With this first clinical, wound-centric resource of miRs and mRNAs, we identified 17 pathologically relevant miRs that exhibited abnormal VU expression and displayed their targets enriched explicitly in the VU gene signature. Intermeshing regulatory networks controlled by these miRs revealed their high cooperativity in contributing to chronic wound pathology characterized by persistent inflammation and proliferative phase initiation failure. Furthermore, we demonstrated that miR-34a, miR-424, and miR-516, upregulated in VU, cooperatively suppressed keratinocyte migration and growth while promoting inflammatory response. By combining miR expression patterns with their specific target gene expression context, we identified miRs highly relevant to VU pathology. Our study opens the possibility of developing innovative wound treatment that targets pathologically relevant cooperating miRs to attain higher therapeutic efficacy and specificity.MicroRNAs (miR), as important epigenetic control factors, reportedly regulate wound repair. However, our insufficient knowledge of clinically relevant miRs hinders their potential therapeutic use. For this, we performed paired small and long RNA-sequencing and integrative omics analysis in human tissue samples, including matched skin and acute wounds collected at each healing stage and chronic nonhealing venous ulcers (VUs). On the basis of the findings, we developed a compendium ( Wound healing is a fundamental biological process (BP) comprising three sequential and overlapping phases, that is, inflammation, proliferation, and remodeling . This deMicroRNAs (miR) represent a group of short (~22 nt) noncoding (nc) ribonucleic acids, incorporating into the RNA-induced silencing complex and binding to the 3′ untranslated region of their target mRNAs, resulting in mRNA destabilization and translational repression . Given tHowever, our insufficient knowledge of the miR-mediated gene regulation in human wounds severely hinders the identification of clinically relevant miRs and their potential therapeutic use. While most previous wound healing-related miR studies rely on in vitro or animal models, only a few have approached miR profiles in human wound tissues or primary cells from patients, including tissues and fibroblasts of diabetic foot ulcers , burn wohttps://www.xulandenlab.com/humanwounds-mirna-mrna), which is the first wound healing-focused miR resource for facilitating the exploration of miR’s clinical application and for aiding in the elucidation of posttranscriptional regulatory underpinnings of tissue repair.In this study, we performed paired small and mRNA expression profiling in the human skin, acute wounds during the inflammatory and proliferative phases, and VU, unraveling time-resolved changes of the whole transcriptome throughout the wound healing process and the unique gene expression signature of a common chronic wound type. The integrative miR and mRNA omics analysis provides a network view of miR-mediated gene regulation in human wounds in vivo and demonstrates the functional involvement of miRs in human skin wound repair at the system level. Importantly, we identified miRs highly relevant to VU pathology, based not only on their aberrant expression but also their targetome enriched in the VU-related gene expression signature. Apart from confirming the in silico findings, the experimental miR expression, targetome, and function validation uncovered that VU-dysregulated miRs could act cooperatively contributing to the stalled wound healing characterized by failed transition from inflammatory-to-proliferative phase, which opens up new possibility for the development of more precise and innovative wound treatment targeting pathologically relevant cooperating miRs to achieve higher therapeutic efficacy and specificity. Additionally, based on this comprehensive analysis of human wound tissues, we built a browsable resource web portal (https://www.xulandenlab.com/humanwounds-mirna-mrna) with more or less rigorous cutoffs. With this unique resource, we dissected further the miR-mediated posttranscriptional regulatory underpinnings of wound repair.To better understand tissue repair in humans, we collected wound-edge tissues from human acute wounds and chronic nonhealing VUs . Tissue https://www.xulandenlab.com/humanwounds-mirna-mrna). Notably, we identified 84% of them as DEGs, suggesting a high consistence between the WGCNA and DE analysis  for classifying miRs according to their coexpression patterns in the 20 sRNA-seq analyzed samples to link the miR expression changes with wound healing progression or nonhealing status at a system level . We idenanalysis .We hypothesized that the coexpression of various miRs could be due to their transcription driven by common transcription factors (TFs). To test this idea, we leveraged TransmiR v2.0 , a databmiRs exert functions through the posttranscriptional regulation of their target mRNAs. Therefore, describing the mRNA expression context would be required for understanding the role of miRs in wound repair . We thusImportantly, our study unraveled a VU molecular signature: downregulated expression of RNA and protein production-  as well as cell mitosis-related (M7) genes, and upregulated expression of genes involved in extracellular matrix organization and cell adhesion (M9). These results were in line with the dermal tissue fibrosis observed in patients with chronic venous insufficiency . MoreoveAmong the multiple gene expression regulatory mechanisms, we aimed to evaluate how miRs could contribute to the protein-coding gene expression in human wound repair. We thus performed a correlation analysis for the miRs and mRNAs that were DE in VU compared to the skin and acute wounds, using the first principal component (PC1) of their expression in each sample. We found significantly negative correlations  between the PC1 of the DE miRs and the DE mRNAs predicted as miR targets, indicating negative regulation of VU-mRNA signature by the aberrantly expressed miRs in VU .Furthermore, we dissected the potential regulatory relationship between the VU-associated miR and mRNA modules. We identified significantly negative correlations between the downregulated miR  and the upregulated mRNA  modules, as well as between the upregulated miR (m8 and m12) and the downregulated mRNA (M5) modules in VU . Among tBased on the above-identified miR–mRNA module pairs, we next searched for individual candidate miRs with their targets enriched for the VU mRNA signature. We observed that the targets of two VU-associated downregulated miRs (miR-144-3p and miR-218-5p) and five m9 miRs  were enriched for the upregulated M9 mRNAs, whereas the targets of three VU-associated upregulated miRs  and seven m8 miRs  were enriched for M5 mRNAs and downregulated mRNAs . These m− epidermal cells consisting of mainly keratinocytes from matched human skin and acute wounds of five healthy donors. With sRNA-seq, we confirmed the in vivo expression of these 11 miRs in the epidermal cells of human skin and wounds (n = 7 per group) and found that the expression of miR-34a-5p and miR-424-5p was upregulated in the wound-edge epidermis from the VUs compared to the acute wounds and the skin, as shown in qRT-PCR analysis . The differences were more significant when we divided the strongest targets into conserved and experimentally validated subtypes, confirming the bioinformatics prediction robustness of the miR targets applied in this study  with miR-34a/c expression levels in the human skin and wound samples  with miR-218-5p expression in the human skin and wounds  of the experimentally validated miR-218-5p, miR-34a-5p, miR-34c-5p, and miR-7704 targets for the VU gene signature . We have samples , suggest samples . Its pre samples  and vali samples  targets d wounds . Interesd wounds . Similard wounds  and valid wounds  miR-7704For miR-96-5p, miR-424-5p, miR-450-5p, and miR-516b-5p, although their predicted targets were significantly enriched  for VU-associated mRNAs , we did In line with the in vivo expression of these eight miRs  in the epidermal cells of human skin and wounds , we alsoTogether, our validation work focusing on epidermal keratinocytes confirmed the robustness and reproducibility of this dataset and highlighted its value as a reference for studying the physiological and pathological roles of miRs in human skin wound healing.From the miR-mediated gene expression regulation networks underpinning VU pathology , we caugIn addition, we performed functional annotations for the genes regulated by the VU-associated miRs identified in the microarray analysis . Both miTo validate miR cooperativity in modulating the key pathological process in VU, we analyzed migration, proliferation, and inflammatory response of keratinocytes with individual miR or miR combination overexpression or inhibition. The microarray data GO analysis  showed tMoreover, our microarray analysis of keratinocytes with miR overexpression showed that the miR-34a-5p and miR-516b-5p combination extended the list of inflammation-related upregulated genes . In partIn summary, our study identified VU signature miRs, for example, miR-34a, miR-424, and miR-516, with cooperativity in inflicting more severe pathological changes . These fOur genome-wide paired analysis of miR and mRNA expression in human healing and nonhealing wounds provides a novel global landscape of the miR regulatory roles in wound biology. A detailed overview of the mRNA expression context at different healing stages or under pathological condition VU allows a more precise understanding about the complex role of miRs in wound repair. The same miR is often described to play different or even opposite roles in different cells, as each cell type has specific gene expression context subjected to the miR-mediated posttranscriptional regulation . TherebyAnother strength of our study is the decryption of time-resolved miR–mRNA expression pattern during human skin wound healing, providing a temporal view to our understanding of the functional miR roles. miRs and their target gene expression contexts change dynamically to support different functional needs during wound repair. Defining an miR as ‘pro-healing’ or ‘anti-healing’ requires specifying its temporal expression pattern. For example, continuous expression of a miR that is beneficial for one healing phase but not the other might also lead to deleterious effects.To understand the molecular mechanisms underlying the miR coexpression patterns, we analyzed the enriched TFs for each miR module with experimentally validated TF-miR regulation data . This anWe summarized the miRNAs that reportedly regulate wound healing and found that most of them also surfaced in our study, supporting the robustness of our profiling data and bioinformatics analysis . TherebyIn addition, certain of these VU-related miRs are involved in skin-related functions but have not yet been linked to wound healing. For example, miR-218-5p regulates hair follicle development , inhibitMoreover, we studied the relationships between the dysregulated miRs in VU, regarding their target repertoire and biological functions and identified miRs that could act cooperatively. Such knowledge is required for developing combined miR therapeutics with increased specificity and efficacy . In the https://www.xulandenlab.com/humanwounds-mirna-mrna) for the research community. This novel, rich resource enabled us to gain a network view of miR-mediated gene regulation during human physiological and pathological wound repair in vivo. With the same sequencing datasets, we have also analyzed circular RNA expression and their potential interaction with miRs and miR targets  to separate the epidermis from each section.