Increased levels of all TIL subsets, except CD4+, were associated with estrogen receptor‐negative tumors (p < 0.001) and high tumor cell proliferation by Ki67 (p < 0.001). Furthermore, high levels of all TIL subsets were associated with high macrophage counts (p < 0.001) and low‐grade stromal elastosis (p ≤ 0.02). High counts of CD3+, CD8+, and FOXP3+ TILs were associated with interval detected tumors . Finally, in the luminal A subgroup, high levels of CD3+ and FOXP3+ TILs were associated with shorter recurrence‐free survival, and high counts of FOXP3+ were linked to reduced breast cancer‐specific survival. In conclusion, higher levels of different TIL subsets were associated with stromal features such as high macrophage counts (CD163+), presence of vascular invasion, absence of stromal elastosis, as well as increased tumor cell proliferation and interval detection mode. Our findings support a link between immune cells and vascular invasion in more aggressive breast cancer. Notably, presence of TIL subsets showed prognostic value within the luminal A category.The tumor microenvironment plays a critical role in breast cancer progression. Here, we investigated tumor‐infiltrating lymphocytes (TILs) and associations with macrophage numbers, tumor stromal elastosis, vascular invasion, and tumor detection mode. We performed a population‐based retrospective study using data from The Norwegian Breast Cancer Screening Program in Vestfold County (2004–2009), including 200 screen‐detected and 82 interval cancers. The number of TILs  and tumor‐associated macrophages (CD163+) was counted using immunohistochemistry on tissue microarray slides. Lymphatic and blood vessel invasion (LVI and BVI) were recorded using D2‐40 and CD31 staining, and the amount of elastosis (high/low) was determined on regular HE‐stained slides. High numbers of all TIL subsets were associated with LVI ( Mammography screening identifies a subset of breast cancers with a better prognosis than expected based on standard histopathologic and molecular prognostic features , 2, 3. TBreast tumors are composed of a heterogeneous population of cells including both tumor cells and the surrounding microenvironment. Stromal components facilitate cancer progression and can be predictive of both prognosis and treatment response , 9, 10. Previously, we reported that deposition of abnormal elastin material (elastosis) in breast cancer stroma is associated with mammographic screen detection, low tumor cell proliferation by Ki67 expression, and a favorable patient prognosis . In a suBased on the importance of the tumor immune microenvironment (TIME) in breast cancer , 18, 19,A population‐based cohort of 285 patients from Vestfold County in Eastern Norway, initially including 202 invasive screen‐detected cancers and 83 invasive interval tumors, was identified. The Norwegian Breast Cancer Screening Program, implemented in 2004, involves biannual mammography in the age‐group 50–69 years. An interval cancer was defined as a cancer diagnosed between two screening sessions. Three patients were excluded: one patient with no residual tumor tissue for analysis, one screen‐detected malignant phyllodes tumor, and one patient with an interval cancer unsuitable for further surgical treatment. Four patients, two screening and two interval cancers, had simultaneous tumors in both breasts. Tumors with the worst prognostic profile, based on the Nottingham Prognostic Index, were selected for inclusion in the study cohort. Finally, 200 screen‐detected and 82 interval cancers were included for further analyses.Information on clinical data, tumor stage, and survival was retrieved from patient records. Last clinical follow‐up was in August 2018. Regarding primary treatment, 204 breast resections (72%) and 78 mastectomies (28%) were performed; 218 patients received radiation therapy (77%), 156 received endocrine treatment (55%), and 77 received chemotherapy (27%); 12 patients lacked information on nonsurgical primary treatment. Subsequent distant metastases were observed in 42 cases (15%), and 32 patients (11%) died of breast cancer during the follow‐up period. The study was approved by the Regional Ethics Committee of Eastern Norway (reference #2018/1102).Three tissue cores (diameter 1.0 mm) were extracted from the invasive edge of tumor samples in paraffin‐embedded blocks and inserted into tissue