Inborn errors of immunity (IEIs) are a group of inherited disorders caused by mutations in the protein-coding genes involved in innate and/or adaptive immunity. Hematopoietic stem cell transplantation (HSCT) is a mainstay definitive therapy for many severe IEIs. However, the lack of HLA-matched donors increases the risk of developing severe immunological complications. Gene therapy provides long-term clinical benefits and could be an attractive therapeutic strategy for IEIs. In this review, we describe the development and evolution of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) gene-editing systems, including double-strand break (DSB)-based gene editing and DSB-free base editing or prime editing systems. Here, we discuss the advances in and issues associated with CRISPR/Cas gene editing tools and their potential as therapeutic alternatives for IEIs. We also highlight the progress of preclinical studies for the treatment of human genetic diseases, including IEIs, using CRISR/Cas and ongoing clinical trials based on this versatile technology. IEIs have been considered rare diseases with an overall estimated prevalence of approximately 1/10,000 to 1/50,000; however, along with the continued discovery of novel IEIs, the prevalence of these diseases appears to have been underestimated by 10-fold, with IEIs actually affecting more than 1 in 1000 to 1 in 5000 births . CurrentPatients bearing a disease-causing mutation that disables the immune system usually suffering from life-threatening infectious disease, early diagnosis and proper management are of paramount importance to prevent the development of complications, especially for those with severe complications. The development of high throughput sequencing technologies and advances in diagnostic methods have resulted in a growing number of IEIs could be identified in pediatric age . The trein situ correction of the pathogenic mutations is needed and gene editing systems provide useful tools to revise GOF mutations. Here, we describe advances in precise genome editing using the lustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) gene editing system and reveal the great potential of CRISPR/Cas-based gene therapy for the treatment of IEIs. In this review, we focus on DNA editing using CRISPR/Cas; thus, RNA editing will not be included.Gene therapy for IEIs has been developed to provide an autologous HSCT option by adding a normal copy of the responsible disease-related gene or correcting the mutation in the patient’s own HSCs. For loss-of-function mutations, viral vector-mediated gene augmentation, which provides the correct copy of defective genes, can be used for gene therapy. Gamma retroviral vectors were used as delivery vectors in early clinical trials for gene therapy of IEIs, including ADA-SCID, X-linked SCID (X-SCID), Wiskott–Aldrich syndrome (WAS), and X-linked chronic granulomatous disease (X-CGD) –27. To p2Targeted genome editing introduces stable genetic modifications at sites of interest using engineered nucleases. Methods for eukaryotic genomic manipulation have evolved rapidly over the past decade. Programmable nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas, are three popular nucleases that have been engineered to induce double-strand breaks (DSBs) at specific endogenous gene loci to enable targeted genome manipulation. DSBs can be repaired by the error-prone non-homologous end joining (NHEJ) pathway, which often results in nucleotide insertions or deletions (indels), thus inactivating the gene, or by the homology-directed repair (HDR) pathway, which enables gene correction or insertion of a transgene when a homologous donor DNA template is provided Figure 12.1via a single RuvC catalytic domain, and it has been widely used for genome editing applications  in various cell types. The editing efficiency of BEs was continuously increased through linker selection, additional UGI appending, nuclear localization sequence modification, codon optimization, and Cas protein or deaminase engineering –78. For 2.2.2in vivo , thus silencing gene expression  have emerged as a genome-editing approach to enable all 12 types of base conversions, base pair insertions or deletions, and even combinations without the generation of DSBs in a targeted way . PEs are33.1CRISPR/Cas systems enable targeted genome manipulation in a programmable and efficient manner and have remarkable potential for correcting mutations underlying human genetic diseases. Recent progress in the development of advanced gene editing approaches with high activity and precision has paved the way for theories to enter practice.HBG1/2) could ameliorate the severe β-globin disorders. Diminishing the expression of the γ-globin transcriptional repressor or attenuating γ-globin-to-β-globin switching through CRISPR/Cas9-mediated gene disruption resulted in increased γ-globin expression, which ameliorated the pathogenic phenotype (−/−mice (in vivo delivery of Cas9:gRNA RNP complex in the humanized transmembrane channel-like 1 (Tmc1) Beethoven (Bth) mouse model ameliorated the pathogenic phenotype (CRISPR/Cas-mediated gene disruption may be useful for removing disease-causing replicated fragments or GOF mutants. Huntington’s disease (HD) is caused by a dominantly inherited CAG repeat expansion in exon 1 of the huntingtin gene (HTT), which can be removed through CRISPR-Cas9-mediated fragment deletion in patient-derived fibroblasts and mouse models , 159. Muhenotype –166. Clihenotype , 168. Lehenotype . The genhenotype . Splicinhenotype . CRISPR/ gene in HSCs and patient-derived HSPCs to treat β-thalassemia and sickle cell disease (SCD)  . Martyn lassemia . Successlassemia . Using s pathway , 183. Fu pathway –186. CRI pathway . However pathway .in vivo using lipid nanoparticles (LNPs) to introduce PCSK9 loss-of-function mutations in living cynomolgus monkeys, which resulted in significant reductions in the blood levels of PCSK9 and LDL cholesterol   . Utilizi 5 (AdV) . Zhi et , X-linked MAGT1 deficiency with increased susceptibility to EBV-infection and N-linked glycosylation defect (XMEN), X-CGD, and WAS. +/− HSPCs with low cytotoxicity  and observed restoration of T cell development . Recentltoxicity . In additoxicity –224.in vitro and in vivo in immunodeficient mice (IPEX syndrome is a monogenic immune disease caused by mutations in FOXP3 that selectively affect the function of regulatory T cells (Tregs). By combining the CRISPR/Cas9 system with a DNA repair homology donor, Goodwin et al. enabled efficient and precise insertion of the FOXP3 cDNA into the endogenous locus of patient derived HSPCs, and the normal differentiation potential of edited HSPCs was maintained both ent mice .ex vivo targeted gene therapy approach using an optimized CRISPR/Cas HDR strategy to insert a therapeutic MAGT1 gene into the endogenous locus, and it presented high genetic correction efficiency and functional rescue in engrafted mice  cells. Brault et al. reported an ted mice .phox protein. CRISPR/Cas9-mediated HDR was employed for CYBB gene correction in HSPCs and functional restoration was achieved  is an X-linked recessive immunodeficiency disease caused by mutations in the WAS gene encoding WAS protein (WASp). Rai et el. inserted a therapeutic WAS cDNA in-frame with its endogenous translation start codon in patient-derived HSPCs observed . Microinobserved . In a probserved .X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIGM) is an IEI caused by mutations in CD40 ligand (CD40L) that affect immunoglobulin class-switch recombination and somatic hypermutation. Utilizing either the TALEN or CRISPR/Cas9 platforms, Kuo et al. integrated a normal copy of the CD40L cDNA with the endogenous CD40L promoter to maintain the physiological expression of CD40L in primary human HSCs and XHIM-patient-derived T cells . In a prX-linked agammaglobulinemia (XLA) is a monogenic IEI caused by a loss-of-function mutation in Bruton’s tyrosine kinase (BTK) gene, and it is characterized by reduced mature B lymphocytes. Gray and colleagues reported that precise integration of a corrective, codon-optimized cDNA along with a truncated terminal intron of the BTK gene into its endogenous locus by CRISPR/Cas-mediated HDR substantially elevated BTK expression to potentially therapeutic levels in cell lines and human HSPCs .SH2D1A. Targeted insertion of SAP cDNA at the first exon of the SH2D1A locus was efficiently achieved using TALEN, CRISPR/Cas9, or CRISPR/Cas12a, and SAP protein expression and SAP-dependent immune function were restored in patient-derived T cells (X-Linked Lymphoproliferative Disease (XLP) is a rare IEI that is caused by a mutation in the signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) protein-coding gene  T cells .Compared with the inefficient HDR-mediated site-specific integration of target genes, NHEJ-based homology-independent targeted integration (HITI) with improved integration frequencies has been utilized to achieve robust site-specific transgene integration at a clinically relevant genetic locus in human mobilized peripheral blood HSPCs . This stBEs enable single-base substitutions without producing DSBs, while PEs enable precise base conversions, insertions, deletions, and even combinations without inducing DSBs, and both of these editors are promising in genetic disease modeling and correction. Compared with BEs, PEs induce more accurate and precise on-target editing, and more importantly, PEs enable multiple types of editing. Gene therapy studies based on BEs or PEs for IEI treatment have not been reported yet. Thus, to identify potential targets for BE- and/or PE-mediated gene correction, disease-causing genes need to be carefully sequenced and sgRNAs and pegRNAs should be designed appropriately to enable efficient editing.3.3ex vivo and in vivo gene therapy.The targeted and efficient delivery of editing reagents plays an important role in gene therapy. Mainstream methods for delivering editing components into HSCs are based on electroporation, viral vectors, or nanoparticles . ElectroCRISPR/Cas components are usually delivered into HSCs in two major forms: the RNP complex of purified Cas protein precomplexed with sgRNAs and “all RNA” delivery with