− and CD45+ epidermal cells were separated by using CD45 microbeads and MACS MS magnetic columns according to the manufacturer’s instructions .The fresh skin and wound tissues were washed in PBS and incubated in dispase II  at 4°C overnight, and the epidermis was separated from the dermis as previously described . After tSnap frozen tissue samples were homogenized with the TissueLyser LT (Qiagen), and total RNA was isolated using the miRNeasy Mini kit (Qiagen). RNA quality and quantity were determined by using Agilent 2100 Bioanalyzer (Agilent Technologies) and Nanodrop 1000 (Thermo Fisher Scientific Inc), respectively.The sRNA-seq libraries were constructed using 3 μg total RNA per sample and NEB Next Multiplex Small RNA Library Prep Set for Illumina (NEB) following the manufacturer’s recommendation. Briefly, total RNA was first ligated to adaptors at the 3′ end by NEB 3′ SR adaptor and 5′ end by T4 RNA ligase followed by reverse transcription into cDNA using M-MuLV Reverse Transcriptase. PCR amplification of cDNA was performed using SR primers for Illumina and index primers. The PCR products were purified, and DNA fragments spanning from 140 to 160 bp were recovered and quantified by DNA High Sensitivity Chips on the Agilent Bioanalyzer. The libraries were sequenced on an Illumina Hiseq 2500 platform  using single-end 50 bp reads, and all samples were run side by side.− epidermal keratinocytes RNA-seq libraries were constructed by following the tutorial of NuGen Ovation Solo RNA-Seq System (Human part no. 0500). Finally, the libraries were sequenced on an Illumina Hiseq 4000 platform, and 150-bp paired-end reads were generated for the following analysis.The long RNA-seq libraries were constructed with a total amount of 2 μg RNA per sample. First, the ribosomal RNA was depleted by Epicentre Ribo-zero rRNA Removal Kit (Epicentre). Second, strand-specific total-transcriptome RNA-seq libraries were prepared by incorporating dUTPs in the second-strand synthesis step with NEB Next Ultra Directional RNA Library Prep Kit for Illumina (NEB). The CD45http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We used mapper.pl module in the miRDeep2 v0.1.3 package  multiple test to estimate the FDR. The DE miRNAs were defined as FDR <0.05 and |log2(fold change)| ≥1.The DESeq2 workflow  was carrplotPCA on the transformed data, in which the variances and size factors were stabilized and corrected. PCA and heatmaps were plotted by using ggplot2 (ComplexHeatmap (https://rstudio.com/).To explore the similarity of each sample, principal component analysis (PCA) was performed by using a DESeq2 built-in function  ggplot2  and CompxHeatmap  packagespickSoftThreshold was then employed to compute the optimized soft-thresholding power based on connectivity, which led to an approximately scale-free network topology , referred to as the PC1 of all miRNAs in each module. Pearson’s correlations  and the corresponding p values between MEs and traits were computed. p values were further adjusted to FDR across all the modules using the BH method. Modules significantly associated with each trait were selected by FDR <0.05 and absolute correlation coefficients >0.4. The module membership  of each miRNA was calculated by the correlations between miRNA expression and ME. MiRNAs with the highest kME values were defined as intramodular hub miRNAs, and networks of hub miRNAs in significant modules were visualized using the Cytoscape v3.7.2 software .Z-scores for each module by permutating 200 times using the same 20 samples as reference and test datasets. Modular preservation is strong if Z-summary >10, weak to moderate if 2 < Z-summary < 10, no evidence of preservation if Z-summary ≤2   , which amoduleEigengenes function of WGCNA package  based on the latest TargetScan v7.2 database |≥ 1), as well as the VU-associated modules’ hub miRNAs, which kME values were greater than the median kME in respective modules . Among these miRNAs’ strongest targets, we selected the ones that were hit by ≥2 miRNAs from m8, m9, m12 modules or miRNAs downregulated in VU; ≥3 miRNAs from m7 module or miRNAs upregulated in VU; ≥4 miRNAs from m3 module, to capture putative module-driving targets. We performed gene set enrichment analyses for these miRs’ targets in VU-specific DE mRNAs (FDR <0.05 and fold change ≥1.5) and VU-associated mRNA modules by using the R function act test . FurtherAAGTGGGATCGAGACATGTAAG-3′) and reverse primer (5′-GGAGACAGCACTCAAAGTAGAA-3′); GAPDH forward primer (5′-GGTGTGAACCATGAGAAGTATGA-3′) and reverse primer (5′-GAGTCCTTCCACGATACCAAAG-3′).To detect miRNA, RNA from human skin and wounds was reverse transcribed using TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific). Individual miRNA expression was then quantified using TaqMan Advanced miRNA Assays (Thermo Fisher Scientific) and normalized with miR-361-5p and miR-423-5p due to their relatively constant expression between human skin and wounds. To detect mRNA, we performed reverse transcription using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Gene expression was examined by SYBR Green expression assays (Thermo Fisher Scientific) and normalized with housekeeping gene B2M and GAPDH. The primer sequences for B2M are forward primer  or negative control using Lipofectamine 3000 (Thermo Fisher Scientific).Adult human epidermal keratinocytes (Thermo Fisher Scientific) were cultured in EpiLife serum-free keratinocyte growth medium supplemented with Human Keratinocyte Growth Supplement (HKGS) and 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific). Adult human dermal fibroblasts (Thermo Fisher Scientific) were cultured in Medium 106 supplemented with Low Serum Growth Supplement (LSGS) and 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific). Cells were incubated at 37°C in 5% COlimma R package (http://www.gsea-msigdb.org/)  (Transcriptome profiling of keratinocytes and fibroblasts transfected with 20 nM miRNA mimics or control mimics for 24 hr (in triplicates) was performed using Affymetrix Genechip system at the Microarray Core facility of Karolinska Institute. Normalized expression data  were exported from Transcriptome Analysis Console (TAC) software and analyzed by the  package . In briedb.org/) , was per package  and visudb.org/) .AB_2687446) overnight at 4°C. The next day, cells were incubated with the secondary antibody conjugated with Alexa 488 (RRID:AB_2535792) for 40 min at room temperature. Cells were mounted with the ProLong Diamond Antifade Mountant with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific). Ki-67 signals were visualized with Nikon microscopy, and positive cells were counted using ImageJ software .Cells transfected with a combination of miRNA and/or control mimics  were fixed in 4% paraformaldehyde for 15 min. Cells were incubated with the Ki-67 antibody  after cells attaching to the plates. Plates were imaged every 2 hr, and pictures were processed and analyzed using IncuCyte ZOOM 2018A software (Essen Bioscience).Human primary keratinocytes transfected with miRNA mimics or inhibitors were plated in Essen ImageLock 96-well plates (Essen Bioscience) at 15,000 cells per well for migration assay. Confluent cell layers were scratched using Essen wound maker to generate wounds. The cells were cultured with EpiLife medium without HKGS. The photographs were analyzed by using the IncuCyte software (Essen Bioscience).U-tests , Wilcoxon signed-rank test , or two-tailed Student’s t-test (parametric). Comparison between more than two groups was performed by one-way analysis of variance (ANOVA with uncorrected Fisher’s LSD). The cell growth and migration assay were analyzed by using two-way ANOVA. p value <0.05 is considered to be statistically significant.Sample size of each experiment is indicated in the figure legend. Data analysis was performed by using R and GraphPad Prism 7 software. Comparison between two groups was performed with Mann–Whitney https://www.xulandenlab.com/humanwounds-mirna-mrna). The analysis source code is available at https://github.com/Zhuang-Bio/miRNAprofiling; copy archived at swh:1:rev:14eaa943d868157d1de14b2e4ffc7f1be2552e15, Raw data of sRNA-seq, long RNA-seq, and microarray performed in this study have been deposited to NCBI’s Gene Expression Omnibus (GEO) database under the accession number GSE174661 and GSE196773, respectively. In addition, the analyzed dataset is presented with an online R Shiny app and can be accessed through a browsable web portal :Limited human-derived specimens and insufficient identification of clinical relevant miRNA hindered development of miRNA based therapy in venous ulcer treatment, this study performed an innovative work to acquire sufficient high-quality miRNA profiles from self-collected human skin specimens, and comprehensively analyzed miRNAs which participate in venous ulcer pathogenesis, and resolved the dynamic change within temporal dimension. The brief experimental validation also makes the conclusions more solid and reliable. While the data presentation form needs to be improved.The project is ambitious and of interest to the medical community in dermatology as the compendium access is free of charge for academic institutions. Moreover, a certain validation of the data has been performed for the identified pathologically relevant miRs in abnormal VU expression.1. The article could be updated on the presentation of clinical data or reference for miRs already found for VU or related dermatological disorders.2. My main criticism is that the compendium database linked to the article is rather difficult to use for clinicians. I would recommend that the authors could propose a virtual visit with an example of miRs and mRNA expression etc, how to look for its level of expression in VU as well as their therapeutic indications.3. Another criticism is that as mentioned in the article, extensive validation should be performed in cell type-specific miR expression data to confirm their findings. Otherwise, it may be difficult to make conclusions based on mixed cell types in biopsies from VU patients.Reviewer #2 (Recommendations for the authors):Liu et al. used biopsies from acute human excisional wounds and from chronic venous ulcers to identify miRNAs and mRNAs that are dysregulated in the ulcer tissue. Bioinformatics analysis of the data identified putative targets of strongly regulated miRNAs, which are also dysregulated in the ulcers. Finally, the authors show that some of the dysregulated miRNAs act cooperatively in keratinocytes to suppress their proliferation and to induce the expression of pro-inflammatory cytokines.The combined analysis of acute and chronic human wounds is a strength. Such biopsies are very difficult to get and therefore, it is particularly appreciated that the authors developed a compendium that will be open to the public. The data analysis is very well done and the results are interesting and important for the field. However, the functional validation of the data is rather limited. There is no information on the cell type that expresses the relevant miRNAs and their targets and no spatial information. Functional studies are limited to keratinocyte proliferation (which is not a major problem in chronic ulcers). Therefore, the data remain speculative and the claims are not fully supported by the available data.1. As mentioned by the authors, the observed changes may well be a consequence of a different cellular composition of acute vs. chronic human wounds. scRNA-seq would be the best way to address this issue, and this has now been done for many complex tissues. While such