microarray (TMA) recipient blocks using a semi‐automated precision instrument . As reported in previous studies , 20, 21,Immunohistochemistry was performed on 4–5 μm standard tumor tissue sections of formalin‐fixed paraffin‐embedded TMAs. All slides were dewaxed with xylene/ethanol before antigen retrieval in a pressure cooker , or in a microwave, in Target Retrieval Solution (TRS) buffer (pH 9) . An endogenous block was applied prior to incubation with the primary antibodies to block for endogenous enzymatic activity. The slides were then incubated for 1 h with mouse monoclonal CD3, CD4, CD8, CD45, and FOXP3 antibodies  followed by incubation with an appropriate HRP‐En Vision (Dako), EnVision Mouse hrp, K4001 for CD8 and FOXP3, EnVision rabbit HRP, and K4003 for CD3 and CD4. Primary antibodies were omitted for the negative controls. Tissue from differentiated breast cancer types were used as positive controls.2), and the numbers of positive cells were counted within one such area per case, similar to what we reported previously for macrophages [Cytoplasmic CD3, CD4, CD8, CD45, and FOXP3 staining was evaluated in a quantitative manner. Staining was examined in the most active areas (hot‐spots), often situated in the periphery of the tumor Figure . Lymphocrophages .A total of 50 immunostained cases were examined by two pathologists (YC and TAK), with kappa values ranging from 0.69 to 0.88 for various lymphocyte populations.Immunohistochemical evaluations of CD163 staining for TAMs, D2‐40 staining for lymphatic vessel invasion (LVI), and CD31 staining for blood vessel invasion (BVI) have been described previously, and data were included for comparisons , 13. EvaAll cases were microscopically reexamined by one of the authors (TAK) using hematoxylin–eosin–safran (HES)‐stained sections from our previous study. Tumors were classified as either invasive carcinoma of no special type or other types. Histologic grading was performed according to the Nottingham criteria . Tumor dElastosis can easily be recognized in standard HES slides as a deposit of gray, fibrillary material, and the histologic evaluation of tumor elastosis was described in our previous paper . In brieP values of <0.05 were considered statistically significant. Associations between categorical variables were assessed by Pearson's chi‐square test. Spearman's rank correlation test was applied when comparing bivariate continuous variables and Spearman's correlation coefficients are reported. When analyzing differences in TIL subsets between phenotypic categories, Kruskal–Wallis test was applied. Univariate survival analysis of time to death due to breast cancer  and time to recurrence for patients without metastases at the time of diagnosis  were performed using the Kaplan–Meier method (log‐rank test for differences). Entry date was the time of diagnosis. Patients who died from other causes were censored at the date of death in the analyses of DSS. A multivariable logistic regression analysis was applied to assess the ability of different TIL subsets to predict aggressive breast cancers . For statistical analysis, TIL scores were dichotomized; high TIL score (upper quartile) and low TIL score (others). A total of 282 patients were available for survival analysis in the current study.All statistical analyses were performed using IBM SPSS Statistics, version 23.0 . Two‐sided r = 0.80, p < 0.001). Median counts of CD3, CD4, CD8, CD45, and FOXP3 are 37, 15, 18, 42, and 4, respectively  , HER2 positivity , high Ki67 , BVI , LVI , and high CD163 TAM . In contrast, the other TIL subsets were not independently associated with these clinicopathologic features, except that, in addition to FOXP3, both CD3 and CD4 were independently associated with CD163 TAM status .When including all TIL subsets  in multivariable logistic regression analyses as explanatory variables, and using histologic grade, ER/PR/HER2 status, Ki67, BVI, LVI, CD163 TAM, elastosis, and detection method as predicted (outcome) variables in separate regression models, we consistently found that FOXP3 independently predicted aggressive tumor features, such as ER negativity . The general pattern was that most LVI−/BVI− tumors (around 80%) had low numbers of infiltrating lymphocytes, whereas a much higher percentage (44–56%) of LVI+/BVI+ tumors were highly infiltrated (Table Table ed Table . LVI+/BVp < 0.001). In