Persistent post-COVID-19 symptoms occurred in more than two-thirds of patients; however, the hospital readmission rate remained low. There was no clinical association between such symptoms and persistently abnormal chest X-rays. Additionally, more than 10% of these patients still required oxygen at home. Female sex, duration of ICU stay, development of ICUAP, and ARDS were independent factors associated with persistent post-COVID-19 symptoms in critically ill patients at 3-month follow-up. Prevention of ICUAP could have beneficial effects in poor health post-COVID 19 syndrome.


Smart luminescent materials, which can respond to the changing of external environment , have always been one of the research hotspots. Mechanochromism refers to the materials whose emission color or intensity can be altered under the stimulation of external mechanical force. This kind of smart materials have been widely used in data storage, information encryption and sensors due to its simple operation, obvious and rapid response. The introduction of metal atoms in metal-organic compounds brings about fascinating metalophilic interactions and results in more interesting and surprising mechanochromic behaviors. In this mini-review, recent advances in mechanochromism of metal-organic compounds, including mono-, di-, multinuclear metal-organic complexes and metallic clusters are summarized. Varies mechanisms are discussed and some design strategies for metal-organic compounds with mechanochromism are also presented. Luminescent materials have always been valued by researchers due to their widely use in data storage, optoelectronic devices, biological imaging and other fields . Metal-oTo date, most of the reported mechanochromic materials are based on organic molecules . TraditiIntermolecular interactions play a vital role in mechanochromic materials. Introducing halogen, or oxygen-containing functional groups into ligands is an important way to obtain mechanochromic complexes. Based on this strategy, R. Bryce et al. rationally designed a neutral iridium (III) complex with reversible mechanochromism . The carApart from traditional interactions, some infrequent interactions can also promote the formation of loose packing modes. Under mechanical stimulation, the change of interactions lead to the change of stacking modes, resulting in the mechanochromism. Pei and Zhao’ group prepared a series of cationic Ir(III) complexes with significant mechanochromic luminescence . For theCompared with the mononuclear complexes, more functional groups can be introduced into the dinuclear metal complexes, and more excellent luminescence properties can be obtained in the dinuclear complexes . For mec3LLCT) was existed in the original crystallic solids of gold(I) complexes, and the aurophilic interactions were formed after grinding, resulting to the formation of metal-metal-to-ligand charge transfer (3MMLCT) states and the extension of the π system, which reduced the energy level of the luminophore and caused red-shifted emission. Notably, this complex possessed the largest red-shifted emission reported so far and provided a facile method to construct infrared light-emitting materials by mechanochromism. On the contrary, some blue-shifted mechanochromic gold(I) complexes were also found. An arylgold (arylisocyanide) complex with hypsochromic mechanochromism was reported in 2018  complexes with mechanochromism, whose emission colors could be red-shifted or blue-shifted . In 2017 in 2018 . Strong A few dinuclear Au(I) complexes with mechanochromism have been reported . In 2002Apart from CA transition, mechano-induced single-crystal-to-single-crystal (SCSC) phase transition was another important mechanism for mechanochromic gold(I) complexes . The phaThe researchers were surprised to find that the neutral-to-ionic (NI) phase transition could contribute to the mechanochromism of Au(I) complexes. In 2015, Baranyai’s group reported a gold(I)-diphosphine complex with reversible mechanochromism . The oriBoth CA and SCSC transitions occured between two phases