a complex analysis can of course not be done in a revision, the authors should at least make an effort to localize the most relevant miRNAs identified in this study and their putative targets in the wound tissue by in situ hybridization or immunostaining (for the targets).2. Along the same line: The authors suggest a few transcription factors that could regulate the differentially expressed miRNAs . To strengthen the data, the authors should perform immunostaining for these transcription factors to determine where they are expressed in acute vs. chronic wounds.3. The functional data are limited to the analysis of the effect of two miRNAs in keratinocyte proliferation. However, it is not clear if these miRNAs are indeed mainly expressed (and regulated) in keratinocytes. In addition, keratinocyte proliferation is not a major problem in chronic wounds – in fact, keratinocytes at the wound edge of chronic ulcers are frequently hyperproliferative . The major problem is the impaired migration . Therefore, the authors should check if some of these miRNAs, which are abnormally expressed in VUs, are expressed in keratinocytes at the wound edge and if they regulate keratinocyte migration in vitro. Finally, the functional data were all obtained after overexpression of miRNAs, while knock-down studies are missing. Even if there is functional redundancy among the miRNAs of interest, it would still be useful to confirm this and to know if the endogenous levels are sufficient to contribute to a certain cellular function.Reviewer #3 (Recommendations for the authors):In this study, Zhuang Liu and colleagues performed paired-RNA sequencing in human skin tissues, including matched acute wounds and chronic non-healing venous ulcers. They identified 17 pathologically relevant miRNAs that exhibited abnormal VU expression and used qPCR to identify some miRNAs. The authors tried to find some targets which have the potential to accelerate wound healing, and this study has done good work on comprehensive analyses, the results are likely to be of interest to the scientific community. However, I have some serious concerns about this research.1. The scientific novelty is limited, several groups have done similar analyses using similar samples before. Also, the sample size is limited in this study so the statistical power is weak to make solid conclusions.2. The conclusion is rather weak because there are no experiments to prove the candidate miRNAs have the function in wound healing at all. Ki67 staining and live-cell imaging only suggest that miR-34a and miR-424 affect keratinocyte cell proliferation.3. The paper is poorly written. Many regulatory network analyses have been done but those results were not clearly explained and it is hard to follow how conclusions were made based on such results.4. Authors need to perform in vivo or in vitro assays to identify miRNA function.5. Authors need to analyse previous data to confirm their findings. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1 (Recommendations for the authors):Limited human-derived specimens and insufficient identification of clinical relevant miRNA hindered development of miRNA based therapy in venous ulcer treatment, this study performed an innovative work to acquire sufficient high-quality miRNA profiles from self-collected human skin specimens, and comprehensively analyzed miRNAs which participate in venous ulcer pathogenesis, and resolved the dynamic change within temporal dimension. The brief experimental validation also makes the conclusions more solid and reliable. While the data presentation form needs to be improved.The project is ambitious and of interest to the medical community in dermatology as the compendium access is free of charge for academic institutions. Moreover, a certain validation of the data has been performed for the identified pathologically relevant miRs in abnormal VU expression.1. The article could be updated on the presentation of clinical data or reference for miRs already found for VU or related dermatological disorders.As suggested by the reviewer, we have added a new Table S3 summarizing the reported miRNAs regulating skin wound healing, analyzed their expression pattern in our datasets , and discussed the potential link between their expression and function patterns (the 4th paragraph in Discussion section). We show that most of the miRNAs that reportedly regulate wound healing also surface in our study, supporting the robustness of our profiling data and bioinformatics analysis.2. My main criticism is that the compendium database linked to the article is rather difficult to use for clinicians. I would recommend that the authors could propose a virtual visit with an example of miRs and mRNA expression etc, how to look for its level of expression in VU as well as their therapeutic indications.We thank the reviewer for this suggestion. We have added detailed virtual and text tutorials to our resource website to make it more user-friendly. Please visit the updated website: http://130.229.28.87/shiny/miRNA_Xulab/.3. Another criticism is that as mentioned in the article, extensive validation should be performed in cell type-specific miR expression data to confirm their findings. Otherwise, it may be difficult to make conclusions based on mixed cell types in biopsies from VU patients.in-silico findings in cell type-specific miR expression data. During revision, we have dedicated major efforts to this aspect :We agree with Reviewer that it is crucial to validate the We purified epidermal keratinocytes from matched human skin and acute wounds of five healthy donors and then performed paired small RNA and long RNA sequencing in these purified cells. From these new experients, we obtained miR and mRNA in vivo expression profiles in keratinocytes of human skin and wounds. In particular, we confirmed the keratinocyte expression of the VU-related miRs and their targets . In addition, with laser capture microdissection (LCM), we isolated epidermal compartments from human skin, acute wounds, and venous ulcers (n=7 per group) and confirmed the VU-related upregulation of hsa-miR-34a-5p and hsa-miR-424-5p occurred in wound-edge keratinocytes by qRT-PCR . These new data provide a rationale for selecting human primary keratinocytes for further functional validation. Next, we validated the miR-mediated gene regulatory network in human primary keratinocytes or fibroblasts for eight miRs by overexpressing each of them and performing microarray analysis with 33 samples . By comparing these in vitro identified miR targets with the VU-in vivo gene expression signature, we identified the most pathological-relevant miR targets  and the related signalling . We then examined the impacts of hsa-miR-34a-5p, hsa-miR-424-5p, and miR-516-5p and their combinations on the major biological processes of keratinocytes during human skin wound healing, i.e., inflammatory response, proliferation, and migration, using both gain- and loss-of-function approaches . Although a complete picture of miR expression and function in each wound cell type is yet to be generated, our validation work focusing on keratinocytes strongly supports the robustness of our profiling data and bioinformatics analysis.Reviewer #2 (Recommendations for the authors):Liu et al. used biopsies from acute human excisional wounds and from chronic venous ulcers to identify miRNAs and mRNAs that are dysregulated in the ulcer tissue. Bioinformatics analysis of the data identified putative targets of strongly regulated miRNAs, which are also dysregulated in the ulcers. Finally, the authors show that some of the dysregulated miRNAs act cooperatively in keratinocytes to suppress their proliferation and to induce the expression of pro-inflammatory cytokines.The combined analysis of acute and chronic human wounds is a strength. Such biopsies are very difficult to get and therefore, it is particularly appreciated that the authors developed a compendium that will be open to the public. The data analysis is very well done and the results are interesting and important for the field. However, the functional validation of the data is rather limited. There is no information on the cell type that expresses the relevant miRNAs and their targets and no spatial information. Functional studies are limited to keratinocyte proliferation (which is not a major problem in chronic ulcers). Therefore, the data remain speculative and the claims are not fully supported by the available data.in-silico findings is crucial, and we have dedicated significant efforts to strengthen this aspect during revision :We appreciate the reviewer's positive comments and constructive suggestions for our work. We agree that the validation of the We purified epidermal keratinocytes from matched human skin and acute wounds of five healthy donors and then performed paired small RNA and long RNA sequencing in these purified cells. With this approach, we obtained miR and mRNA in vivo expression profiles in keratinocytes of human skin and wounds. Particularly, we confirmed the keratinocyte expression of the VU-related miRs and their targets . In addition, with laser capture microdissection, we isolated epidermis from human skin, acute wounds, and venous ulcers (n=7 per group) and confirmed the VU-related upregulation of hsa-miR-34a-5p and hsa-miR-4245p occurred in wound-edge keratinocytes by qRT-PCR analysis . These new data provide a rationale for selecting human primary keratinocytes for further functional validation. Next, we validated the miR-mediated gene regulatory network in human primary keratinocytes or fibroblasts for eight miRs by overexpressing each of them and performing microarray analysis with 33 samples . By comparing these in vitro identified miR targets with the VU-in vivo gene expression signature – we identified the most pathological-relevant miR targets  and the related signalling . We then examined the impacts of hsa-miR-34a-5p, hsa-miR-4245p, miR-516-5p and their combinations on the major biological processes of keratinocytes during human skin wound healing, i.e., inflammatory response, proliferation, and migration, using both gain- and loss-of-function approaches . Although a complete picture of miR expression and function in each wound cell type is yet to be generated, our validation work focusing on keratinocytes strongly supported the robustness of our profiling data and bioinformatics analysis.1. As mentioned by the authors, the observed changes may well be a consequence of a different cellular composition of acute vs. chronic human wounds. scRNA-seq would be the best way to address this issue, and this has now been done for many complex tissues. While such a complex analysis can of course not be done in a revision, the authors should at least make an effort to localize the most relevant miRNAs identified in this study and their putative targets in the wound tissue by in situ hybridization or immunostaining (for the targets).1); however, it remains challenging to analyze miRNAs at the single-cell level. In the few published protocols for single-cell small RNA sequencing, extensive cell handing is needed; therefore, this technology has only been applied to a few cells and is not ready to be used at a scale for analyzing complex dynamics of tissue2. Therefore, bulk small RNA-seq is still a suitable approach for miRNA profiling currently, and our results of analyzing the unique clinical samples will facilitate the research of miRNAs in human skin wound healing.scRNA-seq has been used to analyze human skin and wound tissues  and epidermis  isolated from human skin and wound tissues by RNA-seq and qRT-PCR. These approaches