general, luminal A and luminal B HER2‐negative tumors showed on average low‐grade lymphocytic infiltration in around 80% of tumors , 6% luminal B (HER2 positive), 4% HER2‐type, and 7% triple‐negative tumors in this population‐based cohort (Table rs Table . In contThe median follow‐up time was 138 months (range 108–168). Among the 282 patients, metastases or local recurrence were observed at follow‐up in 42 cases (15%), and 32 patients (11%) died of breast cancer.n = 145), univariate survival analysis indicated that high levels (by upper quartile) of CD3‐ and FOXP3‐positive TILs were associated with reduced RFS .Univariate survival analysis of various TIL subsets across all breast cancer subtypes in the whole cohort did not reveal significant prognostic associations. Among luminal A tumors , but did not show significance in patients who received endocrine therapy. Also, high levels of CD3 and CD4 were associated with reduced RFS in patients who did not undergo chemotherapy , while there was no significant difference in cases with treatment. As expected, treated and untreated cases (for both endocrine treatment and chemotherapy) showed significant differences with respect to tumor diameter, histologic grade, lymph node status, as well as ER and PR positivity.High levels of both CD3 and FOXP3 were associated with reduced RFS in patients who had not received endocrine therapy , absence of stromal elastosis, tumor cell proliferation, and interval detection mode. Our findings suggest an importance of tumor microenvironment factors, and interactions between them, for progression of breast cancer subgroups. Notably, the presence of FOXP3+ lymphoid cells was strongly associated with most factors studied, and was also prognostic within the low‐grade luminal A category. The interplay between cancer cells and their microenvironment to influence cancer progression is a challenging and extremely complicated process, which needs further investigation.YC, TK and LAA contributed to study design, data interpretation, literature search and writing of the manuscript. YC, TAK and HA participated in data collection. YC, TAK and EW performed data analysis and generation of figures. All authors approved the final version of the manuscript.Figure S1. Median CD3, CD4, CD8, FOXP3, and CD45 counts in molecular breast cancer subgroupsTable S1. Counts for different TIL categoriesTable S2. Cross‐correlation between different TIL subsets and tissue macrophagesClick here for additional data file.


EGFR‐mutant non‐small cell lung cancer (NSCLC). However, the effect of EGFR‐TKIs are eventually weakened due to resistance, and there is also a differential efficacy based on EGFR mutation subtypes. The combination of angiogenesis inhibitor (AI) with EGFR‐TKI has shown better efficacy than EGFR‐TKI monotherapy, regardless of the mutation subtypes. Nevertheless, the effect of AI eligibility on overall survival (OS) and progression‐free survival (PFS) remains to be elucidated. Thus, we assessed this impact on patients with NSCLC harboring EGFR mutation.Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) are currently the primary treatment option for patients with EGFR‐mutant NSCLC, who were treated with EGFR‐TKI monotherapy, were retrospectively analyzed for AI eligibility. The patients were categorized into AI‐eligible (AI fit) and ineligible groups (AI unfit).In this study, the data for 450 patients with p = 0.007), and OS was also significantly longer in the AI fit group (median OS = 33.0 months) compared to that in the unfit group . Multivariate analysis indicated that AI ineligibility was associated with shorter PFS and poor prognosis. Also, in the AI fit group, there was no significant difference in the PFS between EGFR L858R mutation and EGFR exon 19 deletion .The median PFS of the AI fit group was 12.9 months, compared to 9.6 months in the unfit group  eligibility on epidermal growth factor receptor mutant non‐small cell lung cancer. AI eligibility was associated with longer progression‐free survival and overall survival. Selection bias may affect previous data on the efficacy of AI combination therapy. Treatment with EGFR‐tyrosine kinase inhibitors (TKIs) has enabled significantly prolonged outcomes for patients compared to conventional platinum‐based combination chemotherapy. EGFR‐TKIs became the standard first‐line regimen for patients with NSCLC harboring