Cas mRNA with or without chemically modified sgRNAs. Hendel et al. reported that chemically modified sgRNAs could enhance genome editing in both human primary T cells and HSPCs; use of RNA- or RNP-based delivery method further improved editing . Protect3.4In terms of safety, CRISPR/Cas gene therapy avoids the need for semi-random integration of viral vectors, thereby minimizing the risk of integration-activated oncogenesis. One of the major concerns associated with CRISPR/Cas gene therapy is the potential off-target effects; many efforts have been made for off-target prediction, characterization, and reduction in both cell lines and human HSPCs , 248. Of3.5ex vivo and in vivo. Ex vivo gene therapy infuses cultured cells into patients after editing by CRISPR/Cas, and in vivo gene therapy delivers CRISPR/Cas gene editing reagents into patients to facilitate gene modification at pathogenic sequences. The current active CRISPR/Cas-based clinical trials are listed in CRISPR/Cas gene therapy has been considered a promising strategy to treat many incurable genetic and nongenetic disorders, including hereditary blood diseases, ocular diseases, neuromuscular disorders, metabolic disorders, cancers, and acquired immunodeficiency syndrome. Currently initiated clinical trials based on CRISPR/Cas technology are both The first in-human CRISPR/Cas clinical trial was performed at Sichuan University’s West China Hospital to treat advanced non-small cell lung cancer by targeting the PD-1 gene using CRISPR/Cas9 . According to the reported results, the safety and feasibility were proven, and they demonstrated that a more advanced gene editing strategy to improve efficacy is required in future trials . Severalin vivo gene therapy trial was initiated by Editas Medicine and Allergan to treat genetic ocular disease LCA10 by eliminating the disease-causing CEP290 mutant . In another CRISPR/Cas-based in vivo clinical trial , NTLA-2001 comprised LNP-encapsulated CRISPR/Cas9 elements targeting TTR to treat ATTR amyloidosis by reducing the concentration of TTR in serum. The safety and pharmacodynamic effects of single escalating doses of NTLA-2001 were evaluated in six patients with hereditary ATTR amyloidosis, and the results demonstrated mild adverse events and dose-dependent pharmacodynamics, and a significant reduction in serum TTR protein was observed (CRISPR/Cas-based clinical trials have been initiated for the treatment of genetic diseases. The first Cas9-mediated observed . In addi4The positive results of clinical trials of viral-based gene addition therapy suggest that autologous HSC gene therapy could provide sustained clinical benefits in patients with IEIs. However, gene addition approaches are not amenable for cases where the physiological regulation of genes is required to avoid transgene-related toxicity and genes with large sizes are difficult to deliver. The CRISPR/Cas gene editing system enables permanent, precise, and flexible gene editing without the drawbacks of semi-random genomic insertion, thus emerging as an important new tool for genetic manipulation of HSCs . Advancein vitro increases the risk of losing the multilineage potential of HSCs (Although clinical trials of CRISPR/Cas gene therapy are in progress, their application for IEIs is still in its infancy, and many issues still need to be addressed to enable safe and effective clinical application. A critical limitation of the gene therapy approach for IEIs is the broad spectrum of disease categories as well as the varying mutations, which require the development of personalized medicine approaches depending on the specific mutation a patient carries . To ensu of HSCs . Exposur of HSCs , 251.The advent and evolution of BE and PE variants with efficient and precise editing holds tremendous promise for the development of novel gene therapies for IEIs. Moreover, the implementation of decentralized manufacturing will enable the translation of gene-modified cell therapies from basic research to hospital-based clinical trials.XL, GL, XH designed the manuscript, XL drafted the manuscript, XL, YL prepared figures for the manuscript, FZ, GL, KL, and XH reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.


To compare the benefits and harms of drug treatments for adults with type 2 diabetes, adding non-steroidal mineralocorticoid receptor antagonists (including finerenone) and tirzepatide /glucagon-like peptide-1 (GLP-1) receptor agonist) to previously existing treatment options.Systematic review and network meta-analysis.Ovid Medline, Embase, and Cochrane Central up to 14 October 2022.Eligible randomised controlled trials compared drugs of interest in adults with type 2 diabetes. Eligible trials had a follow-up of 24 weeks or longer. Trials systematically comparing combinations of more than one drug treatment class with no drug, subgroup analyses of randomised controlled trials, and non-English language studies were deemed ineligible. Certainty of evidence was assessed following the GRADE  approach.https://matchit.magicevidence.org/230125dist-diabetes).The analysis identified 816 trials with 471 038 patients, together evaluating 13 different drug classes; all subsequent estimates refer to the comparison with standard treatments. Sodium glucose cotransporter-2 (SGLT-2) inhibitors  and GLP-1 receptor agonists  reduce all cause death; non-steroidal mineralocorticoid receptor antagonists, so far tested only with finerenone in patients with chronic kidney disease, probably reduce mortality ; other drugs may not. The study confirmed the benefits of SGLT-2 inhibitors and GLP-1 receptor agonists in reducing cardiovascular death, non-fatal myocardial infarction, admission to hospital for heart failure, and end stage kidney disease. Finerenone probably reduces admissions to hospital for heart failure and end stage kidney disease, and possibly cardiovascular death. Only GLP-1 receptor agonists reduce non-fatal stroke; SGLT-2 inhibitors are superior to other drugs in reducing end stage kidney disease. GLP-1 receptor agonists and probably SGLT-2 inhibitors and tirzepatide improve quality of life. Reported harms were largely specific to drug class . Tirzepatide probably results in the largest reduction in body weight . Basal insulin  and thiazolidinediones  probably result in the largest increases in body weight. Absolute benefits of SGLT-2 inhibitors, GLP-1 receptor agonists, and finerenone vary in people with type 2 diabetes, depending on baseline risks for cardiovascular and kidney outcomes (This network meta-analysis extends knowledge beyond confirming the substantial benefits with the use of SGLT-2 inhibitors and GLP-1 receptor agonists in reducing adverse cardiovascular and kidney outcomes and death by adding information on finerenone and tirzepatide. These findings highlight the need for continuous assessment of scientific progress to introduce cutting edge updates in clinical practice guidelines for people with type 2 diabetes.PROSPERO CRD42022325948. People with type 2 diabetes face an elevated risk of cardiovascular and kidney disease, resulting in impaired quality of life and reduced life expectancy. In the light of increased recognition of these risks and the failure of intensive glycaemic control to provide substantial risk reduction, regulatory agencies and researchers have increasingly shifted away from a glucose centric paradigm. Instead, reductions in cardiovascular disease and chronic kidney disease are now, through new and effective treatment options, priority treatment objectives.Recently, two novel agents have become available to treat patients with type 2 diabetes: finerenone, a non-steroidal mineralocorticoid receptor antagonist, and tirzepatide, a dual glucose dependent insulinotropic polypeptide (GIP)/GLP-1 receptor agonist. In two randomised trials looking at the cardiovascular outcomes of finerenone, findings suggested cardiovascular and kidney benefits in people with type 2 diabetes and chronic kidney disease,BMJ Rapid Recommendations for diabetes drugs, we designed this network meta-analysis to inform professional societies and healthcare systems in updating their health technology assessments, clinical practice guidelines, and other decision support.Clinicians now face the challenge of guiding their patients with type 2 diabetes in whether to add SGLT-2 inhibitors, GLP-1 receptor agonists, or finerenone and tirzepatide to their ongoing therapeutic regimens. Expanding on our previous large network meta-analysisThe taskforce of the guideline workshop , an international multidisciplinary team including endocrinologists, cardiologists, and nephrologists,Eligible randomised controlled trials compared drugs used to treat adults with type 2 diabetes. We considered the following drug classes: SGLT-2 inhibitors, GLP-1 receptor agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors, thiazolidinediones, sulfonylureas, metformin, α-glucosidase inhibitors, meglitinides, insulins, dual GIP/GLP-1 receptor agonists, and non-steroidal mineralocorticoid receptor antagonists. Appendix 1.3 describes the detailed drug names and definitions of control arms . Eligible trials had a follow-up of 24 weeks or longer. Trials systematically comparing combinations of more than one drug treatment class with no drug treatment, subgroup analyses of randomised controlled trials, and non-English language studies were deemed ineligible.We used comprehensive literature search strategies from previously published network meta-analyses in Ovid Medline, Embase, and Cochrane Central to 14 October 2022 (see appendix 1.1 for search strategies). The search added terms for non-steroidal mineralocorticoid receptor antagonists and dual GIP/GLP-1 receptor agonists. The search included reference lists of other identified systematic reviews evaluating cardiovascular and kidney outcomes associated with drugs of interest.The study performed a pilot test for the study selection process before screening. Pairs of reviewers  independently screened identified hits at the title and abstract and full text levels, with discrepancies resolved by a senior reviewer (SL).Using a standardised extraction form, the paired trained reviewers  independently extracted the