allow us to detect many miRNAs and their putative target mRNAs simultaneously and gain an overview of the miR-mediated gene regulation in wound-edge keratinocytes. Also, we have localized one of the most relevant miRNAs identified in this study  and its putative target LGR4 expression  to wound2. Along the same line: The authors suggest a few transcription factors that could regulate the differentially expressed miRNAs . To strengthen the data, the authors should perform immunostaining for these transcription factors to determine where they are expressed in acute vs. chronic wounds.4et al.) and shown that GATA3 expression was detected throughout the epidermis of healthy skin but was completely absent in all VUs tested, confirming marked suppression of the main regulators of stem cell niche quiescence in non-healing edges of VUs4. KLF4 has been known for its crucial role in establishing skin barrier function5, and we have shown the downregulation of KLF4 expression in human VUs by microarray analysis in our previous study with an independent patient cohort6. As suggested by the reviewer, we are currently performing the immunostaining of KLF4 in human wound tissues, which will be completed for the revised version of the manuscript.We have performed immunofluorescence staining of GATA3 in our previous study  in keratinocytes.In the revised manuscript, by isolating keratinocytes  and epidermal compartments  from human wound tissues, we confirmed the expression and change of hsamiR-34a-5p and hsa-miR-424-5p in keratinocytes. We then examined the impacts of hsa-miR-34a5p and hsa-miR-424-5p on the major biological processes of keratinocytes during human skin wound healing, i.e., migration, inflammatory response, and proliferation .In addition, keratinocyte proliferation is not a major problem in chronic wounds – in fact, keratinocytes at the wound edge of chronic ulcers are frequently hyperproliferative . The major problem is the impaired migration . Therefore, the authors should check if some of these miRNAs, which are abnormally expressed in VUs, are expressed in keratinocytes at the wound edge and if they regulate keratinocyte migration in vitro.As suggested by the Reviewer, we have performed scratch wound healing assays, showing that simultaneous overexpression of miR-34a-5p and miR-424-5p delayed keratinocyte migration, whereas inhibition of endogenous miR-34a-5p or miR-424-5p enhanced keratinocyte motility . These new data suggest that miR-34a-5p and miR-424-5p, two miRs upregulated at the VU wound-edge keratinocytes , suppress keratinocyte migration, contributing to VU pathology.Finally, the functional data were all obtained after overexpression of miRNAs, while knock-down studies are missing. Even if there is functional redundancy among the miRNAs of interest, it would still be useful to confirm this and to know if the endogenous levels are sufficient to contribute to a certain cellular function.Besides overexpression of miRNAs, we also knock-down hsa-miR-34a-5p and hsa-miR-424-5p expression by transfection of miR-specific inhibitor to human primary keratinocytes in the revised manuscript . We found that inhibition of either hsa-miR-34a-5p or hsa-miR-424-5p enhances keratinocyte migration, suggesting that their functions are not redundant.Reviewer #3 (Recommendations for the authors):In this study, Zhuang Liu and colleagues performed paired-RNA sequencing in human skin tissues, including matched acute wounds and chronic non-healing venous ulcers. They identified 17 pathologically relevant miRNAs that exhibited abnormal VU expression and used qPCR to identify some miRNAs. The authors tried to find some targets which have the potential to accelerate wound healing, and this study has done good work on comprehensive analyses, the results are likely to be of interest to the scientific community. However, I have some serious concerns about this research.1. The scientific novelty is limited, several groups have done similar analyses using similar samples before. Also, the sample size is limited in this study so the statistical power is weak to make solid conclusions.We appreciate the constructive suggestions from the Reviewer. Although the strategy of paired miRNA and mRNA analysis has been used in other diseases , to the best of our knowledge, our study is the first attempt to apply this strategy to human wounds. We have performed a literature search on PubMed and summarized all the published microRNA profiling data of human skin wounds , which aOur study has at least three strengths that enhance its novelty: (1) The human in vivo wound healing model allows us to monitor the in vivo dynamics of gene expression in human wound tissues and epidermal cells across the healing process. (2) The acute wounds were matched with venous ulcer in donor age and wound site – which has been rarely achieved in previous studies, although both aging and body locations are important factors affecting gene expression. (3) Paired small and long RNA-seq allows more accurate integrative multi-omics analysis. Our work leads to a novel and easy-to-use resource that can be used to prioritize clinical-relevant miRNAs for more in-depth functional studies and to evaluate the clinical relevance of previous findings of miRNAs studied in non-human models – such efforts are highly demanded in research community.7. Pastar I. et al. have summarized published studies using human wound tissues and stated each study's sample size . Pastar I. et al. commented that 'Traditional clinical trials that are testing efficacy typically involve hundreds of patients. Most clinical research sample size collections are markedly smaller, which often challenges their value. Therefore, one has to make a fundamental distinction between clinical trial and clinical research that is not just semantic. As described above, clinical research that focuses on deciphering molecular and cellular mechanisms that inhibit healing use comprehensive multipronged strategies involving modern technologies, and the type of research is often limited by rather moderate research funds and an allotted amount of time to complete it. Therefore, it is unrealistic to expect that the sample size of clinical research studies should be compared to that of clinical trials that are testing efficacy of therapies and interventions.' 7Regarding the sample size, this is a common challenge in the studies with human wound tissuesin-silico analysis , our study generated transcriptomic data of human wound tissues with relatively high robustness and reproducibility. Such efforts add a novel dimension to the current knowledge mostly relying on non-human models, and would be a valuable resource for further mechanistic studies of miRs with high translational potential.After revision, we have analyzed tissue samples from 22 healthy donors  and 19 venous ulcer patients in this study, which sample size is bigger than most existing studies using human wound tissues. Together with rigorous research design  and fortified efforts in experimental validation of the 2. The conclusion is rather weak because there are no experiments to prove the candidate miRNAs have the function in wound healing at all. Ki67 staining and live-cell imaging only suggest that miR-34a and miR-424 affect keratinocyte cell proliferation.We agree with the reviewer that the functional validation of the identified miRs is a key, and we have dedicated major efforts to strengthen this aspect during the revision :In keratinocytes and epidermis isolated from human skin and wound tissues, we confirmed the keratinocyte expression of some VU-related miRs and their targets by RNA-seq and qPCR , which provide a rationale for the selection of human primary keratinocytes for functional validation. We next validated the miR-mediated gene regulatory network in keratinocytes or fibroblasts for 8 miRs by overexpressing each of them and performing microarray analysis with 33 samples . By comparing these in vitro identified miR targets with the VU-in vivo gene expression signature – we unravelled the most pathological-relevant miR targets  and their related biological processes . We then examined the impacts of hsa-miR-34a-5p, hsa-miR-424-5p, miR-516-5p and their combinations on the major biological processes of keratinocytes required for skin wound healing, including migration (scratch assays with live-cell imaging), proliferation , and inflammatory response (chemokine expression), using both gain- and loss-of-function approaches . Of note, many candidate miRNAs identified in this study have been shown to regulate wound healing in previous studies with in vitro and animal models , supporting the robustness and reproducibility of our profiling study. Together, our study provides an open resource for the research community that could evaluate the clinical relevance of previous findings of miRNAs studied in non-human models and prioritize the clinical-relevant miRNAs for more in-depth functional studies.3. The paper is poorly written. Many regulatory network analyses have been done but those results were not clearly explained and it is hard to follow how conclusions were made based on such results.Besides adding a significant amount of new experimental data (as mentioned above), we have substantively revised our manuscript by re-writing many parts of the text, as well as changing current figures and adding new figures and tables. We have also sent this manuscript to Enago for a professional language editing. We hope the updated version is clearer and easier to follow. We look forward to more suggestions to improve this manuscript further.4. Authors need to perform in vivo or in vitro assays to identify miRNA function.nd comment of Reviewer 3. In the updated manuscript, we have examined the impacts of hsa-miR-34a-5p, hsa-miR-424-5p, miR-516-5p and their combinations on the major biological processes of keratinocytes required for skin wound healing, including migration (scratch assays with live-cell imaging), proliferation , and inflammatory response (chemokine expression), using both gain- and loss-of-function approaches .Please see our response to the 25. Authors need to analyse previous data to confirm their findings.th paragraph in Discussion section). We found that most of the miRNAs that reportedly regulate wound healing also surfaced in our analysis, supporting the robustness and reproducibility of our profiling data and bioinformatics analysis.In response to this excellent suggestion, we summarised the reported miRNAs regulating skin wound healing (Table S3), analyzed their expression pattern in our datasets , and discussed the potential link between their expression and function patterns .Li, D.bioRxiv, 2020.2007.2014.202051 (2020).Nielsen, M.M. & Pedersen, J.S. miRNA activity inferred from single cell mRNA expression. , et al. MicroRNA-34 Family Enhances Wound Inflammation by Targeting LGR4. J Invest Dermatol 140, 465-476 e411 (2020).Wu, J., et al. Deregulation of epidermal stem cell niche contributes to pathogenesis of nonhealing venous ulcers. Wound Repair Regen 22, 220-227 (2014).Stojadinovic, O.Nature Genetics 22, 356-360 (1999).Segre, J.A., Bauer, C. & Fuchs, E. Klf4 is a transcription factor required for establishing the barrier function of the skin. , et al. Deregulation of keratinocyte differentiation and activation: a hallmark of venous ulcers. J Cell Mol Med 12, 2675-2690 (2008).Stojadinovic, O.Exp Dermatol 27, 551-562 (2018).Pastar, I., Wong, L.L., Egger, A.N. & Tomic-Canic, M. Descriptive vs mechanistic scientific approach to study wound healing and its inhibition: Is there a value of translational research involving human subjects?