an EGFR mutation. Yet, the benefits of EGFR‐TKIs eventually fade because of acquired resistance to TKI treatment. Additionally, previous EGFR‐TKI monotherapy studiesEGFR mutation subtypes and have suggested that tumors with exon 19 deletion (19 del) exhibit a higher sensitivity to EGFR‐TKI treatment compared to those with EGFR an exon 21 point mutation (L858R). Thus, novel treatment regimens for NSCLC harboring EGFR mutations are needed to prolong the survival time and reduce the imbalance in the effectiveness between EGFR subtypes.Lung cancer is one of the major causes of cancer‐related deaths in the world. Although the prognosis remains limited compared to that of other types of cancers, the evolution of molecular targeted therapy has drastically improved the prognosis, especially in driver mutation‐positive non‐small cell lung cancer (NSCLC). Epidermal growth factor receptor  plus erlotinib combination therapy showed significantly prolonged progression‐free survival (PFS) compared with those who received erlotinib monotherapy.EGFR mutation, especially the L858R subtype.Recently, the EGFR‐TKI plus angiogenesis inhibitors (AIs) combination therapy has demonstrated better efficacy than EGFR‐TKI monotherapy in patients with EGFR mutation is also unclear. Thus, in this study, we aimed to assess the prognostic impact of AI eligibility criteria on patients with NSCLC with EGFR mutation and compare the effectiveness of EGFR‐TKI monotherapy in patients with an L858R mutation to that in those with exon 19 deletion in the AI‐eligible population.However, because of the unique eligibility criteria for AIs, some scientists express concerns about a selection bias in AI combination clinical studies and believe that a better prognosis can be achieved even without the administration of AIs.22.1EGFR mutations in tumor tissues were detected using cobas® EGFR mutation test v2 (Roche Molecular Systems), CycleavePCR™ Assay , or scorpion arms assay (DxS). Tumor response was evaluated using Response Evaluation Criteria in Solid Tumor criteria version 1.1.Patients with an EGFR‐mutant (L858R or 19 del) NSCLC, who started first‐line EGFR‐TKI treatment at Shizuoka Cancer Center between 2002 and 2019 were collected retrospectively. We excluded patients whose Eastern Cooperative Oncology Group (ECOG) performance status (PS) was 3 or 4, who had been treated by any AIs, and patients with symptomatic brain metastasis. We defined the enrolled patients as the ALL group. In the ALL group, we defined patients as AI‐ineligible if they met at least one of the following conditions: (1) A history of tumor exposure in the bronchus or of producing bloody sputum; (2) a major vessel infiltration (MVI) by the tumor, diagnosed by a radiologist; (3) a history of cardiovascular disease (CVD), including thrombotic diseases, ischemic heart diseases, or congestive heart failure; (4) treatment by chemoradiotherapy before initiation of EGFR‐TKI monotherapy. Since there were no cases with active peptic ulcer disease before treatment, we did not include active peptic ulcer disease as the classifying condition. Next, we evaluated the effects of first‐line EGFR‐TKI monotherapy in both AI‐eligible (AI fit) and ineligible (AI unfit) groups. We also evaluated the efficacies of EGFR‐TKI monotherapy in patients with exon 19 deletion or L858R mutation in the AI fit and unfit groups. PFS was defined as the time from the start of EGFR‐TKI monotherapy to death, disease progression, or censoring at the last follow‐up examination. A considerable number of cases showed several months or years of a treatment‐free period due to EGFR‐TKI toxicity before disease progression or initiation of sequential treatment. Therefore, patients who started new therapy without confirmation of tumor progression were censored at the time of the latest tumor assessment before the new therapy was initiated. We defined overall survival (OS) as the interval between the initiation of EGFR‐TKI therapy, and death from any cause or censoring at the last follow‐up. This study was authorized by the institutional review board of Shizuoka Cancer Center.2.2U test for all continuous variables, and Fisher's exact test for the categorical data. Clinical evaluation of PFS and OS was conducted using the Kaplan–Meier method. We used the Log‐rank test to compare the cumulative survival in each group and Cox's proportional hazards