following data: Study characteristics ; Baseline characteristics of included participants , duration of diabetes, and complications or comorbidities including cardiovascular diseases, chronic kidney diseases, and obesity); Interventions ; and Outcomes . A senior reviewer (SL) resolved discrepancies. We prioritised study reported intention-to-treat results or modified intention-to-treat results over per protocol results.Pairs of reviewers independently assessed the risk of bias . The Cochrane risk-of-bias tool, modified by the CLARITY group at McMaster University, informed risk-of-bias assessments2, a sustained percentage decline in estimated glomerular filtration rate of at least 40% or a doubling of serum creatinine, or kidney related death.We judged the following outcomes as critical: all cause death, cardiovascular death, non-fatal stroke, end stage kidney disease, and amputation; and the following outcomes as important: non-fatal myocardial infarction, admission to hospital for heart failure, body weight change, health related quality of life, severe hypoglycaemia, severe gastrointestinal events, genital infection, ketoacidosis due to diabetes, and hyperkalaemia leading to admission to hospital. We evaluated the impact on end stage kidney disease using a composite of long term dialysis, kidney transplantation, a sustained estimated glomerular filtration rate <15 mL per minute per 1.73 mWe measured the binary outcomes using odds ratios. For continuous outcomes, we measured health related quality of life as standardised mean difference, and body weight in kg as mean difference.We conducted random effect network meta-analysis using a frequentist graph theoretical approach with the weighted least square estimator and Moore-Penrose pseudoinverse.We performed sensitivity analyses, including a bayesian network meta-analysis adjusted by trial durationFor trial and aggregated patient characteristics measured as continuous variables, we performed the following four meta-regressions:Proportion of patients with established cardiovascular diseases .Mean patients’ estimated glomerular filtration rate at baseline (hypothesising a larger relative effect in reducing death and cardiovascular and kidney outcomes in patients with lower estimated glomerular filtration rate).Mean patients’ body mass index at baseline (hypothesising a larger relative effect in reducing death and cardiovascular and kidney outcomes in patients with higher body mass index).Trial follow-up length (hypothesising a larger relative effect in reducing death and cardiovascular and kidney outcomes in studies with longer follow-up).The credibility of any apparent subgroup effect  was rated using the ICEMAN tool.Following GRADE  guidance, evidence from direct comparisons started as high certainty evidence and could be rated down for risk of bias, inconsistency, indirectness, and publication bias.To categorise the relative impact of interventions, we chose the null effect as the decision threshold and standard treatments as the reference intervention.To better inform clinical decision making, we estimated the anticipated absolute effects of all drugs on the cardiovascular, kidney, and safety outcomes. If valid, we adopted baseline risk estimates applied in a clinical practice guideline that included a systematic review of risk prediction models.https://matchit.magicevidence.org/230125dist-diabetes). This tool also allows end users to compare any of the treatment options, including change of comparator .Given the complexity of presenting 9770 estimates of effect from this network meta-analysis, we elected to primarily present relative and absolute estimates of effect, certainty, and more detailed network meta-analysis results  through an interactive GRADE summary of findings table, the MATCH-IT tool , missing outcome data (26%), and allocation concealment (25%) (appendix 3). The evidence did not suggest global publication bias and intransitivity for any outcome (appendix 4.7), nor did the results suggest relevant global inconsistency or incoherence in outcomes except for health related quality of life, body weight change, and amputation .https://matchit.magicevidence.org/230125dist-diabetes) provides an interactive view of the anticipated absolute effects for all populations at varying risks of cardiovascular and kidney outcomes. These data are also available in appendix 6 and - with even more details, in another interactive online tool . Below, we summarise the relative effects for cardiovascular, kidney, and harm outcomes for all drugs and provide examples of anticipated absolute effects for selected outcomes; all subsequent estimates refer to the comparison with standard treatments. The MATCH-IT tool  and GLP-1 receptor agonists  reduce all cause mortality, and cardiovascular death . Non-steroidal mineralocorticoid receptor antagonists probably reduce all cause mortality  and possibly reduce cardiovascular death . Metformin possibly reduces all cause mortality  and might have little or no effect on cardiovascular death. DPP-4 inhibitors probably have little or no effect on cardiovascular death (moderate certainty). Sulfonylureas possibly increase all cause mortality (low certainty) and might have little or no effect on cardiovascular death. Other drugs might have little or no or uncertain effect on mortal outcomes —as, probably, do GLP-1 receptor agonists  and, possibly, metformin . GLP-1 receptor agonists are the only drug class that convincingly reduces non-fatal stroke . Other drugs might have little or no or uncertain effects on non-fatal myocardial infarction or stroke, relative to standard treatments  decrease admission to hospital for heart failure as, probably, do GLP-1 receptor agonists  and finerenone . SGLT-2 inhibitors and finerenone are among the most effective drugs in this regard and SGLT-2 inhibitors are probably superior to GLP-1 receptor agonists (moderate certainty). Thiazolidinediones probably increase admission to hospital due to heart failure . Metformin and other drugs might have little or no effect, or uncertain effects , GLP-1 receptor agonists , and finerenone  probably reduce end stage kidney disease. We rated down the certainty of evidence to moderate owing to indirectness, a result of our composite outcome of end stage kidney disease driven by variable reporting of kidney outcomes in the trials. SGLT-2 inhibitors are among the most effective drugs and are possibly superior to GLP-1 receptor agonists and finerenone (low certainty). Other drugs might have little or no effect, or uncertain effects on end stage kidney disease, relative to standard treatment . SGLT-2 inhibitors, GLP-1 receptor agonists, and tirzepatide probably improve health related quality of life with standardised mean differences ranging from 0.17 to 0.39 (moderate certainty), which did not surpass the minimal important difference . DPP-4 inhibitors probably have little or no effect, and other drugs might have little or uncertain impact on health related quality of life  and basal bolus insulin  probably increase the risk of severe hypoglycaemic events (moderate certainty), with likely smaller increases in risk with basal insulin , bolus insulin , and DPP-4 inhibitors , with and without the contamination of other treatments. Meglitinides and thiazolidinediones may increase the risk of severe hypoglycaemic events (low certainty). SGLT-2 inhibitors and GLP-1 receptor agonists do not increase the risk of severe hypoglycaemic events (high certainty). Finerenone is probably associated with fewer severe hypoglycaemia than the standard treatments . Other drugs might have little to no effect compared with standard treatments  and GLP-1 receptor agonists  probably increase the risk of severe gastrointestinal adverse events (moderate certainty). Other drugs might have little or no effects compared with standard treatments . Sulfonylureas may reduce the risk of a genital infection . Other drugs might have little to no effect compared with standard treatments ; other drugs do not ; other drugs do not . With an estimated baseline risk of 0.2%, treatment with non-steroidal mineralocorticoid receptor antagonists in 1000 patients for five years probably results in 10 additional events  and all sensitivity analyses confirmed the robustness of our findings (appendix 8).This network meta-analysis comprehensively summarises the benefits and harms of available drug treatments for type 2 diabetes, including the two recently available drugs finerenone and tirzepatide. Among all drug classes, SGLT-2 inhibitors, GLP-1 receptor agonists, and finerenone show benefits in reducing all cause mortality, admission to hospital due to heart failure (SGLT-2 inhibitors and—probably—finerenone are the most effective drug treatments), and end stage kidney disease (SGLT-2 inhibitors are the most effective drug treatments). Only GLP-1 receptor agonists convincingly reduce non-fatal stroke. SGLT-2 inhibitors, GLP-1 receptor agonists, and tirzepatide improve health related quality of life, but did not reach the threshold for minimal important differences, suggesting trivial effects. As illustrated in the MATCH-IT tool, the absolute benefits of these drugs vary greatly in people with type 2 diabetes depending on baseline risks for cardiovascular and kidney outcomes Our work represents the most comprehensive systematic review and network meta-analysis assessing all pertinent drug classes for type 2 diabetes treatment. An international multidisciplinary team shaped the study question and protocol, optimising its relevance to current clinical practice. Our study incorporated current and rigorous approaches to network meta-analysiss and GRADE assessment.Limitations of our systematic review and network meta-analysis are largely driven by the available evidence. Firstly, for many outcomes that are important to patients, we found low to very low certainty evidence for the oldest as well as the newest classes of drugs, including metformin, sulfonylureas, tirzepatide, and non-steroidal mineralocorticoid receptor antagonists. Secondly, this network meta-analysis cannot answer a highly relevant question: what are the benefits and harms of coadministration of SGLT-2 inhibitors, GLP1 