Pseudomonas aeruginosa harboring Verona-integron–encoded metallo-β-lactamase were identified in a city in western Texas, USA. A faucet contaminated with the organism, identified through environmental sampling, in a specialty care room was the likely source for infection in a subset of patients.During June 2017–November 2019, a total 36 patients with carbapenem-resistant  Pseudomonas aeruginosa (VIM-CRPA) and other carbapenemase-producing organisms (CPOs) are emerging public health threats. CPOs cause infections that are often extensively drug-resistant and associated with substantial rates of illness and death. By colonizing faucet aerators and wastewater plumbing systems, CPOs can spread rapidly within healthcare facilities, including to patients  in the 6 months before VIM-CRPA was isolated, but patients did not have overlapping hospital stays or common procedures. We assessed water sources and plumbing in hospital A to identify potential VIM-CRPA reservoirs. VIM-CRPA is uncommon in the United States; <150 isolates are reported to CDC annually . The 2 clinical laboratories that served hospitals in city A began submitting all CRPA for mechanism testing in June 2017  and April 2018 . During July 2017–January 2019, a total of 36 patients with VIM-CRPA isolated from clinical cultures were identified from city laboratories; 21 (58%) had been admitted to hospital A for >1 night in the 6 months before culture collection. Beginning in June 2017, the Texas Department of State Health Services asked clinical laboratories to voluntarily submit clinical P. aeruginosa–colonized patients. On the basis of common exposure to room A, we considered the potential for an environmental reservoir.We reviewed medical records from hospital A of patients with VIM-CRPA. Median patient age was 57 (range 9–84) years; 57% were male. VIM-CRPA was isolated from wounds in 9 (43%) patients, respiratory sources in 7 (33%), and urine in 5 (24%). Most patients primarily received care on either medical/surgical units  or intensive care units . Among persons who had been hospitalized at hospital A in the previous 6 months, VIM-CRPA–positive specimens collected by hospital day 2 were classified as healthcare-associated, community-onset ; those collected on or after the third hospitalization day were considered hospital-onset . No patients overlapped on the same unit at the same time, but 3 were placed in the same room in ICU 1, room A, over a 2-year period. A single point-prevalence screening of patients in ICU 1 in October 2018 did not identify additional We conducted an environmental investigation focused on water supplies and other sites conducive to biofilm formation. We collected 85 samples from plumbing fixtures and environmental surfaces in patient care areas as well as from water intake and storage areas, and evaluated for the presence of VIM-CRPA . We identified VIM-CRPA in 3 sites in room A related to a sink: the drain, bulk water (1 L of tap water), and the interior surface of a faucet serving as the water source for the reverse osmosis unit of portable dialysis machines , panel AThe dialysis faucet in room A was installed in October 2017. After we identified VIM-CRPA from associated samples, it was disassembled, revealing a rubber gasket trapped in the gooseneck. We collected swab samples from the interior of the disassembled faucet, valve, and core; we then instilled water into the fixture, agitated it using a sonic device, and filtered it onto culture medium with 4 rinses from the faucet, connection supply line, core, and gasket. We cultured VIM-CRPA from the gasket, faucet, and water supply line , panel BWe considered that portable dialysis machines attachable to the contaminated faucet could spread VIM-CRPA to other dialysis hook-ups and sink drains where effluent reverse osmosis water was discharged. We cultured bulk water and swab samples from the sink in the biomedical room where dialysis machines were cleaned, the dialysis machine connector, and the tubing from the reverse osmosis unit of the dialysis machine connected to the contaminated faucet to test for CPOs, but identified no VIM-CRPA. After removing the dialysis faucet and adopting measures intended to mitigate spread of organisms from premise plumbing (We performed whole-genome sequencing on 33 VIM-CRPA clinical isolates from 20 patients at multiple locations in hospital A and 13 environmental isolates from room A (The mechanism by which the faucet became contaminated is unknown. We considered it might have been through the water supply, considering a recent report of carbapenemase-producing organisms, although not VIM-producing, in US municipal water systems (We could not ascertain relative contributions of the faucet interior and upper part of the sink drain toward patients acquiring VIM-CRPA. Although none of the patients underwent dialysis, the faucet was not labeled for dialysis use and may have been used for hand hygiene and other purposes. Infection prevention efforts at hospital A focused on improving sink hygiene, including removing patient care supplies from sink splash zones and regularly cleaning splash zones to prevent future transmission to patients from wastewater plumbing. During the 18-month period after the sink hygiene interventions began in October 2018, another 2 cases were identified, but in units unrelated to the specialty care unit. We identified VIM-CRPA from a dialysis faucet, in water from that faucet, and from the associated sink drain in an ICU room where VIM-CRPA infections of the same strain developed in 3 patients. Although healthcare facility wastewater plumbing and faucet aerators are well-documented reservoirs of CPOs, our findings highlight the importance of considering other plumbing sources as well. Pseudomonas aeruginosa in acute care hospital specialty care unit, Texas, USA. Additional information about investigation of carbapenemase-producing


Is there any difference in uveitis outcomes between patients with inactive disease given recommendations for early and deferred non–messenger RNA (mRNA) COVID-19 vaccination?In this open-label, randomized clinical trial involving 543 patients, recommendation for early vaccination resulted in an increased incidence of self-reported worsening of symptomatic uveitis compared with deferred vaccination, but no differences were observed in disease and visual prognosis at 3 months.These findings suggest that recommendation for early non-mRNA COVID-19 vaccination in patients with inactive uveitis may lead to subjective uveitis symptoms but no adverse effects on disease and visual prognosis at 3 months. This randomized clinical trial compares recommendations for early and deferred non–messenger RNA COVID-19 vaccination with respect to ocular outcomes among patients with inactive uveitis. Improper host response to COVID-19 vaccines could trigger immune-mediated adverse events. The question remains whether COVID-19 vaccination should be postponed until complete remission in patients with uveitis, a preexisting immune-related condition.To compare recommendations for early and deferred COVID-19 vaccination with respect to uveitis outcomes.This open-label, randomized clinical trial at a large, specialized teaching center for uveitis care in China enrolled unvaccinated patients with inactive uveitis between August 10, 2021, and February 22, 2022, with follow-up to June 6, 2022.Participants were randomly assigned to receive recommendation for early or deferred COVID-19 vaccination after complete remission of uveitis. Non–messenger RNA (non-mRNA) COVID-19 vaccines were available in China during the trial.The primary outcome was the time to symptomatic uveitis worsening during 3 months of follow-up. Secondary outcomes included uveitis activity and best-corrected visual acuity at 3 months.P = .01 by log-rank test). Changes in anterior chamber cells, vitreous haze, and best-corrected visual acuity from baseline to month 3 appeared similar in the 2 groups in the evaluable population after the month 3 in-person visit.Of the 543 participants , 262 were recommended for early vaccination and 281 for deferred vaccination. By month 3, 109 patients (41.6%) in the early group had been vaccinated compared with 14 (5.0%) in the deferred recommendation group. In the intention-to-treat population, the time to symptomatic uveitis worsening was shorter in the early group than in the deferred group (hazard ratio, 1.68 ; In this randomized clinical trial of patients with inactive uveitis, recommendation for early non-mRNA COVID-19 vaccination resulted in a higher incidence of self-reported symptomatic uveitis worsening with possible reporting bias compared with recommendation for deferred vaccination, but no adverse effects were observed in disease and visual prognosis at 3 months. These findings would be useful to guide the individual timing choices of non-mRNA COVID-19 vaccination in this clinically vulnerable population.ChiCTR2100049467Chinese Clinical Trial Registry:  Guidance from clinicians would be expected to have an impact on public health, such as affecting the coverage of COVID-19 immunization, the course of population immunity, and the protection of special groups. However, evidence supporting which timing treating clinicians should recommend is lacking. In this randomized clinical trial involving patients with inactive uveitis, we compare recommendations for early and deferred non–messenger RNA (mRNA) COVID-19 vaccination with respect to uveitis outcomes.Uveitis is an immune-related disorder characterized by vision-threatening intraocular inflammation, which has a wide association with systemic inflammatory conditions.10 The trial protocol  reporting guideline.This open-label randomized clinical trial was conducted at The First Affiliated Hospital of Chongqing Medical University, Chongqing, China. The hospital has set up a large, specialized teaching center for uveitis care and has treated 15 373 patients with uveitis across mainland China as of 2018.found in  was appr25 Uveitis inactivity was defined as an anterior chamber cell grade of 0.5+ or less, a vitreous haze grade of 0.5+ or less, and no active inflammatory choroidal or retinal vascular lesions in both eyes. Anterior chamber cells were graded according to the Standardization of Uveitis Nomenclature criteria on a range from 0 to 4, with higher values indicating greater severity of anterior segment inflammation of the eye.25 Vitreous haze was graded based on the National Eye Institute criteria adapted by the Standardization of Uveitis Nomenclature working group on a scale from 0 to 4, with higher values indicating worse activity of posterior segment inflammation.26 We excluded individuals in complete remission of uveitis, defined as an inactive condition lasting for 3 months or more after discontinuing all treatments.25 We also excluded those with any known vaccine contraindications, including a history of anaphylaxis to any component of the vaccine, a body temperature higher than 38.5°C, and pregnancy. All participants were Chinese, and ethnicity data were routinely collected from their resident identity cards .Eligible participants were 12 years or older, had not yet received COVID-19 vaccines, and were diagnosed with any form of uveitis in an inactive status.Participants underwent simple randomization to receive the recommendation for early or deferred COVID-19 vaccination in a 1:1 ratio. Random assignment sequence was computer-generated, and individual