analysis for the multivariate analysis. All p values were two‐sided, and p < 0.05 was considered significant. Statistical analyses were performed using EZR , which is a converted version that added frequently‐used biostatistical functions to an original R commander (version 1.6‐3).Patient characteristics were compared between the AI fit and unfit groups and patients with L858R and exon 19 deletion, using the Mann–Whitney 33.1EGFR major mutation‐positive patients had been treated with first‐line EGFR‐TKIs monotherapy during the study term  and age (p = 0.019).This study included 293 female patients (65.1%), and the median age at the initiation of EGFR‐TKI therapy was 71 years (range 31–92). Most patients were diagnosed with stage IV disease (69.8%), and 28.0% relapsed after the surgery. Around 50% of the enrolled patients had a history of smoking, and 45.8% had an L858R mutation. Most patients underwent gefitinib treatment (60.7%), followed by erlotinib (18.9%), osimertinib (13.6%), and afatinib (6.9%). As per the four factors of AI eligibility, 113 patients were defined as AI‐ineligible, of which 51 patients had a history of tumor exposure in the bronchus or of producing bloody sputum, 17 had MVI, 45 had a history of CVD, and 16 had been treated with chemoradiotherapy before starting the EGFR‐TKI treatment. Both groups (AI fit group and AI unfit group) showed similar patient characteristics regarding sex, smoking history, 3.2p = 0.007; Figure p = 0.018), stage , and PS  were significantly associated with PFS  (Kaplan–Meier estimate), the PFS was significantly better in the AI fit group (median PFS = 12.9 months) than in that the AI unfit group , age , sex , PS , stage , and EGFR subtype  were indicated as prognostic factors (Table p = 0.027), and was indicated to be a significant prognostic factor for the OS .In the multivariate analysis of the OS, AI eligibility , and PS 0–1  were associated with a significantly longer PFS; however, the efficacy of EGFR‐TKI in the EGFR mutation subtypes was not significant ; however, multivariate analysis indicated that age , PS , and stage  were significant prognostic factors in the AI fit group, demonstrating that the EGFR mutation subtype  with an L858R mutation and 55.2% (186/337) with an exon 19 deletion. Median PFS in the AI fit subgroup was 11.4 months for L858R mutation and 13.8 months for exon 19 deletion; although it did not show a significant difference, 19 del resulted in a longer PFS , and the OS was 20.0 and 17.6 months, respectively , PS , and a history of tumor exposure in the bronchus or producing bloody sputum  were related to a shorter PFS , ECOG PS, and clinical stages are related to EGFR mutation‐positive NSCLC prognosis.EGFR‐mutant NSCLC.EGFR mutation. Therefore, we believe that there is a possibility that the results described in previous AI combination studies may have overestimated median PFS data or median OS data because of the selection bias due to AI eligibility. Our results also explain why erlotinib monotherapy in the NEJ026 study resulted in a relatively longer median PFS (13.3 months) and OS (46.2 months) than other erlotinib monotherapy studies ,Several factors, such as sex, history of smoking, EGFR mutation subtype has been considered as an important stratified factor in recent clinical trials for patients with NSCLC harboring EGFR mutations.de novo T790M resistance mutation, tumor heterogeneity, difference in protein structure, and EGF‐induced tyrosine phosphorylation patterns.EGFR subtype was not an independent factor for both PFS and OS. Our results imply that the prognosis of EGFR subtypes is comparable for patients that are potentially eligible for AI, irrespective of the AI used. Similar results can be observed in the NEJ 026 and RELAY studies, which demonstrated an equivalent median PFS in subgroups of EGFR mutations in the erlotinib monotherapy group.Previous phase III studies and meta‐analyses have shown that patients with an exon 19 deletion benefit more from EGFR‐TKI treatment than those with an L858R mutation. Accordingly, EGFR subtypes.In this study, we also assessed the effects of all four factors of an AI fit condition. The multivariate analysis demonstrated that among the four factors, a history of tumor exposure in the bronchus or producing bloody sputum significantly affected both PFS and OS. This factor was likewise associated with PFS