receptor agonists, and finerenone? Nevertheless, observational studies and post hoc analyses of trials suggest cardiovascular and kidney benefits of the combination of SGLT-2 inhibitors, finerenone, and GLP-1 receptor agonists.Finerenone is the first non-steroidal mineralocorticoid receptor antagonist likely to reduce all cause mortality and admission to hospital for heart failure and improve kidney outcomes in people living with type 2 diabetes. Finerenone is an alternative to SGLT-2 inhibitors and GLP-1 receptor agonists in patients with concomitant chronic kidney disease, but results provide only indirect evidence for other populations.Although finerenone results in a fivefold relative increase in hyperkalaemia leading to admission to hospital, the absolute numbers of admissions to hospitals were very low. The absolute effects of hyperkalaemia  are, for most patients with type 2 diabetes and chronic kidney disease, of less importance than the benefits of 16 fewer deaths, 21 fewer admissions to hospital for heart failure, and 14 fewer cases of end stage kidney disease. Nevertheless, for patients at elevated baseline risk of hyperkalaemia, clinicians should closely monitor serum potassium when prescribing finerenone. Such patients include those taking drug treatments that could elevate serum potassium such as angiotensin converting enzyme inhibitors, angiotensin receptor blockers, and angiotensin receptor-neprilysin inhibitors.Tirzepatide, the only dual GIP/GLP-1 receptor agonist currently available, improves quality of life and reduces body weight with a greater effect than any other drugs or drug classes. GLP-1 receptor agonists also reduce body weight, with semaglutide being the most effective drug in people with type 2 diabetes, as previously established in patients with overweight and obesity.Our results raise concerns about the use of some well established drugs for glucose lowering in adults with type 2 diabetes. In particular, sulfonylureas may increase all cause mortality (low certainty), and thiazolidinediones probably increase admission to hospital due to heart failure (moderate certainty). Clinicians should be cautious in prescribing these drugs, especially to those at higher baseline risks for such outcomes. An additional new finding in our review is that DPP-4 inhibitors, in addition to sulfonylurea and insulin, show a probable intermediate increased risk of severe hypoglycaemia (moderate certainty). Since laboratory studies do not support the hypoglycaemic risk caused by the monotherapy of DPP-4 inhibitors,Consistent with previous systematic reviews,Keeping pace with the growing number of published randomised trials in adults with type 2 diabetes, this network meta-analysis finds highly heterogeneous benefits and drug specific harms across 13 drug classes. Beyond confirming the substantial cardiovascular and kidney benefits of SGLT-2 inhibitors and GLP-1 receptor agonists—with absolute effects highly dependent on patient risk profiles—we find that finerenone, the drug recently made available, displays quite similar benefits to SGLT-2 inhibitors and GLP-1 receptor agonists. Tirzepatide shows superior benefits on weight loss than SGLT-2 inhibitors and GLP-1 receptor agonists. These results and other key findings of our comprehensive systematic review highlight the need for continuous assessment of scientific progress to introduce cutting edge updates in clinical practice guidelines for people with type 2 diabetes.Sodium glucose cotransporter-2 (SGLT-2) inhibitors and glucagon-like peptide-1 (GLP-1) receptor agonists have proven benefits in cardiovascular and kidney outcomes, with some notable differences in outcomes such as heart failure and strokeRecent randomised trials report both cardiovascular and kidney benefits with finerenone, a novel non-steroidal mineralocorticoid receptor antagonist, and improvements in quality of life and weight loss with tirzepatide, a dual glucose dependent insulinotropic polypeptide (GIP)/GLP-1 receptor agonistCompared with standard treatments, adding finerenone probably reduces all cause mortality, admission to hospital for heart failure, and end stage kidney disease, while adding tirzepatide could reduce body weightCompared with standard treatments, findings indicate that adding SGLT-2 inhibitors or GLP-1 receptor agonists reduces all cause mortality, cardiovascular death, non-fatal myocardial infarction, admission to hospital for heart failure, and end stage kidney disease, while adding only GLP-1 receptor agonists reduces non-fatal strokeCompared with standard treatments, adding metformin possibly reduces all cause mortality and non-fatal myocardial infarction, adding sulfonylureas possibly increases all cause mortality, and adding thiazolidinediones probably increases admission to hospital due to heart failure


Twenty percent to 40% of patients are affected by angina after percutaneous coronary intervention (PCI), which is associated with anxiety, depression, impaired physical function, and reduced quality of life. Understanding patient and procedural factors associated with post-PCI angina may inform alternative approaches to treatment.Two hundred thirty patients undergoing PCI completed the Seattle Angina Questionnaire (SAQ-7) and European quality of life–5 dimension–5 level (EQ-5D-5L) questionnaires at baseline and 3 months post-PCI. Patients received blinded intracoronary physiology assessments before and after stenting. A post hoc analysis was performed to compare clinical and procedural characteristics among patients with and without post-PCI angina (defined by follow-up SAQ-angina frequency score <100).P<0.001) and EQ-5D-5L health index scores . Pre-PCI fractional flow reserve (FFR) was lower among patients who had no post-PCI angina . Percentage change in FFR after PCI had a moderate correlation with angina frequency score at follow-up . Patients with post-PCI angina had less improvement in FFR . There were no between-group differences in post-PCI FFR, coronary flow reserve, or corrected index of microcirculatory resistance. Patients with post-PCI angina had lower SAQ-summary scores  and EQ-5D-5L index scores  at follow-up.Eighty-eight of 230 patients (38.3%) reported angina 3 months post-PCI and had a higher incidence of active smoking, atrial fibrillation, and history of previous myocardial infarction or PCI. Compared with patients with no angina at follow-up, they had lower baseline SAQ summary scores (69.48±24.12 versus 50.20±22.59, Larger improvements in FFR following PCI were associated with less angina and better quality of life at follow-up. In patients with stable symptoms, intracoronary physiology assessment can inform expectations of angina relief and quality of life improvement after stenting and thereby help to determine the appropriateness of PCI.https://www.clinicaltrials.gov; Unique identifier: NCT03259815.URL:  Angina after percutaneous coronary intervention (PCI) is associated with long-term anxiety, depression, and impairment of both physical function and quality of life.Persistence or recurrence of angina after PCI may affect 20% to 40% of patients during short-to-medium-term follow-up.Patients with post-PCI angina reported more frequent angina and poorer quality of life at baseline yet had physiologically less severe disease and, accordingly, tended to achieve less improvement in coronary physiology metrics than those who were free from angina post-PCI.The degree of improvement in patient-reported outcome measures following PCI is not associated with post-PCI coronary physiology values. It relates instead to the pre-PCI value and the change from baseline. Lower pre-PCI values and larger increases in fractional flow reserve were associated with higher patient-reported outcome measures scores at follow-up.Intracoronary physiology assessment can inform expectations of angina relief and quality of life improvement after stenting and thereby help to determine the appropriateness of PCI intended to alleviate symptoms.Smilowitz and SetoSee Editorial by 1 PCI achieves greater reductions in myocardial ischemia than optimal medical therapy alone.2 The greater the degree of ischemia in a myocardial territory, the greater the improvement in symptoms following PCI.3 Patients with moderate or severe ischemia randomized to an initial invasive strategy in the ISCHEMIA trial  had greater improvement in angina-related health status than those assigned to the conservative strategy, and larger differences were observed in patients who actually had anginal symptoms at baseline.4 Nevertheless, persistence or recurrence of angina after PCI is well-recognized and may affect 20% to 40% of patients during short-to-medium-term follow-up.5,6 Total health care costs in the first year after an index PCI can be up to 1.8× greater for patients with angina or chest pain after stenting with cost differentials continuing out to 36 months post-PCI.7 Understanding patient factors associated with post-PCI angina may support different approaches to revascularization.8 Conflicting data exist regarding the association between invasive coronary measurements and patient-reported outcome measures at follow-up.11 TARGET-FFR  was a randomized controlled trial designed to assess the efficacy of a post-PCI physiology-guided incremental optimization strategy versus standard angiographic guidance.12 In this analysis, the incidence and associates of angina at 3 months post-PCI were examined.Angina after percutaneous coronary intervention (PCI) is associated with long-term anxiety, depression, impaired physical function, and quality of life.13 In brief, 260 patients undergoing standard-of-care PCI for either chronic or medically stabilized acute coronary syndromes provided informed consent to be randomized 1:1 to a control group or the experimental physiology-guided incremental optimization strategy. All patients completed questionnaires on anginal symptoms (Seattle Angina Questionnaire [SAQ-7]) and health status  at baseline and were contacted to repeat this assessment 3 months after their procedure. The questionnaires were administered by telephone or mail by a research nurse blinded to the randomized group allocation and the physiology