allocations were known by telephone from a designated staff who kept the sequence and had no involvement in other parts of the trial. Patients and treating clinicians were aware of the trial assignment.29 Patients’ COVID-19 vaccination status was verified via access to the Health Code System managed by the local health administration, where vaccine information about date, site, and type of each dose administered were recorded in real time.Patients in the early vaccination group were recommended to receive COVID-19 vaccines as soon as possible, while patients in the deferred vaccination recommendation group were recommended to receive COVID-19 vaccines after the complete remission of uveitis. Our clinic did not provide COVID-19 vaccination service. We assessed patients’ uveitis status and only gave the recommendation to patients. Non-mRNA COVID-19 vaccines were free of charge and available in the community. During the trial, 2 inactivated SARS-CoV-2 vaccines, CoronaVac (Sinovac Biotech) and BBIBP-CorV (Sinopharm), and 1 protein subunit vaccine, ZF2001 , were available under emergency use authorization among people 12 years or older in China.Routine follow-up encounters occurred every month by telephone, and an in-person clinic visit was scheduled at month 3. At the telephone follow-up encounters, patients were asked about adherence to the recommendation, reasons for nonadherence, experiences of systemic adverse events, and uveitis symptoms, including the presence and duration of new-onset eye redness, eye pain, decreased vision, light sensitivity, and floaters. These queries were administered in a standardized telephone interview from the trial call center. Information on missed encounters could be completed in subsequent encounters provided that participants were brought back. In addition, all participants were repeatedly encouraged to follow the assigned vaccination recommendation during the telephone call. If the aforementioned symptoms or systemic adverse events were reported, patients would be instructed to return to the study site early or visit local hospitals for further clinical evaluation. We asked patients to provide their medical records from their local hospitals if any. Patients would receive best medical judgment and proper treatment, and intervention with trial recommendations would be terminated. At the month 3 in-person visit, patients underwent additional clinical evaluations, including slitlamp biomicroscopy, ophthalmoscopy, measurement of best-corrected visual acuity (BCVA), and other auxiliary examinations according to clinical needs.The primary outcome was the time to worsening of symptomatic uveitis, defined by 1 of the following new-onset symptoms occurring in at least 1 eye and lasting for at least 2 days: eye redness, eye pain, decreased vision, light sensitivity, or floaters, as confirmed by blinded review by an adjudication committee. The adjudication committee consisted of 3 independent clinicians, and the outcome was achieved with a majority of 2. Key secondary outcomes included proportion of COVID-19 vaccination, proportion of adherence to recommendation, proportion of each worsening symptom, proportion of 2-grade increase in anterior chamber cells from baseline to month 3, proportion of 2-grade increase in vitreous haze from baseline to month 3, change in BCVA from baseline to month 3, and incidence of systemic adverse events. The original protocol also included secondary outcomes concerning clinically confirmed uveitis worsening, hospitalization, and treatment, which were determined based on in-person clinical evaluation when patients returned to the study site or visited local hospitals with symptomatic uveitis worsening. During the execution of the trial, most cases with worsening symptoms did not seek medical services and assessment at our study center, nor did they provide medical records to show their local hospital visits, possibly due to their mild diseases, self-care, remote living, or COVID-19 travel restrictions. Therefore, these secondary outcomes were not reported in terms of data unavailability.P = .05. The target of enrollment was not met because of the absence of eligible unvaccinated patients in the later period of trial. A decision was made to suspend enrollment on February 28, 2022, as the full vaccination rate improved to 86.6% in China.30Assuming that the cumulative incidence would be 10% in the deferred vaccination group during the month 3 follow-up and the proportion dropping out in both groups would be 15%, we estimated that an overall sample of 1314 participants would provide 80% power to detect a hazard ratio (HR) for early vaccination recommendation of 1.50 for the primary outcome compared with deferred vaccination recommendation, at a 2-sided significance level of 32 We performed 1 prespecified sensitivity analysis with multiple imputation for nonresponse and several post hoc analyses , and R, version 3.5.0 .Binary outcomes are presented as numbers with proportions. We calculated 95% CIs for between-group differences in proportions using the Wilson score method.A total of 543 patients were formally assessed for eligibility and enrolled. The median age was 35  years; 304 patients (56.0%) were female and 239 (44.0%) were male. Two hundred sixty-two patients were recommended for early COVID-19 vaccination and 281 for deferred vaccination. The baseline characteristics were similar between 2 groups . Before By month 3, 109 patients (41.6%) of the patients assigned to early vaccination recommendation had received at least 1 dose of non-mRNA COVID-19 vaccine compared with 14 (5.0%) of those assigned to a deferred vaccination recommendation . The fulP = .01 by the log-rank test) (Symptomatic uveitis worsening occurred in 51 patients (19.5%) recommended for early COVID-19 vaccination and in 34 (12.1%) recommended for deferred COVID-19 vaccination . In the intention-to-treat analysis, the time to symptomatic uveitis worsening was shorter in the early vaccination group than in the deferred vaccination group  . The finnk test) , prespecnk test) , sensitink test) , and as-nk test) . The hignk test) . Among tnk test) , the priPatients in the early vaccination group more frequently reported new onset of floaters as one of the reasons for symptomatic uveitis worsening . The ocular conditions, including changes in anterior chamber cells, vitreous haze, and BCVA from baseline to month 3, appeared similar between the 2 groups in the month 3 in-person evaluable population , even afThis open-label, randomized clinical trial showed that recommendation for early non-mRNA COVID-19 vaccination to patients with inactive uveitis led to earlier self-reported symptomatic uveitis worsening than the recommendation for deferred vaccination. We found no evidence that uveitis activity and visual acuity at 3 months differed substantially according to recommendation type.21 and in particular provides randomized evidence for such an effect. However, our findings suggest that COVID-19 vaccination might temporarily affect the symptoms but would not modify the disease and visual prognosis at 3 months. A reasonable explanation may be that uveitis worsening after COVID-19 vaccination appears to be acute and transient, usually responds to corticosteroid therapy, and may be quickly controlled.37This trial extends prior observations linking COVID-19 vaccination with an increased risk of newly active or worsening uveitisAs a subjective measure, the primary outcome could at least partially reflect subjective feelings about the disease condition and certain levels of quality of life. However, participants were aware of their vaccination status, and those concerned about vaccination could have an exacerbated experience of their symptoms. Our trial detected the significant effects of recommending early COVID-19 vaccination on patient’s symptoms. However, the between-group mean difference  in incidence of symptomatic uveitis worsening was subtle, and it would be possible to have reporting bias from people who received COVID-19 vaccines as large as that would negate the currently reported significant difference in the primary outcome. More importantly, no adverse effects were observed in disease and visual prognosis at 3 months. The inconsistency between the primary and secondary outcomes also suggested the possibility of reporting bias incurred by psychological factors related to subjective assessment of the primary outcome. These findings highlight that, in clinical practice, more psychological reassurance should be provided to patients before or after COVID-19 vaccination, especially those worried about the adverse effects of COVID-19 vaccine. The timing choices for COVID-19 vaccination in inactive uveitis may be primarily based on the individual risk of SARS-CoV-2 infection and severe COVID-19 illness as well as patient’s expectations and preferences rather than on anticipated differences in the disease and visual prognosis of uveitis.38 However, such a short-term follow-up compromised the representativeness of long-term disease and visual prognosis. In addition, due to the vaccine availability in China, inactivated vaccines made up 95.9% of those administered, and mRNA vaccines were not included in the trial, which might limit the generalizability of the findings.This study has some limitations. First, interpretation of the outcomes was limited by the open nature of the trial, especially when interpreting results for a subjective outcome in the context of COVID-19 vaccination. Moreover, all symptoms were based on self-report, and we lacked data regarding clinical evaluation to confirm disease worsening. The possibility that the greater reporting bias in patients receiving COVID-19 vaccines may lead to the slightly higher incidence rather than the vaccine increasing inflammation cannot be ruled out. Second, our sample size fell short of expectations due to the lack of eligible unvaccinated patients in China, although this sample size was powered to detect the difference in the primary outcome. Third, a number of patients were not included in the month 3 in-person evaluable population. Nevertheless, this population was only related to several secondary outcomes, in which data showed materially small between-group differences in the point estimate. Moreover, results were similar after accounting for data missing with inverse probability weighting. Fourth, we could not precisely define which uveitis worsening was caused by COVID-19 vaccination. Instead, we only examined the excess risk of symptomatic worsening in the early vaccination group compared with the deferred one, with randomization leading to the balanced background risk between the 2 groups. To mitigate the interference of other factors unrelated to vaccination, we chose a 3-month observation period for vaccine uptake and uveitis worsening because a later onset of uveitis flare was less likely to be related to COVID-19 vaccination.In this randomized clinical trial of patients with inactive uveitis, recommendation for early non-mRNA COVID-19 vaccination resulted in a higher incidence of self-reported symptomatic uveitis worsening with possible reporting bias compared with recommendation for deferred vaccination, but no adverse effects were observed in disease and visual prognosis at 3 months. These findings would be useful to guide the individual timing choices of non-mRNA COVID-19 vaccination in this clinically vulnerable population.