and OS in the L858R subgroup, but not in the 19 del subgroup. The presence of bloody sputum before treatment is reported to be a strong prognostic factor for advanced non‐squamous NSCLC.The limitations of this study must be acknowledged. First, since this is a single‐center, retrospective study, there is a possibility of an unintentional selection bias. Further, unlike previous phase III studies and meta‐analyses of EGFR‐TKI treatment, the PFS in the ALL group was equivalent between 19 del and L858R subgroups, which might have affected the result for the AI fit and unfit groups.5EGFR mutation. Moreover, the impact of AI eligibility may differ based on the EGFR subtype, which could, in turn, result in an unexpected selection bias in clinical trials and an overestimation of results. Since there is a possibility that such a selection bias affected previous trials evaluating the efficacy of EGFR‐TKI in combination with AI, the results of these trials should be carefully evaluated henceforth.Our study showed that the eligibility for AIs resulted in a longer PFS and OS and suggested that selection bias for AI eligibility could impact the selection of patients with NSCLC harboring an H. K, T. K, A. N, M. Y, N. N, T. M, and E. M. has nothing to disclose. H. K. reports grants and personal fees from Chugai Pharmaceutical Co, Ltd, personal fees from Ono Pharmaceutical Co, Ltd, personal fees from Boeringer Ingelheim, personal fees from Eli Lilly K.K, personal fees from Kyowa Hakko Kirin Co. Ltd, personal fees from Bristol‐Myers Squibb, personal fees from MSD, grants and personal fees from Novartis Pharma K.K, grants and personal fees from Daiichi‐Sankyo Co. Ltd, grants and personal fees from AstraZeneca K.K, personal fees from Pfizer, and personal fees from Taiho Pharma outside the submitted work. N. M. reports personal fees from AstraZeneca KK, Pfizer Japan Inc, personal fees from Chugai Pharmaceutical Co. Ltd, grants and personal fees from Boehringer Ingelheim, personal fees from MSD K.K, personal fees from Taiho Pharmaceutical, and personal fees from Ono Pharmaceutical Co. Ltd outside the submitted work. H. K. reports personal fees from Eli Lilly K.K, personal fees from Taiho Pharmaceutical, and personal fees from AstraZeneca outside the submitted work. S. O. reports personal fees from Chugai Pharmaceutical Co. Ltd, personal fees from Ono Pharmaceutical Co. Ltd, personal fees from Taiho Pharmaceutical Co. Ltd, personal fees from Daiichi Sankyo Co. Ltd, personal fees from Amgen K.K., personal fees from AstraZeneca K.K, and personal fees from Novartis Pharma K.K outside the submitted work. K. W. reports grants and personal fees from Chugai Pharmaceutical CFo. Ltd, personal fees from Taiho Pharmaceutical, personal fees from Boehringer Ingelheim, personal fees from Eli Lilly K.K, personal fees from Ono Pharmaceutical, personal fees from MSD, grants and personal fees from Astrazeneca, grants from Novartis, and grants from Abbvie outside the sFDrubmitted work. T. N. reports grants from Ono Pharmaceutical Co. Ltd, grants from Pfizer US. Inc, and grants from Mochida Pharmaceutical Co. Ltd outside the submitted work. H. Murakami reports grants and personal fees from AstraZeneca, grants and personal fees from Chugai pharma, grants and personal fees from Takeda, grants and personal fees from Daiichi Sankyo, grants from Abbvie, grants from IQvia, personal fees from Ono Pharmaceutical, personal fees from Bristol‐Myers Squibb Japan, personal fees from MSD, personal fees from Pfizer, personal fees from Novartis, personal fees from Lilly Japan, and personal fees from Taiho Pharmaceutical outside the submitted work. T. T. reports grants and personal fees from AstraZeneca KK, Pfizer Japan Inc, grants and personal fees from Eli Lilly Japan K.K, grants and personal fees from Chugai Pharmaceutical Co. Ltd, grants and personal fees from Ono Pharmaceutical Co. Ltd, grants and personal fees from MSD K.K, grants and personal fees from Boehringer Ingelheim Japan Inc, grants and personal fees from Pfizer Japan, Inc, and personal fees from Roche Diagnostics K.K outside the submitted work.This study was approved by the institutional review board of Shizuoka Cancer Center (IRB No. J2020‐170‐2020‐1), and performed under the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The need for informed consent was waived owing to the retrospective nature of the study.