results. A summary of the study protocol and additional detail on the patient questionnaires is provided in methods in the Supplemental Material. The study was approved by an institutional review committee, and patients gave informed consent.The data that support the findings of this study are available from the corresponding author upon reasonable request. The rationale, study design, and primary outcome of the TARGET-FFR controlled trial have been published previously.8 Prior to PCI, in addition to patient-reported SAQ scores, anginal symptoms were also assessed and adjudicated by a physician with a Canadian Cardiovascular Society (CCS) score of class I or above defining the presence of angina at baseline. This definition was used for subgroup analyses of patients with anginal symptoms at baseline.The presence of angina post-PCI was defined by a patient-reported follow-up SAQ-angina frequency (SAQ-AF) score of <100. Patients with a follow-up of SAQ-AF score=100 were classified as having no angina.14Clinical outcomes at a median of 3 years post-PCI were assessed by electronic health record linkage. The primary clinical outcome was target vessel failure, a composite end point comprising cardiovascular death, target vessel myocardial infarction, and target vessel revascularization.t test was used to compare patient-level characteristics with continuous variables. Categorical variables were compared using the χ2 test without continuity correction. Whenever appropriate, a Fisher exact test was used instead. Follow-up patient-reported outcome measures scores stratified by FFR tertiles were analyzed with an ANCOVA model on the parameter’s follow-up value adjusted for FFR tertiles and baseline value. Relationship between variables was assessed using Spearman correlation coefficient. Analyses were performed using SAS 9.4 and SPSS Statistics 28.Continuous variables are presented as mean±SD and categorical data as counts and percentages. A 2-sample Tables S1 through S5). Five of eighty-eight (5.7%) patients with post-PCI angina did not have angina (physician-adjudicated CCS class ≥1) at baseline. There were no periprocedural myocardial infarctions within this subgroup and mean post-PCI physiology values compared favorably to the other subgroups (Table S2).Of 260 participants, 230 (88.5%) provided follow-up SAQ-AF scores 3 months  post-PCI. For the purposes of this analysis, patients were stratified by the presence of post-PCI angina. Eighty-eight (38.3%) of 230 patients had post-PCI angina as determined by a follow-up SAQ-AF score <100. Expanded subgroup analyses with additional stratification by the presence of angina at baseline are included in the . Additionally, in those patients reporting any angina after PCI, the mean change in the EQ-5D-5L Weighted Health Index was effectively zero (−0.001 units), indicating they felt their quality of life had not improved after stenting (Table S7). Among patients who had angina at baseline (CCS class I and above), FFR and coronary flow reserve (CFR) correlated with the absolute patient-reported outcome measures scores at follow-up. FFR had a moderate, positive correlation with follow-up SAQ-AF scores. CFR had a similar, albeit weaker, correlation. This reflected negative correlations with the pre-PCI SAQ-AF score rather than positive correlations with post-PCI values (Tables S8 through S11). Pre-PCI and change in FFR (delta FFR) both also had significant, albeit somewhat weaker, correlations with the other SAQ domains and the EQ-5D-5L weighted health index score (Tables S8 and S9). The magnitude of change in pressure ratios was predicated by the pre- rather than post-PCI value and patients reporting angina post-PCI tended to have achieved smaller changes in FFR following PCI . There was no correlation between index of microcirculatory resistance  and patient-reported outcome measures (Tables S12 and S13).Patients with post-PCI angina had significantly lower SAQ scores, both at baseline and follow-up, compared with those free from angina , however, the study was not powered to detect a difference in clinical outcomes.The rate of target vessel failure at a median (interquartile range) follow-up of 3 (0.9) years was 1.7% (4/230) with no significant difference between groups  of patients in the drug-eluting stent arm had physician-adjudicated angina or angina-equivalent symptoms at 1-year follow-up.As the impact of anginal symptoms is inherently subjective, we concluded that attempting to define a level of post-PCI angina that might be acceptable to patients or represent a clinically meaningful improvement would be arbitrary and ultimately futile. Improved but persistent symptoms may be considered a success by some patients yet be completely unacceptable to others, so we therefore, determined a complete absence of patient-reported angina after PCI to represent the gold standard. The SAQ-AF domain asks patients to report the frequency of “chest pain, chest tightness, or anginal attacks” over the preceding 4 weeks with a score <100 indicating at least one anginal episode within that period. Accordingly, we adopted a follow-up SAQ-AF score of <100 as the definition of post-PCI.Approximately 6% of participants reporting angina after stenting did not have symptoms prior to their intervention which highlights the importance of ascertaining the indication for and appropriateness of PCI with patients prior to embarking upon the procedure, particularly for those who are asymptomatic.Patients reporting post-PCI angina had a higher burden of cardiovascular risk factors, including cigarette smoking, prior myocardial infarction, and atrial fibrillation at baseline. The higher rates of oral anticoagulants and concomitant lower rate of antiplatelets prescribed at baseline among patients reporting post-PCI angina are likely just commensurate with the higher incidence of atrial fibrillation in this group. As there was no difference in the incidence of the heart failure or hypertension, the higher rate of diuretic therapy may relate to prescriptions for dyspnea thought to represent an angina-equivalent symptom.Table S7) and had significantly lower SAQ quality of life scores compared with those who were angina free (Table S6). These findings are contrary to a previous report concluding that preprocedural angina frequency is the most important prognostic indicator of quality of life after PCI.15Patients with post-PCI angina had more severe symptoms at baseline (higher incidence of both CCS class 2 and 3 angina and self-reported rates of daily and weekly angina) and were prescribed more antianginal agents with greater use of oral and sublingual nitrates than those who were angina-free postprocedure. Patients experiencing any angina post-PCI reported effectively no improvement in quality of life as assessed by the mean change in their EQ-5D-5L weighted health index score  than those who were angina-free at follow-up. There were no differences in absolute post-PCI physiology values between groups, and among patients with angina at baseline, there was no correlation between post-PCI values and patient-reported outcome measures at follow-up. Coronary microvascular dysfunction has been proposed as a potential mechanism for persistent angina post-PCI.9 A subsequent analysis of pooled data from the FAME  1 and 2 trials reported that larger improvements in FFR with PCI were associated with an increased probability of improvement of at least 2 CCS classes at 1-month follow-up but did not find any correlation between pre-PCI FFR values and symptom improvement.11 Latterly, another pooled analysis of FAME 1 and 2 data found that lower pre-PCI FFR, higher delta FFR and higher percentage delta FFR were associated with significantly larger change from baseline EQ-5D index score at both 1 month and 1 year post-PCI.10A physiology-stratified analysis of the ORBITA trial  assessed paired SAQ and EQ-5D-5L data from 189 patients and found that pre-PCI FFR and the instantaneous wave-free ratio (iFR) did not predict the effect of placebo-controlled PCI on anginal symptoms or quality of life.Figure S1, pre-PCI values drove the magnitude of the change. Larger change in FFR following PCI was associated with higher patient-reported outcome measures scores at follow-up. In keeping with these findings, significantly lower absolute and percentage change values for FFR were observed in the post-PCI angina group. After multivariate analysis, delta FFR was found to be an independent predictor of post-PCI angina. Patients with a higher symptom burden and lower quality of life scores at baseline are more likely to report post-PCI angina, particularly where PCI can only achieve a small improvement in physiology metrics, such as in those with diffuse patterns of coronary disease and borderline or gray-zone pre-PCI values. Accordingly, this analysis from the TARGET-FFR randomized trial supports the concept that intracoronary physiology assessment can inform expectations of angina relief and quality of life improvement after stenting and thereby help to determine the appropriateness of PCI intended to alleviate symptoms. Patients with physiologically severe lesions can expect a larger improvement in intracoronary pressure ratios following PCI which is associated with a higher likelihood of angina relief and improved quality of life.In the present analysis, among patients who had angina at baseline (CCS class I and above), pre-PCI, absolute and percentage delta FFR values had significant correlations with patient-reported outcome measures at follow-up. Post-PCI values had no correlation with angina or quality of life at follow-up indicating that, as demonstrated in the Figure and TARGET-FFR was a single-center study with a relatively homogeneous PCI practice, including high rates of lesion predilatation and high-pressure stent postdilatation. A larger multicenter trial incorporating a wider range of PCI strategies and techniques may have had a different outcome. The study was not powered for clinical outcomes and the incidence of target vessel failure at a median follow-up of 3 years remained low.The magnitude of FFR improvement following PCI correlated with angina status and quality of life at follow-up and