Moreover, brequinar facilitated myeloid maturation and inhibited immune-suppressive features in human bone marrow culture systems. These findings advance the concept of MDSC differentiation therapy in immuno-oncology.While immune checkpoint inhibitors (ICIs) have transformed the therapeutic landscape in oncology, they are effective in select subsets of patients. Efficacy may be limited by tumor-driven immune suppression, of which 1 key mechanism is the development of myeloid-derived suppressor cells (MDSCs). A fundamental gap in MDSC therapeutics is the lack of approaches that target MDSC biogenesis. We hypothesized that targeting MDSC biogenesis would mitigate MDSC burden and bolster tumor responses to ICIs. We tested a class of agents, dihydroorotate dehydrogenase (DHODH) inhibitors, that have been previously shown to restore the terminal differentiation of leukemic myeloid progenitors. DHODH inhibitors have demonstrated preclinical safety and are under clinical study for hematologic malignancies. Using mouse models of mammary cancer that elicit robust MDSC responses, we demonstrated that the DHODH inhibitor brequinar (a) suppressed MDSC production from early-stage myeloid progenitors, which was accompanied by enhanced myeloid maturation; (b) augmented the antitumor and antimetastatic activities of programmed cell death 1–based (PD-1–based) ICI therapy in ICI-resistant mammary cancer models; and (c) acted in concert with PD-1 blockade through modulation of MDSC and CD8 Howevfunction –7. A majfunction .MDSCs have received considerable attention as a dominant cellular constituent of an immune-suppressive network , 3. MDSCWe and others have demonstrated that MDSCs emerge from hematopoietic progenitors in the BM, particularly early myeloid progenitors such as granulocyte-monocyte progenitors (GMPs) –22. We hSeveral groups have demonstrated that inhibiting the enzyme dihydroorotate dehydrogenase (DHODH), a rate-limiting step in pyrimidine biosynthesis, unexpectedly and profoundly enforces the terminal differentiation of AML cells, mainly toward neutrophils –28. DHODOne such DHODH inhibitor is brequinar (BRQ). A study by Sykes et al. demonstrated that BRQ, an orally available DHODH inhibitor, had potent in vitro and in vivo anti-AML activity . In thathttps://doi.org/10.1172/JCI158661DS1). Since pyrimidine synthesis is the specific target of BRQ  synthesis pathway in GMPs from tumor-bearing mice compared with GMPs from non-tumor-bearing (NTB) controls  subset (– neutrophils are associated with tumor progression (+Ly6ChiLy6G–), with or without BRQ or Lef treatment  subset . Mature gression . ConsistARG1), NOS2, PDL1, TGFB1, and VEGFA   , and Arg values) . As withWe extended our studies to test whether BRQ exerted antitumor activity and/or reduced MDSC development or function in vivo. BRQ was chosen over Lef for the in vivo studies, given its higher potency, aqueous solubility, and relative ease of i.p. administration , 45. We + cells within splenic PMN-MDSCs from BRQ-treated mice compared with the PMN-MDSCs from vehicle control–treated mice. Similarly, we observed increased levels of CD101 expression, as measured by MFI values . Interestingly, during this analysis, we observed an increase in the MFI values for the Ly6G and Ly6C markers on the PMN-MDSCs , data in this model are reported as the percentage of mice with evidence of metastases (Since both tumor models can metastasize to the lung (a common site for human TNBC spread), we examined the impact of mono- or combination therapy on spontaneous lung metastasis. The lungs from either 4T1- or E0771.ML-bearing mice were histologically quantified for metastatic lesions. Interestingly, we observed that BRQ treatment, either as a single agent or combined with anti–PD-1 mAb, significantly decreased lung metastasis , whereastastases . Togethetastases . As with+ T cell dependent  on target, (b) MDSC dependent, and (c) CD8ependent ? First, ependent , 45. Theependent , B and F+Gr-1+ cells) isolated from the spleens of –/–Irf8 mice, a model that generates high numbers of suppressive splenic MDSCs, even in the absence of tumor implantation. This observation was derived from our earlier work demonstrating that IRF8 is an integral negative regulator of MDSC biogenesis in the BM . 4T1-bearing mice were then treated with combination therapy. The depletion of CD8+ T cells significantly negated the effects of the combination therapy and restored tumor growth  compartment of the TME, priming cells to respond to ICI therapy.We next investigated the presence and activation status of CD8 the TME , buildin the TME . As with control . Howeverand ICOS . The incer Ki-67 . These d+ granulocytes in the BM, but no significant effect on BM monocyte or myeloid DC populations  in 4T1-bearing mice treated with vehicle or BRQ using the same schedule used for the TME analysis , A and Bulations , C and Dulations .Given that BRQ had a modest quantitative effect on GMPs, we investigated whether BRQ had a qualitative effect on GMPs by testing whether BRQ inhibited the ability of GMPs to differentiate into MDSCs. GMPs sorted from the BM of 4T1-bearing mice were treated with vehicle or BRQ and cultured in vitro with G-CSF to drive differentiation toward PMN-MDSCs (GMP-derived MDSCs) . Treatme+ BM cells isolated from NTB mice and 4T1-bearing mice treated with vehicle or BRQ  with NTB mice (NTB-GMPs) . We. We+ BM TB-GMPs) . We obseTB-GMPs)  and provTo identify potential molecular mechanisms of action for BRQ, we focused on pathways where BRQ reversed tumor-driven effects on the GMP population (based on the Veh-GMP versus NTB GMP comparison) and that were known to be involved in MDSC biology or hematopoiesis . OverallCHOP and DNAJB11 showed decreased expression in MDSCs within the TME of 4T1-bearing mice treated with BRQ compared with mice treated with vehicle (CDC42 was increased in MDSCs within the TME of 4T1-bearing mice treated with BRQ. These results were consistent with the single-cell RNA-Seq (scRNA-Seq) data and suggest that the effect of BRQ on gene expression in the BM progenitors was maintained in MDSCs within the TME.We also examined pathways that were upregulated in BRQ-GMPs compared with Veh-GMPs. Here, we unexpectedly found that pathways regulated by members of the RhoGTPase family  were upregulated in BRQ-GMPs compared with Veh-GMPs . RhoGTPa vehicle . ConversTo compare the differentiation trajectories of BM progenitors in 4T1-bearing mice treated with BRQ compared with those treated with vehicle, we performed pseudotime analysis on the scRNA-Seq data sets. We observed no marked difference in the trajectories between the treatment conditions . We thenhi and the latter population being SSClo  suppressed pathways, such as the UPR, that are known inducers of MDSC activity in the BM, potentially revealing mechanisms of MDSC biogenesis. We propose a model in which the ability of BRQ to enhance ICI efficacy can be mediated through an immune mechanism by which BRQ suppresses MDSC function that in turn enables the activation of CD8+ T cells  inhibited the immune-suppressive activity of murine MDSCs, which was accompanied by enhanced myeloid maturation, especially affecting the PMN-MDSC subset; (b) showed significant antitumor and antimetastatic activities when combined with the ICI anti–PD-1 mAb in ICI-resistant mouse mammary TNBC models; (c) acted in the BM and reversed tumor-induced MDSC biogenesis originating from early myeloid progenitors; (d) boosted the antitumor activity of anti–PD-1 mAb via MDSC- and CD8 T cells , B and C–/–Irf8 MDSCs as a model system) was able to reverse the antitumor efficacy of the BRQ plus anti–PD-1 mAb combination. Immunophenotyping showed that BRQ treatment did not have profound effects on the numbers of MDSCs, suggesting that the primary effect of BRQ was qualitative as opposed to quantitative. Thus, we hypothesized that BRQ treatment reduces the MDSC burden through a promotion of maturation rather than elimination, which is supported by our observation that BRQ treatment resulted in signatures associated with mature neutrophil function in BM progenitors. This hypothesis is strengthened by our findings that BRQ treatment increased the frequency of segmented neutrophils (a morphologic marker of maturation) and upregulated the expression of CD101, a recently discovered marker of mature granulocytes that is associated with reduced protumorigenic function  were unaffected by BRQ treatment, also allay potential concerns that treatment with BRQ, through its known mechanism of action as an inhibitor of pyrimidine synthesis, could suppress proliferation or antitumor activity of CD8+ T cells.Our observation that the adoptive transfer of MDSCs was able to inhibit the effects of the combined BRQ and anti–PD-1 mAb regimen also supports the idea of a functional link between the reduction of MDSC function and restoration of a functional CD8+ T cell response in the context of our preclinical cancer models, DHODH inhibitors are being used clinically in the context of autoimmune disorders to dampen autoimmune T cell activity. For example, 2 different DHODH inhibitors are approved for the treatment of rheumatoid arthritis (Lef) and multiple sclerosis (MS) (teriflunomide)  –69. Withunomide)  showed aAlthough single-agent BRQ did not inhibit primary tumor growth in our preclinical models, single-agent BRQ was able to inhibit lung metastases. This observation is consistent with prior studies from our group and others demonstrating that a reduction of MDSCs has a significant effect on metastatic, but not necessarily primary, tumor growth , 70, 71.The ability of BRQ to suppress MDSC biogenesis at the progenitor stage may result from these pathways working in combination. Thus, the ability of BRQ to upregulate pathways involved in hematopoiesis  may work in concert with the downregulation of pathways that promote MDSC development and function –63. The ICIs have shown clinical benefits across multiple types of cancer, but these benefits have been largely limited to a subset of patients. Breast cancer, including TNBC, has demonstrated significant resistance to ICIs. The preclinical results reported here suggest that the use of DHODH inhibitors (such as BRQ) can overcome this resistance in part through the inhibition of MDSC biogenesis. The applicability of this approach to the treatment of patients is supported by our observation that BRQ hindered the in vitro development of human MDSC-like populations. However, it is important to point out that these human studies made use of BM biospecimens from healthy individuals and not from patients with cancer. Thus, this work served as a proof of concept to demonstrate that BRQ can act on human myeloid cells and that future studies are warranted to investigate these objectives in detail in patients with cancer. The extensive experience in the clinical use of BRQ gained from prior and current clinical trials  suggests that this approach is feasible , 75–77. Detailed methods are provided in the The RNA-Seq data shown in All studies involving mice were approved under protocols 1108M and 1117M and conducted in accordance with the IACUC of Roswell Park Comprehensive Cancer Center. All human BM specimens were deidentified, and the studies using these specimens were approved under protocol BDR 134520 as nonhuman subject research and conducted in accordance with the IRB of Roswell Park Comprehensive Cancer Center.SHC, MJN, and SIA supervised and designed the study. SHC, RAZ, JP, AMA, EDK, BM, and PKS performed the experiments. SHC, RAZ, AMA, EDK, JW, MJN, and SIA analyzed the data. JB and PKS provided essential reagents. SHC, AMA, JW, MJN, HY, and SIA performed and verified statistical analyses. SHC, AMA, PKS, JW, MJN, and SIA wrote the manuscript. JB, MO, and DBS provided feedback and helped edit the manuscript. SHC is listed first as co–first author because of the larger role he played in the original conceptualization. RAZ is listed first as co-second author because of the larger role he played in data analysis.