and caused bicolor mechanochromism. The existence of another metastable state enabled the complexes to exhibit multicolor mechanochromic properties . Wang etThe photoluminescence properties of Cu(I) complexes are greatly affected by cuprophilic interaction. However, it is difficult to predict the formation or destruction of Cu-Cu interactions, so it is urgent to develop a simple method to construct mechanochromic copper complexes. Liske’ group investigated some Cu(I) complexes and came up with a new mechanism for mechanochromism . They thIn the aforementioned Cu(I) complexes, the emission colors could not be returned the original states, showing irreversible mechanochromism. In order to obtain reversible mechanochromic materials based on Cu(I) complexes, flexible and rigid ligands were introduced to prompt the formation of loose stacking modes. Yu et al. reported a steric bis(pyrazol-1-yl)borohydrate-containing Cu(I) complex with reversible mechanochromism . The stu4− anion  complexes. It is crucial to discuss the relationship between structural changes such as Cu-Cu distance and luminescence performance. In 2018, the mechanochromisms of dinuclear Cu(I) complexes bearing N-heterocyclic carbene (NHC) ligands were studied by Lu’s group . The sti− anion . The des3ML/3HLCT) excited states to cluster-centred (3CC) excited states. In 2017, Xin’s group found a pillared-layered copper(I) MOF, whose mechanochromic performance was resulted from the weak bond contraction of the copper halide layer  complexes with mechanochromic properties resulting from CA phase transition have been reported successively. Xie et al. reported a carbazole-based cyclic trinuclear Cu(I) complex with abnormal pressure-induced phosphorescence enhancement (PIPE) . The resde layer . Apart fde layer .3π-π*/3MLCT to 3MLCT. This example proposed a new mechanism of mechanochromic properties and provided a new design strategy to tune phosphorescence by switching triplet excited states.Pt (II) complexes have unique photophysical properties due to their multiple excited states, such as LE , MLCT , LLCT (ligand-to-ligand charge transfer), and ILCT . These excited states would be changed under the stimuli of force, showing mechanochromism. In 2020, Zhu et al. reported Pt (II) complexes with mechanochromic properties . The emiZinc is abundant and inexpensive compared with other noble metals, so it is more attractive to design zinc complexes with mechanochromic properties, which would facilitate the development of low-cost sensor materials. In 2018, Song et al. designed a Zn(II) schiff-based complex . The comThis paper reviewed the recent advances in mechanochromism of metal-organic compounds such as Ir(III), Au(I), Cu(I), Pt (II) and Zn(II) complexes. Considering the structures of complexes, mono-, di-, multi-nuclear complexes and even clusters, metal-organic frameworks (MOF), and coordination polymers have been summarized according to the type and number of metal atoms. Considering the mechanochromic properties of complexes, various mechanisms were outlined: CA and SCSC phase transitions, NI transition, the pore structure and switching the triplet excited states. By exploring the structure-property relationship of these complexes, several effective design strategies have been proposed: 1) Introduce heteroatom-containing groups to form multiple intermolecular interactions especially the metallophilic interactions; 2) Introduce large flexible groups to form the loose packing modes; 3) Construct the AIE complexes. Despite this, mechanochromic materials still lacked a clear theory because of the difficulty in characterizing the amorphous phase. The current mechanochromic materials suffered from problems that low crystallinity, poor controllability, and low sensitivity. In the future, high-performance mechanochromic materials based on organic-metal compounds that can be used in sensors and anti-counterfeiting fields still need to be further explored. We expect that this paper can provide valuable reference for the development of mechanochromic materials.