was an independent predictor of the presence of post-PCI angina. In patients with stable symptoms, intracoronary physiology assessment can aid prediction of angina relief and quality of life improvement after stenting and thereby help to determine the appropriateness of PCI.Endowment funds at the Golden Jubilee National Hospital (NHS Golden Jubilee), Glasgow, United Kingdom, with support from the British Heart Foundation (Research Excellence Award RE/18/6/34217).Dr Collison received consultancy fees from Abbott. Dr Mizukami received consultancy fees from Zeon Medical Inc; research grants from Boston Scientific; speaker fees from Abbott, Cathworks, Boston Scientific. Dr Collet received research grants from Biosensors, Coroventis Research, Medis Medical Imaging, Pie Medical Imaging, CathWorks, Boston Scientific, Siemens, HeartFlow, Abbott; consultancies HeartFlow, OpSens, Abbott, Philips Volcano. Dr Ford received consulting fees from BioExcel; honoraria from Abbott, Boehringer, Novartis. Dr Watkins received honoraria from Abbott, AstraZeneca, Biosensors, Boston Scientific, Daiichi Sankyo, GE Healthcare, ShockWave Medical. Dr Robertson received honoraria from AstraZeneca, Abbott. Dr O’Boyle received honoraria from AstraZeneca, Boston Scientific, Novartis. Dr McEntegart received consulting fees from Abbott, Boston Scientific, ShockWave Medical; honoraria from International Medical Device Solutions, Medtronic. Prof Berry received institutional grants/contracts from Abbott, AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, HeartFlow, Novartis, Siemens Healthcare; consulting fees from Abbott, AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, HeartFlow, Menarini, Novartis; honoraria from Abbott, AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, HeartFlow, Philips, Valo Health. Prof Oldroyd received honoraria from Abbott, Biosensors International, Boston Scientific; institutional research grant from Boston Scientific which supported the present manuscript; full-time employee of Biosensors International since May 2020. The other authors report no conflicts.Supplemental MethodsFigure S1Tables S1–S13


Panel A, dotted red line) was used, and inter-atrial septal (IAS) incision was finally closed with a 4-0 prolene suture. Post-operative transthoracic echocardiogram (TTE) revealed a residual mild left-to-right shunt at the level of IAS. During the following years, the patient remained asymptomatic. About 7 years after cardiac surgery, TTE showed an increase of inter-atrial shunt. Transoesophageal echocardiography (TEE) revealed intimal layer debris on the right side of IAS , expanding into the right atrium (RA). A significantly prominent eustachian valve was involved by dehiscence at its insertion point on the IAS . Small hyperechoic points (red circles) denoted the residual of stitches. Colour Doppler mode revealed left-to-right shunt originating from multiple sites, probably along the suture line , and exiting into the RA above the eustachian valve . Whether the blood flowing from inferior cava vein towards IAS might have contributed to damage IAS at a ‘locus minoris resistentiae’ is unknown. This is the first report of IAS dehiscence as a consequence of incomplete closure of bi-atrial trans-septal incision. TEE is essential to formulate diagnosis, and volumetric rendering  allows to better understand the spatial relationships between cardiac structures.A 70-year-old woman underwent surgical replacement of aortic valve and mitral valve by bio-prostheses and annuloplasty of tricuspid valve due to rheumatic disease. A bi-atrial trans-septal approach (European Heart Journal - Cardiovascular Imaging online.Funding: None declared.Data availability: The data underlying this article will be shared on reasonable request to the corresponding author.jead226_Supplementary_DataClick here for additional data file.


Hospitalized patients have an increased risk of developing hospital-acquired sacral pressure injury (HASPI). However, it is unknown whether SARS-CoV-2 infection affects HASPI development. To explore the role of SARS-CoV-2 infection in HASPI development, we conducted a single institution, multi-hospital, retrospective study of all patients hospitalized for ≥5 days from March 1, 2020 to December 31, 2020. Patient demographics, hospitalization information, ulcer characteristics, and 30-day-related morbidity were collected for all patients with HASPIs, and intact skin was collected from HASPI borders in a patient subset. We determined the incidence, disease course, and short-term morbidity of HASPIs in COVID-19(+) patients, and characterized the skin histopathology and tissue gene signatures associated with HASPIs in COVID-19 disease. COVID-19(+) patients had a 63% increased HASPI incidence rate, HASPIs of more severe ulcer stage , and HASPIs more likely to require debridement  compared to COVID-19(-) patients. Furthermore, COVID-19(+) patients with HASPIs had 2.2x increased odds of a more severe hospitalization course compared to COVID-19(+) patients without HASPIs. HASPI skin histology from COVID-19(+) patients predominantly showed thrombotic vasculopathy, with the number of thrombosed vessels being significantly greater than HASPIs from COVID-19(-) patients. Transcriptional signatures of a COVID-19(+) sample subset were enriched for innate immune responses, thrombosis, and neutrophil activation genes. Overall, our results suggest that immunologic dysregulation secondary to SARS-CoV-2 infection, including neutrophil dysfunction and abnormal thrombosis, may play a pathogenic role in development of HASPIs in patients with severe COVID-19. The skin is among the organs affected by COVID-19, with manifestations including various localized or diffuse exanthems and retiform or acral purpura associated with thrombotic vasculopathy . Skin mahemistry –8.Sacral ulcers are a leading cause of cutaneous-related patient morbidity in hospitalized patients , 10. TheDespite the frequent presence of common HASPI risk factors, our clinical experience is that some COVID-19 patients acquire HASPIs that develop and progress more rapidly than usual, which may be associated with retiform purpura , 15. BasWe found that COVID-19(+) patients are at an increased risk of developing HASPIs and HASPI-associated morbidity compared to COVID-19(-) patients, even when controlling for baseline risk factors for ulceration. A large percentage of sacral skin biopsies from the borders of HASPIs of COVID-19(+) patients displayed thrombotic vasculopathy (TV), with significantly more thrombosed vessels per tissue section compared to sacral skin samples from PU in COVID-19(-) patients. Furthermore, the transcriptional profile differences in sacral biopsies from COVID-19(+) patients with TV support abnormal thrombosis and enhanced neutrophil extracellular trap (NET)-driven inflammation in sacral skin of COVID-19(+) patients compared to sacral skin specimens from PU in COVID-19(-) patients. These findings implicate thrombotic and inflammatory responses to SARS-CoV-2 infection in the promotion of cutaneous vascular thromboses, tissue ischemia, and eventual cutaneous ulceration.To understand the risk of HASPI development in COVID-19 disease, the incidence of sacral ulcers was compared between COVID-19(+) and COVID-19(-) patients in our study cohort , longer hospitalization length , and more severe COVID-19 disease course, including increased requirement of mechanical ventilation or death (p<0.001) as compared to COVID-19(+) patients without HASPIs  patients with and without HASPIs were compared in relation to their comorbidities , congestive heart failure and diabetes) was performed to assess impact of co-morbidities on HASPI development and hospitalization course  and a higher frequency of requiring intubation  compared to COVID-19(-) patients (vs. 9.0% Stage 3 or 4 ulcer p=0.003).Clinical and demographic characteristics in the subset of COVID-19(+) (49 patients) and COVID-19(-) (244 patients) hospitalized patients who developed HASPIs were compared to examine the impact of COVID-19 on disease course and ulcer phenotypes Table 1.vs. 813 ng/mL, p=0.007). Other laboratory parameters, including maximum D-dimer concentration, maximum white blood cell (WBC) count, and neutrophil-to-lymphocyte ratio did not significantly differ between the two groups  and COVID-19(-) patients. COVID-19(+) patients had significantly higher maximum ferritin concentrations compared to COVID-19(-) patients  patients were compared to similar tissue specimens from COVID-19(-) patients with PUs. Qualitative assessments revealed that biopsies of COVID-19(+) patients with HASPIs could be broadly categorized into 2 groups: histologic features consistent with thrombotic vasculopathy  or histologic features consistent with PUs (33%); Table 3.vs. 59.5 days). There were no significant differences observed in the maximum D-dimer levels and the absolute neutrophil counts between the three groups; however, not all subjects had D-dimer levels reported, especially in COVID-19(-) patients. Histologically, COVID-19(+) TV biopsies had a significantly greater number of vascular thromboses in the superficial and deep dermis, but not in the subcutis, and significantly more leukocytoclasis in the upper dermis, compared to COVID-19(+) PU and COVID-19(-) PU histologic sections  patients with HASPIs led us to hypothesize that immunologic dysregulation due to SARS-CoV-2 infection may contribute to the pathogenesis of HASPIs in these patients. To explore this, the differentially expressed gene (DEG) profile of the tissue specimens from the TV and PU COVID-19(+) groups were compared to the COVID-19(-) PU controls. Dimensionality reduction using principal component analysis (PCA) revealed that the COVID-19(+) TV and PU groups segregated into separate clusters that were distinct from the COVID-19(-) PU control group (Among 44 DEGs that were unique to the COVID-19(+) TV group .HLA-DRB3, CXCL13, PTPN22 and AHR were identified as genes that were commonly down-regulated in both COVID-19(+) TV and COVID-19(+) PU groups compared to the COVID-19(-) PU control group. These genes are key players in antigen processing and T and B cell homeostasis. Only CIITA, a master regulator of