How effective is vaccination against COVID-19 after recovery from prior SARS-CoV-2 infection?In this cohort study of more than 95 000 Rhode Island residents from March 2020 to December 2021, including residents and employees of long-term congregate care (LTCC) facilities, completion of the primary vaccination series after recovery from COVID-19 was associated with 49% protection from reinfection among LTCC residents, 47% protection among LTCC employees, and 62% protection in the general population during periods when wild type, Alpha, and Delta strains of SARS-CoV-2 were predominant.These findings suggest that among people who have recovered from COVID-19, subsequent completion of the primary vaccination series reduced the risk of reinfection by approximately half. This cohort study assesses the association of vaccination with protection against SARS-CoV-2 reinfection among long-term congregate care residents and employees and the general population previously infected with SARS-CoV-2 in Rhode Island. The benefit of vaccination for preventing reinfection among individuals who have been previously infected with SARS-CoV-2 is largely unknown.To obtain population-based estimates of the probability of SARS-CoV-2 reinfection and the effectiveness associated with vaccination after recovery from COVID-19.This cohort study used Rhode Island statewide surveillance data from March 1, 2020, to December 9, 2021, on COVID-19 vaccinations, laboratory-confirmed cases, hospitalizations, and fatalities to conduct a population-based, retrospective study during periods when wild type, Alpha, and Delta strains of SARS-CoV-2 were predominant. Participants included Rhode Island residents aged 12 years and older who were previously diagnosed with COVID-19 and unvaccinated at the time of first infection, stratified into 3 subpopulations: long-term congregate care (LTCC) residents, LTCC employees, and the general population . Data were analyzed from October 2021 to January 2022.Completion of the primary vaccination series, defined as 14 days after the second dose of an mRNA vaccine or 1 dose of vector virus vaccine.The main outcome was SARS-CoV-2 reinfection, defined as a laboratory-confirmed positive result on a polymerase chain reaction (PCR) or antigen test at least 90 days after the first laboratory-confirmed positive result on a PCR or antigen test.Overall, 3124 LTCC residents , 2877 LTCC employees , and 94 516 members of the general population  met eligibility criteria. Probability of reinfection at 9 months for those who remained unvaccinated after recovery from prior COVID-19 was 13.0%  among LTCC residents, 10.0%  among LTCC employees, and 1.9%  among the general population. Completion of the primary vaccination series after infection was associated with 49%  protection among LTCC residents, 47%  protection among LTCC employees, and 62%  protection in the general population against reinfection, adjusting for potential sociodemographic and clinical confounders and temporal variation in infection rates.These findings suggest that risk of SARS-CoV-2 reinfection after recovery from COVID-19 was relatively high among individuals who remained unvaccinated. Vaccination after recovery from COVID-19 was associated with reducing risk of reinfection by approximately half. A prior infection of SARS-CoV-2 confers some immunity to individuals who are not vaccinated,4 but this natural immunity wanes over time. Additionally, it has been shown that the JNJ-78436735 (Janssen), mRNA-1273 (Moderna), and BNT162b2 (Pfizer-BioNTech) vaccines offer substantial protection against COVID-19 infection and remain effective against newer variants.7Current evidence suggests that individuals who experience a SARS-CoV-2 infection are susceptible to reinfection.8 Similar results have been reported in larger cohort studies in Israel and United Kingdom.10 Our primary goal was to quantify the effectiveness associated with vaccination against reinfection among individuals who had been previously infected with SARS-CoV-2 prior to vaccination. Secondary goals were to estimate the probability of reinfection at 3, 6, and 9 months after recovery from primary infection and to assess the associations of measurable risk factors with reinfection. Our analysis has particular relevance to the ongoing discussion about vaccination requirements for individuals who have recovered from COVID-19.A prior case-control study of Kentucky residents found that individuals who had been previously infected with completion of the primary vaccination series (CPVS) were better protected against reinfection than individuals who remained unvaccinated.STROBE) reporting guideline for observational studies.This cohort study was classified as exempt from review and informed consent by the Rhode Island Department of Health (RIDOH) institutional review board because we used existing data that were deidentified. This study follows the Strengthening the Reporting of Observational Studies in Epidemiology  residents of long-term congregate care (LTCC) settings, which included nursing homes and assisted living facilities, (2) employees of LTCC settings, and (3) the general population . LTCC residents were analyzed separately owing to the high risk of morbidity and mortality in this population, and LTCC employees were analyzed separately because they worked for extended periods of time in these high-transmission environments. We did not include group home residents or employees , other congregate setting residents or employees , and individuals with unknown congregate status in this study. Other congregate settings included predominantly correctional facilities. After exclusions, our analysis sample included 3124 LTCC residents, 2877 LTCC employees, and 94 516 members of the general population.The study database was assembled by linking data from 4 surveillance systems maintained by RIDOH: COVID-19 vaccinations, cases, hospitalizations, and fatalities. COVID-19 vaccination doses administered were identified based on the Rhode Island Child and Adult Immunization Registry (RICAIR) data system. In Rhode Island, positive polymerase chain reaction (PCR) and antigen (prior to widespread availability of home-based rapid antigen tests) tests are reported to RIDOH, and new cases are investigated to collect health and demographic information. Using RIDOH’s statewide Hospital Incident Reporting System, hospitals report to RIDOH patients who are admitted to an inpatient bed and are being placed on COVID-19 precautions. RIDOH derives data for laboratory-confirmed COVID-19-associated fatalities from 3 sources: the Office of the State Medical Examiners, vital records death certificate data, and the Hospital Incident Reporting System.11 starting from the date of the positive test results for the first infection. After the 90-day recovery period, individuals who have remained unvaccinated were considered eligible to enter the risk cohort for COVID-19 reinfection. Individuals were then followed from entry time to time of second infection. Individuals who died after entry or did not experience a second infection by December 9, 2021, were considered censored . The tier classification is based on community characteristics, such as population density, sociodemographic characteristics, and COVID-19 burden. Within each model, the interaction of age and time of entry into the risk cohort for reinfection were captured using cubic splines with df = 4. Cubic splines were used to capture nonlinear associations. Of note, case investigations for residents of LTCC settings generally occurred through a single point of contact at each facility, rather than individual discussions with residents who were diagnosed with COVID-19, leading to heterogeneity in the level of detail on sociodemographic and clinical characteristics. For each of the 3 subpopulations, we estimated vaccine effectiveness (VE) associated with preventing COVID-19 reinfection as VE = 1 – AHR,15 where AHR is the adjusted hazard ratio estimated by the proportional hazards model.We used the Cox proportional hazards regression model to estimate vaccine effectiveness, adjusting for age (years), sex assigned at birth , race and ethnicity , previous hospitalization during the first infection, symptom status during the first infection , and time of entry into the risk cohort for reinfection relative to the beginning of the pandemic in Rhode Island (in weeks). Race and ethnicity were included because of previously documented disparities in infection risk.Our sample included all cases from March 2020 to December 2021, meaning that the VE estimates partially depend on data from a period during which vaccination was not yet available. To assess the robustness of our findings to sample selection, we conducted a supplementary sensitivity analysis in which we restricted to individuals whose first infection occurred during or after December 2020, when vaccines first became available.Statistical analyses were conducted with R statistical software version 4.1.2  using the ‘survival’ package for Kaplan-Meier and Cox proportional hazards model. Data were analyzed from October 2021 to January 2022.Between March 1, 2020, and December 9, 2021, 100 517 Rhode Island residents aged 12 years and older had recovered from SARS-COV-2 infection and remained unvaccinated at the end of their 90-day recovery period. During the subsequent study follow-up period, a total of 56 888 people (56.6%) received at least 1 dose of a vaccine , including 2077 of 3124 LTCC residents (66.4%), 2133 of 2877 LTCC employees (74.1%), and 52 687 of 94 516 members of the general population (55.7%). Sociodemographic and clinical characteristics of the subpopulations are shown in The cumulative probabilities of reinfection as a function of time since recovery from the primary infection for each subpopulation are shown in eTable 1 in the The findings of the proportional hazards regression model fit to each subpopulation are presented in eTable 3 in the The proportional hazards model was also used to identify factors associated with risk of reinfection . The findings of our statewide cohort study suggest that vaccination after recovery from prior COVID-19 was associated with substantial benefit for preventing subsequent reinfection, with risk reduced by nearly half for all subpopulations. Among LTCC residents and employees, for whom the probability of reinfection at 9 months after primary infection among those who remained unvaccinated was at least 5-fold greater than in the general population (10%-13% vs 2%), this is a particularly notable finding.8 using surveillance data from Kentucky, which found that being unvaccinated was associated with approximately 2-fold higher odds of reinfection compared with being fully vaccinated. A cohort study by Hammerman et al9 using electronic medical record data from Israel estimated VE among people who had recovered from prior COVID-19 to be higher (approximately 60% for those aged ≥65 years and 82% for those aged 16-64 years). Another study of health care workers from the United Kingdom found that infection-acquired immunity waned after a year but was consistently higher than 90% for individuals who were subsequently vaccinated.10 Given that most hospitalizations and fatalities in our cohort were associated with the first infections, we were not able to conduct formal statistical inference about VE associated with preventing more severe reinfections; however, the observed incidence rates of reinfection-associated COVID-19 hospitalization and death were lower among individuals who were vaccinated compared with individuals who were unvaccinated.Our findings are consistent with a case-control study by Cavenaugh et alOur findings highlight the continued importance of vaccination, including after recovery from prior COVID-19, among LTCC residents and employees, who are likely at increased risk of reinfection owing to their older age and/or greater exposure to the virus in LTCC settings. Employees and some residents or clients of other settings with greater COVID-19 exposure  may similarly experience high risk of reinfection and benefit from vaccination after recovery from COVID-19.AHR).Our study has several key strengths. The database contains statewide information on positive PCR test results, most positive antigen test results, and nearly all vaccine doses administered in Rhode Island during the study period. The database also has information about whether the individual with positive test results is a resident or employee of a LTCC, a critical subpopulation with respect to SARS-COV-2 infection risk. A second major strength is the prospective nature of the analysis: we were able to follow individuals prospectively in time and calculate VE in terms of relative incidence . We may have incomplete information on reinfections identified through home-based antigen testing, but this is expected to be minimal because home-based testing was uncommon until the final weeks of our analysis period. Additionally, our VE estimates could be biased to the extent that testing propensity differs between individuals who are vaccinated and those who are not; for example, individuals who have been previously vaccinated may be less likely to seek testing for a mild infection, leading to an overestimate of VE. Another limitation is that RIDOH’s immunization database does not include most vaccination doses administered by federal entities or out-of-state, which may lead to some misclassification of vaccination status. However, we expect this bias to be conservative, as erroneously classifying some individuals who had been vaccinated as unvaccinated should attenuate our VE estimates. Additionally, children ages 5 to 11 years were not included in our study because they were not eligible for vaccination until the final month of the analysis period. This is an important subgroup for future research. Furthermore, a substantial percentage of LTCC residents were missing data on race and ethnicity (65%) and symptom status during the first infection (80%), owing to heterogeneity in the level of detail reported by each facility, which limited our ability to account for potential confounding by these characteristics.The findings of this cohort study suggest that risk of reinfection after recovery from COVID-19 was relatively high among individuals who remained unvaccinated, including the general population and LTCC residents and employees. Vaccination after recovery from COVID-19 was associated with substantial benefit, reducing risk of reinfection by approximately half. Individuals who have recovered from COVID-19 and remain unvaccinated should be encouraged to complete vaccinations, as they are eligible, to reduce their risk of reinfection.