Immunomodulatory therapies have fueled interest in targeting microglial cells as part of the innate immune response after infection or injury. In this context, the colony-stimulating factor 1 (CSF-1) and its receptor (CSF-1R) have gained attention in various neurological conditions to deplete and reprogram the microglia/macrophages compartment. Published data in physiological conditions support the use of small-molecule inhibitors to study microglia/macrophages dynamics under inflammatory conditions and as a therapeutic strategy in pathologies where those cells support disease progression. However, preclinical and clinical data highlighted that the complexity of the spatiotemporal inflammatory response could limit their efficiency due to compensatory mechanisms, ultimately leading to therapy resistance. We review the current state-of-art in the field of CSF-1R inhibition in glioma and stroke and provide an overview of the fundamentals, ongoing research, potential developments of this promising therapeutic strategy and further application toward molecular imaging. Inflammation is a biological process triggered by injuries, infections and damages suffered by the cells that disrupt tissue homeostasis. Together with the innate and adaptive immune responses, they are discussed as essential factors in the onset and progression of many neurological conditions . TherefoMicroglial cells serve as regulators of homeostasis in the central nervous system and represent the first line of defence against infection and injury . They arThey play a significant role in neuronal plasticity and synaptic connections , 16. Thetmem119, Selplg and Slc2a5 markers were highly expressed in postnatal microglia compared to embryonic microglia, indicative of cell maturation. It was suggested that the four postnatal clusters might be related to different cell functions -derived macrophages, including morphologies, surface markers and other characteristics . That exThe colony-stimulating factor-1 receptor (CSF-1R), also known as macrophage colony-stimulating factor (M-CSF) receptor, is a transmembrane tyrosine kinase receptor found at the cell surface of microglial cells, bone-marrow-derived macrophages, monocytes, and other cell types . The CSF-1/CSF-1R axis regulates cell survival, proliferation, differentiation, and functions of the mononuclear phagocytes , 26.-/- deficient mice  and includes PI3K-AKT and AMPK pathways implicated in macrophages differentiation. Both cytokines promotes macrophages survival, differentiation and proliferation but show different ability to polarize macrophages . They shent mice .A low level of CSF-1 stimulates microglia survival and inhibits protein degradation, while increased CSF-1 expression, as observed in inflammatory conditions, was associated with cell proliferation and enhanced migration . AdditioMany CSF-1R inhibitors have been developed . Among them, PLX5622 (Plexxikon Inc.) is a potent inhibitor of the kinase activity (KI = 5.9 nM) showing high selectivity over other kinases , 30. X-rvia apoptosis  microglial cells and non-microglial (Iba-1-) cell populations within the first days of repopulation , drug withdrawal resulted in a rapid re-emergence of symptoms, leading eventually to peak scores comparable to those in control EAE mice, associated with an increase in microglia number 5-6 days after drug withdrawal . MoreoveImmunotherapies represent a promising approach for treating cancer. Despite favorable results obtained treating different tumor types –50, theyreg) cell response , they acquire a pro-tumorigenic phenotype under the influence of the tumour cells. Similarly, tumour-associated macrophages (TAMs) are differentiated from monocytes precursors recruited from the systemic reservoirs to the tumour in response to cytokines and chemoattractants released by tumour cells, including the CSF-1 ligand , ultimatresponse . Additioresponse . Thereforesponse .Efficient targeting of TAM using small molecule CSF-1R inhibitors was assessed in many tumour models, including solid tumours and breast cancers , 62. Hig+ Treg influx in response to macrophage depletion  anti-inflammatory effect, while detrimental pro-inflammatory microglia seem to dominate at later stages . In expePLX5622 and derivatives have been investigated in transient and permanent middle cerebral artery occlusion (MCAo) rodent models to understand how microglia depletion prior to stroke may affect disease outcomes. From the preventive studies, data confirmed that microglia might confer protection against injury at an early stage . Effect + macrophages (-80%) at day 4 post ischemia  and increasing levels of growth factors (IGF-1) in some cases. Additionally, CSF-1R depletion prior to and after MCAo reduces the pro-inflammatory astrocytic reactivity  with no change in astrocytes number  dynamics. Interestingly, the decrease in activated microglia/macrophages number in remote areas such as the contralateral side may moderate the impact of spreading depression and ultimately global inflammation, as observed by the global decrease in pro- and anti-inflammatory markers expression in both hemispheres at late stage -PET imaging has been widely used to study inflammation dynamics, immune cell activation and/or microgliosis in preclinical and clinical studies. Despite several well-known caveats, TSPO-PET imaging allows assessing global inflammation, visualizing areas of immune cell infiltration and defining tissue heterogeneity –98. Moreex vivo. Therefore, we supported the use of TSPO-PET imaging as a microglia-targeting therapy readout in stroke.Recently, our group reported the