MHC class II genes, was differentially expressed and down-regulated in the COVID-19(+) PU group compared to the COVID-19(-) PU controls. Taken together, this data suggests that innate immune responses are primarily dysregulated in COVID-19(+) TV pathology in comparison to COVID-19(-) PUs. In contrast, antigen processing and T and B cell signaling mechanisms are potentially downregulated in both COVID-19(+) TV and PU groups.To better characterize the transcriptional signatures in skin tissue from the three ulcer groups, hierarchical clustering of the top up- and down-regulated genes in each disease category was performed  patients may be a direct, local response to viral infection or a result of systemic immune responses. Although a majority of the COVID-19(+) TV tissue samples were positive for the presence of a limited set of SARS-CoV-2 transcripts  TV patients compared to COVID-19(+) PU and COVID-19(-) PU controls. Genes that were highly upregulated in COVID-19(+) TV were downregulated in both PU groups indicative of similar mechanisms of pathogenesis in PU. Upregulation of genes involved in neutrophil dysfunction and pro-inflammatory response mechanisms, specifically genes involved in response to viral infection, suggests that transcriptional differences observed in the COVID-19(+) TV group might be a result of SARS-CoV-2 infection  and account for the unique histopathological findings of thrombotic vasculopathy  TV group compared to the COVID-19(-) PU control group, indicating hampered adaptive immune responses in the COVID-19(+) TV group (To understand the effect of infection and thrombotic vasculopathy in the COVID-19(+) TV group compared to the COVID-19(-) PU control group, the top 10 enriched Gene Ontology (GO) biological pathways in the COVID-19(+) TV and COVID-19(-) PU control groups were identified, followed by a sub-analysis to determine the significantly altered transcripts within these pathways Figure 3The analysis was expanded to uncover transcriptional signatures in the above three biological pathways up-regulated in COVID-19(+) TV specimens compared to COVID-19(-) PU control specimens  cells were significantly higher in the COVID-19(+) TV group compared to the other disease groups  TV samples relate to regulation of immune cell populations, cell type scores were calculated to identify differences in the skin immune cell landscape in the tissue specimens from the three HASPI groups. Neutrophils and CD56e groups Table 3.Neutrophils have been implicated as key players in mechanisms contributing to immunothrombosis in COVID-19 disease –19. GiveNext, the dataset was surveyed for all genes that are involved in immune cell chemotaxis and extravasation  patients to study the role of mature and immature neutrophil subsets in COVID-19 disease pathogenesis . We utilImmature neutrophils, or low-density granulocytes, have been shown to undergo uncontrolled NET formation in several autoinflammatory and autoimmune diseases, contributing to disease pathology . Genes avs. PADI4) and neutrophil subsets that potentially contribute to disease pathology (immature vs. mature neutrophil subsets) differ, possibly due to local SARS-CoV-2 infection.Finally, NETs in skin tissue from all three HASPI groups were visualized by immunofluorescent staining. Rare NETs were detected in the COVID-19(+) PU group  patients. Overall, COVID-19(+) patients had a 63% increased incidence of developing a HASPI compared to COVID-19(-) patients Table 2.This study identified that COVID-19(+) patients are at risk of more severe HASPIs and more often require surgical debridement compared to COVID-19(-) patients 2. In paImportantly, when controlling for age, co-morbidities, and other variables COVID-19(+) patients with HASPIs were more likely to have a severe hospitalization course compared to COVID-19(+) patients without HASPI. These results support that development of a HASPI is a poor prognostic sign in the setting of COVID-19. However, it is unclear whether a severe hospitalization course increases risk of HASPI development, or vice versa. Regardless, this finding aligns with the hypothesis that HASPIs in COVID-19(+) patients likely arise as a result of both intrinsic and extrinsic disease factors.A comparison of laboratory parameters revealed COVID-19(+) HASPI patients had significantly higher maximum levels of serum ferritin compared to COVID-19(-) HASPI patients Table 1.Surprisingly, maximum D-dimer levels were not significantly higher in COVID-19(+) patients with HASPIs compared to COVID-19(-) patients with HASPIs in our cohort. However, a high percentage of COVID-19(-) patients had no D-dimer levels drawn, suggesting a selection bias towards evaluating D-dimer levels in those patients who may have had clinical features supporting a thrombotic state Table 1.SARS-CoV-2-induced thrombotic events in the skin microcirculation represent another possible contributor to increased HASPI risk in COVID-19 patients. Thrombotic events in the skin and other organs of patients with COVID-19 are well-described . AlthougIn our cohort, 25 patients had skin specimens of their HASPIs available, 15 of which were biopsies performed on COVID-19(+) patients (TV and PU). We compared the histologic, cellular and molecular characteristics of these specimens in detail to each other and to those in COVID-19(-) PU control skin tissue. Significantly higher numbers of vascular thromboses and leukocytoclasis were observed in skin tissue from the COVID-19(+) TV group compared to the other groups, supporting the role of thrombotic vasculopathy as a potential contributor to subsequent ulceration in some COVID-19(+) patients.Interestingly, vascular thromboses were detected occasionally in our COVID-19(-) PU control specimens. In fact, vascular thromboses have been described in skin biopsies of HASPIs in COVID-19(-) patients, and vascular occlusion has long been hypothesized to play an important pathogenic role in the development of PUs , 27. HowAlthough the majority of skin specimens from COVID-19(+) patients showed a TV pattern, one-third showed a PU histologic pattern with far less vascular thromboses compared to TV skin tissue. Although not statistically significant, mean maximum D-dimer levels were higher in the COVID-19(+) TV group compared to the COVID-19(+) PU group. This raises the possibility that there may also be a threshold D-dimer level, above which there is increased risk of vascular thrombosis in cutaneous vessels under pressure during viral infection, which might contribute to the unique histologic observations in this study.Transcriptional analyses on skin specimens from the sites of sacral ulcer formation evaluated the inflammatory landscape in skin tissue during SARS-CoV-2 infection in comparison to sacral ulcers obtained from uninfected patients. This type of analysis has not been previously performed. Our results showed that COVID-19(+) TV skin is associated with up-regulation of genes related to innate immune responses and viral defense responses, and down-regulation of T-cell mediated immune responses Figure 4Recently, Yang et al.  examinedCytokines, such as IL1A and IL27, which have been shown to be higher in patients with more severe COVID-19 disease –37, were+dim NK cell, CD45+ cell, and mast cell signatures, we focused our subsequent efforts on specifically understanding the role of neutrophils in the ulcer phenotypes with and without SARS-CoV-2 infection since neutrophils have been shown to play an important role in COVID-19 disease pathogenesis, particularly in relation to thromboses and multi-organ damage (high (PD-L1+) mature neutrophils in TV skin tissue suppress T cell responses due to the inhibitory interaction between PD-L1 and PD-1, resulting in stunted adaptive immune responses. In support of this, mature CD274 (PD-L1+) neutrophils have been shown to exert suppressive effects on T cell function in various other diseases like cancer and HIV infection (Cell type scoring demonstrated that neutrophils were enriched in COVID-19(+) TV skin compared to COVID-19(+) PU and COVID-19(-) PU skin. While we also observed changes in CD56n damage , 41. A sn damage . In our nfection , 43.vs. PU skin, indicating that neutrophils and NETs potentially contribute to cutaneous thrombotic vasculopathy during severe COVID-19 infection. While NETs were also observed in COVID-19(-) PU (controls), the baseline levels in 5 patients were nearly zero, indicating that exacerbated NET formation might be a consistent feature of TV in COVID-19(+) patients. In addition to significantly higher NETs in COVID-19(+) TV vs. COVID-19(+) PU skin, COVID-19(+) TV skin also had more intense neutrophil elastase and CD61 staining than COVID-19(+) PU skin, correlating with the known ability of NETs to propagate inflammation and thrombosis  TV rombosis . The degOur study has several advantages and limitations. Advantages include the establishment of a comprehensive COVID-19 registry at our institution that allowed identification of all COVID-19(+) patients admitted to our institutional hospitals, as well as a relatively large cohort utilized for analysis. Additionally, the standard protocol to photograph ulcers allowed objective confirmation that patients were accurately labeled as having HASPIs. More importantly, our results correlate clinical findings with histopathologic and molecular results. The comparison of COVID-19(+) patients with HASPIs to both COVID-19(+) patients without HASPIs and COVID-19(-) patients with HASPIs has not previously been done, especially with quantification of genes in sacral skin under pressure in all 3 groups. We were also able to corroborate the contribution of NETs to local tissue inflammation indicated by our transcript analyses using immunofluorescence imaging.Limitations include the retrospective nature of our study and possibility that electronic medical records did not always contain accurate documentation of data points (i.e. date of ulcer onset). One important data point that we could not completely characterize was skin color due to absence of images and/or incomplete disclosure of ethnicity for several patients in our cohort. However, we believe it is notable that our COVID-19(+) HASPI cohort had a large number of black patients. As black patients have been found to be at increased risk for severe COVID-19 compared to other ethnic groups, our ethnicity data further supports a link between severe COVID-19 and HASPI development, and suggests clinicians should be particularly meticulous in monitoring black COVID-19(+) patients for HASPI development , 46. AddIn conclusion, we show that COVID-19(+) patients are at an increased risk of HASPIs compared to COVID-19(-) patients, and COVID-19(+) patients with HASPIs develop ulcerations of more severe stage that more often require debridement than COVID-19(-) patients. Furthermore, we show that thrombotic vasculopathy was prominent in sacral skin of COVID-19(+) patients with HASPIs compared to controls, and TV tissue displayed increased expression of DEGs known to contribute to abnormal thrombosis and ischemia. Overall, our results implicate SARS-CoV-2 infection as a contributing factor to HASPIs in at least some COVID-19 patients.Unlike other reports of skin manifestations in COVID-19 patients, our overall findings support the skin is yet another organ system that may sustain significant damage secondary to COVID-19. Recognizing risk of HASPIs and the potential role of SARS-CoV-2 infection in COVID-19 patients is important, as they may contribute to significant future morbidity, healthcare expenditure, and even mortality in patients who recover from COVID-19. Further research is needed to uncover detailed pathogenic factors leading to increased HASPI risk in COVID-19(+) patients, as well as how to optimally prevent them from occurring. For now, healthcare workers should be especially attentive to potential early signs of HASPI development in patients hospitalized with COVID-19, especially those with serious disease, and be meticulous with currently proven preventative clinical interventions.Patients with HASPIs were identified as having been hospitalized for greater than 5 days within our institution between March 1 and December 31, 2020, with an ICD-10 diagnosis of sacral ulcer or photographic documentation of sacral ulceration at some point during hospitalization (Patients that did not undergo a polymerase chain reaction (PCR)-based COVID-19 test at admission or during hospitalization, those hospitalized for <5 days, and those with sacral ulcers already present at hospital admission were excluded from our study. Additionally, stage 1 ulcer patients were excluded as these types of ulcers are difficult to reliably detect and are not routinely documented by our wound care teams. Some patients were hospitalized more than once during our defined study period. However, only the hospitalization associated with an initial development of a HASPI was included for our study purposes.Among the 143,223 patients hospitalized at our institution during our defined study period (March 2020-December 2020), 58,766 patients met our inclusion/exclusion criteria and comprised our study cohort , HASPI ulceration risk , presence of specific medical comorbidities ) index hospitalization information  admission, intubation/mechanical ventilation requirements), laboratory results ), HASPI characteristics , and 30-day HASPI-related morbidity information .The Braden risk assessment score is a validated clinical tool calculated by nursing staff throughout our institution as part of routine patient skin assessments at admission and every 12 hours thereafter. This standardized risk scale assigns numerical ratings (ranging from 1-4) to six domains, including sensory perception, moisture, activity, mobility, nutrition, and friction/shear forces . The sum2) of sacral ulceration during the index hospitalization. Ulcer stage was defined as most severe stage of ulcer  recorded during hospitalization according to National Pressure Injury Advisory Panel (NPIAP) classifications. Severe stage of HASPI was defined as either Stage 3 or Stage 4 HASPI according to NPIAP staging criteria. These specific stages were selected as they are considered hospital ‘never events’ by the NPIAP due to their associated, independent risk of increased morbidity and mortality . Time to ulcer formation was measured in days from admission to first mention and/or photographic documentation of HAPSI in the patient’s EMR, whichever occurred first. Ulcer size was defined as the largest recorded area  stained tissue specimens by a board-certified dermatopathologists (APF and JDM). For COVID-19(+) patients, 4mm biopsies from  areas of sacral purpura were obtained. For control COVID-19(-) specimens, which were typically submitted to pathology during debridement, a search of Cleveland Clinic pathology archives was performed to find specimens from PUs. H&E sections of all available specimens were reviewed to identify those appropriate to use as control specimens. Any specimens with histologic features suggestive of secondary infection, extensive epidermal ulceration, or prominent fibrosis (supporting significant chronicity) were excluded. Ten specimens were found to have histologic features suggestive of inherent pressure-induced changes. For these, a 4mm wide area was demarcated and examined to be consistent with the area examined in COVID-19(+) tissue. When possible, areas with intact epidermis were chosen in COVID-19(-) specimens to also reduce variability with tissue examined from COVID-19(+) patients. Histopathologic features that were evaluated included neutrophil infiltrate, vascular thrombosis, total number of thromboses, intraluminal neutrophils and leukocytoclasis Table 3.RNA was extracted from Formalin-Fixed and Paraffin Embedded (FFPE) tissue biopsies, using the RNeasy FFPE kit (Qiagen) with deparaffinization solution (Qiagen). Total RNA concentrations were determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). One-hundred nanograms of RNA was then hybridized with the NanoString Human Autoimmune Profiling Panel and spike in Coronavirus Panel Plus probe sets (NanoString Technologies). Transcriptional analysis of immune related genes and SARS-CoV-2 genes in tissue biopsies was conducted using the NanoString nCounter Pro Analysis System (NanoString Technologies). Raw data was initially processed using the nSolver Analysis Software (V.4.0). Quality control and normalization was performed using the 6 positive control, 8 negative control, and 20 housekeeping probes. Samples flagged for failing quality control were removed. Remaining data was assessed for cell type scoring using nSolver Advanced Analysis (V.2.0) and exported for subsequent analysis in R software.vs Control results against PU vs Control. Resulting residuals for principal component analysis (PC1 and PC2) were plotted using ‘ggplot2’ package (version 3.3.5). For pathway analysis, DEGs were separated into up- and down-regulated gene lists (fold change > 0 and fold change < 0 respectively). Package ‘gprofiler2’ (version 0.2.1) to find up- and down-regulated pathways. GO pathways were filtered to remove redundant pathways to generate the data by GO’s hierarchically structured database. Using ordered p-values from pathway analysis, the top 10 up-regulated and down-regulated pathways were obtained and p-values were log10-transformed for Analysis was performed using R (version 4.1.0). Venn diagram was generated utilizing DEGs from TV Paraffin embedded 5 μm tissue sections were sectioned by the Cleveland Clinic Histology Core. Sections were deparaffinized and blocked using blocking buffer  for 1 hour at room temperature. Qualitative evaluation of presence of NETs in the tissue was performed using antibodies for anti-neutrophil elastase  and Anti-Histone H3 . Primary antibody dilutions were made in the blocking buffer  and added to the sections for an overnight incubation at 4°CC. Next day, slides were washed in 1X PBS (3X) and secondary antibodies were added to the sections for 1 hour at room temperature . After the 1 hour incubation, slides were washed in 1X PBS (3X) and mounted with DAPI . Images were obtained using an inverted Leica SP8 confocal microscope using objective lens 40X oil/1X zoom factor. Five fields/tissue section were imaged for the purpose of quantitative assessment of presence of tissue NETs in the epidermis as well as the dermis. Images obtained from various disease groups were qualitatively assessed for presence of high, medium-low and no tissue NETs. Area of NETs was quantified using Image J software (version 1.53r 21).Categorical variables are presented as count (percentage) and continuous variables as median (interquartile range). Wilcoxon rank-sum was utilized for two-sample continuous variable comparisons. Chi-square and Fisher’s Exact tests were utilized to compare nominal categorical data across groups. Cochrane-Armitage exact trend test was utilized to compare univariate ordinal categorical data. Multivariable logistic and multivariable Cox proportional hazards regression were used to model binary and time-to-event outcomes, respectively. Multivariable linear regression was used to model wound size. Potential for multicollinearity was explored using variance inflation factor analysis. One-way ANOVA was utilized for analysis of tissue NETs with post-hoc Tukey analysis. An a level of 5% (two-sided) was utilized for all comparisons. Statistical analysis was performed using SAS JMP Pro (version 15) and Prism (version 9.3.1).www.ncbi.nlm.nih.gov/geo/, GSE214647.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by Cleveland Clinic Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.JN, SJ, AF and CM conceptualized and designed the study. JN, SJ, KC, AF and CM acquired funding for the study. JN, SJ and AP carried out laboratory-based investigations. JN, SJ, AP, RM, JG, AW, SS, JJ, LK, AN, JM, EM-C, SG, KC, AF, CM carried out clinical-based investigations and analysis of data including statistical analysis. AF and CM supervised the study. JN, SJ, AP and RM created the original figures and tables. AP and RM performed analysis of the Nanostring data. JN and SJ wrote the original draft of the manuscript. JN, SJ, AP, AF and CM wrote, reviewed and edited the manuscript. The order of authors was based on their sequential roles in the study. All authors contributed to the article and approved the submitted version.