suitability of 18F-DPA-714 PET imaging, used as a biomarker of TSPO-dependent neuroinflammation and immune cell activation, to track the immunomodulatory effect of long-term PLX5622 administration in the post ischemic phase . We demoIt should be noted that none of the TSPO radioligands is cell-specific or function-specific. Among the emerging targets, the purinergic metabotropic 12 receptor (P2Y12R) PET tracer is an attractive imaging biomarker to study microglial function , 101. P2As part of the same receptor family, the purinergic P2X7 receptor is found to be specifically upregulated in pro-inflammatory activated microglial cells in response to high ATP release. This receptor mediates cytokine and chemokines release, regulates T lymphocytes survival and differentiation Di . A cliniGlioma and stroke are two complex pathological conditions, both inducing strong and chronic inflammatory and immunological responses following alterations of the immune balance. However, innate, and adaptive immune response differs in major aspects: origin, progression, and disease-induced phenotype. The comparison of both diseases represents a very interesting avenue of research as they represent the two extremes of neurological conditions. Stroke on the one hand with a strong pro-inflammatory stimulus  and on the other hand gliomas with a strong anti-inflammatory immunosuppressive microenvironment. The comparison thus supports evidence generation for the role of CSF-1R across neurological diseases.CSF-1R inhibition studies demonstrate that many discernible subpopulations of microglial cells co-exist in the inflammatory environment and are differentially sensitive to CSF-1R inhibition. In both glioma and ischemia, a resistant population of CSF-1R-independent microglia/macrophages were observed after long-term PLX5622 treatment. Their contribution to compensatory mechanisms or therapy resistance still needs further research. One line of investigation focuses on the enhanced communication with other immune players, triggering modulation of the peripheral immune cell infiltration or activation of resident cells. Still, numerous compensatory and resistance mechanisms seem to be implemented by immunological responses beyond microglia/macrophages cells, limiting the efficiency of CSF-1R inhibition as a monotherapy. Therefore, research supports its use as a combination therapy to synergize the therapeutic effects of other immunomodulatory approaches.in vivo TSPO dynamics upon therapy. Advances in microglia-targeting immunotherapy should boost the development of microglia-specific PET radioligands. This could support further clinical trials for glioma and stroke patients, improve personalized management and understand the prognostic value of multimodal imaging in microglia-targeting therapeutic approaches.Recently, we validated the use of TSPO-PET in preclinical studies assessing the therapeutic effect of new microglia-targeting treatments. TSPO-PET employing 18F-DPA-714 allows visualization of microglia/macrophages depletion-repopulation and areas of immune cell infiltration. However, a better knowledge of the therapeutic effects on other immune cell populations after short-term or long-term CSF-1R inhibition would improve our understanding of the All authors conceptualized and wrote this manuscript. All authors contributed to the article and approved the submitted version.This work was partly funded by the Horizon 2020 Programme under grant agreement n°675417 (PET3D) and the Herbert-Worch-Stiftung. Additionally, this work was supported by a Collaboration Grant of the Medical Faculty of the University of Bonn between CIO UKB and Johanniter Hospital. This review was supported by the Immune-Image consortium. The Immune-Image project receives funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 831514 (Immune-Image). The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA.The author BZ is currently employed by F. Hoffman-La Roche Ltd.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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After the age of 65, one’s ability to walk while performing an additional cognitive task  is predictive of both future falls and cognitive decline. However, while it is well-known that older adults exhibit diminished dual-task performance, the time course of age-related dual-task decline has not been established. We thus conducted an analysis of data collected within the ongoing Barcelona Brain Health Initiative, a prospective population-based study characterizing the determinants of brain health maintenance in middle-aged adults. Cognitively-unimpaired participants (n=655) aged 40-65 years without neuro-psychiatric disease completed laboratory-based trials of walking normally (single-task) and walking while performing a verbalized serial subtraction task . A smartphone-based gait assessment application was used to capture data and derive both the mean stride time (ST) and stride time variability  of each trial. The dual-task costs (DTC) to each gait metric were obtained by calculating the percent change from single- to dual-task conditions. We categorized participants into five groups according to age . Age group did not have an effect on single-task gait outcomes (p>0.51). However, the oldest age group, as compared to each of the other groups, exhibited greater DTC to both ST and STV (p<0.03). These results indicate that dual-task walking performance in particular may begin to diminish in late middle age even in the absence of detectable cognitive issues, DTC may offer a sensitive metric to age-related change in cognitive function.