The optimal first‐line approach for patients with symptomatic atrial fibrillation (AF) remains unclear. We compared the efficacy and safety of cryoballoon ablation (CBA) and antiarrhythmic drugs (AADs) in the initial management of symptomatic AF.CBA is superior to AAD as initial therapy for symptomatic AF.We searched the EMBASE, PubMed, and Cochrane Library databases for randomized controlled trials (RCTs) that compared CBA with AAD as first‐line treatment for AF from the date of database establishment until March 18, 2021. The risk ratio (RR) with a 95% confidence interval (CI) was used as a measure of treatment effect.p < .00001; I2 = 0%). There was a significant difference in the rate of symptomatic atrial arrhythmia recurrence ; however, the rate of serious adverse events was similar between the two treatment groups . Transient phrenic nerve palsy occurred in four patients after the CBA procedure.Three RCTs that enrolled 724 patients in total were included in this meta‐analysis. Majority of the patients were relatively young and had paroxysmal AF. CBA was associated with a significant reduction in the recurrence of atrial arrhythmia compared with AAD therapy, with low heterogeneity (RR, 0.59; 95% CI, 0.49–0.71; The current meta‐analysis suggests that CBA is more effective than AAD as initial therapy in patients with symptomatic paroxysmal AF. Serious iatrogenic adverse events are uncommon in CBAs. RFA applies radiofrequency currents in a point‐by‐point mode, whereas CBA uses low‐temperature energy on the balloon in a one‐step mode, causing cellular necrosis by tissue heating and freezing, respectively. A previous meta‐analysis of three randomized controlled trials (RCTs) demonstrated that RFA was more effective than AAD as an initial treatment strategy for paroxysmal AF.2No ethical approval and patient consent were required.2.1ClinicalTrials.gov for further potential information. There were no restrictions on publication year or language.This meta‐analysis was conducted based on the preferred reporting items for systematic reviews and meta‐analyses (PRISMA) guidelines.2.2Studies that met the following criteria were included: (a) RCTs that evaluated CBA versus AAD as initial therapy for symptomatic AF and (b) studies that reported efficacy and/or safety outcomes. Observational studies, reviews, editorials, and letters were excluded. The Cochrane bias risk assessment tool was used to evaluate bias in the included trials.2.3Two authors (M.‐Y.J. and F.‐W.Y.) independently extracted relevant data from the included studies and reached an agreement on all items. Disagreements were resolved through discussion. The following data were recorded from each trial: study name, year of publication, sample size per group, mean age of patients, proportion of males per group, number of patients who crossed over from the AAD group to the CBA group or vice versa, and duration of follow‐up. Additionally, underlying diseases and cardiac function parameters of subjects were recorded, including hypertension, diabetes mellitus, coronary artery disease, previous thromboembolic events , left atrial diameter, and left ventricular ejection fraction.2.4The primary outcomes of this study were recurrences of atrial arrhythmia and symptomatic atrial arrhythmia. The secondary outcomes were serious adverse events (SAEs), additional ablation after initial treatment failure with either CBA or AAD, percentage of crossover to the alternative treatment, thromboembolic events, bradycardia, syncope, chest pain, vascular access site hemorrhage, and pericardial complications .2.5I2 statistic was used to analyze the heterogeneity, which was divided into high (I2 ≥ 75%), moderate (I2 > 25% and <75%), and low (I2 ≤ 25%).p < .05. RevMan software (version 5.3.5) was used for all analyses.The risk ratio (RR) and 95% confidence interval (CI) were calculated to compare the effects of the combined therapy. The Cochrane 33.1The PRISMA flowchart systematically describes the literature selection process Figure . First, The three trials were multicenter and included 724 patients, with 365 in the CBA group and 359 in the AAD group.3.2p < .00001; I2 = 0%; Figure p < .0001; I2 = 0%; Figure Three trials comprising 724 patients reported recurrence of atrial arrhythmia during follow‐up. CBA was associated with a significant reduction in the recurrence of atrial arrhythmia compared with AAD, with low heterogeneity , stroke (n = 1), symptomatic PV stenosis (n = 1), and tamponade (n = 7).The comparative outcomes between RFA and AAD cannot be extrapolated to studies on CBA versus AAD as first‐line therapy. Unlike in comparisons between CBA and AAD groups, there is no significant difference in the recurrence rate of symptomatic AF between RFA and AAD groups.It is worth noting that due to the potential extra‐cardiac toxicity of amiodarone, it was excluded from use in all included RCTs. However, amiodarone is the most effective drug for restoring and maintaining normal sinus rhythm.5Although this meta‐analysis only included RCTs, there were certain limitations. First, our analysis was based on study‐level data, not on data from individual participants, which could have greatly increased the validity of the comparison. Second, publication bias was not carried out due to the small number of qualified studies. Third, the included studies were methodologically inconsistent, particularly in the detection of arrhythmias during follow‐up. Using intermittent rather than continuous monitoring may have led to an overestimation of the treatment success rates in both groups. Fourth, the current study was not registered at the International Prospective Register of Systematic Reviews (PROSPERO), although we strictly followed the steps in conducting a systematic review. Finally, the follow‐up duration in the current trial was limited to 12 months, so we were unable to determine the long‐term impact of early ablation on AF progression. Further investigations are warranted.6Our meta‐analysis suggests that CBA is superior to AAD as a first‐line therapy for the prevention of recurrent atrial arrhythmia in relatively young patients with symptomatic paroxysmal AF and normal cardiac structure. Serious CBA‐related complications were observed to be uncommon. However, due to the open‐label design, the study limitations include small sample sizes, short follow‐up duration, and failure to reflect real‐world populations in the included RCTs. Thus, the results should be interpreted with caution. Larger patient populations and longer follow‐up periods are required to determine the beneficial role of early CBA in preventing degenerative processes that may lead to later recurrence of atrial arrhythmia.The authors report no conflicts of interest.Data S1. Supporting Information.Click here for additional data file.


N‐methyl‐d‐aspartate receptor (NMDAR) encephalitis are important to achieve a better prognosis. The study aims to summarize the real‐world perspectives of anti‐NMDAR encephalitis patients in China via electronic medical records (EMRs).Early recognition and intervention of patients with the anti‐Using EMRs of patients from 2013 to 2019 from West China Hospital in China, a retrospective research was conducted to demonstrate the temporary rank of clinical characteristics and disease prognosis of anti‐NMDAR encephalitis. The modified Rankin Scale (mRS) scores were used to divide the anti‐NMDAR‐encephalitis into two groups (poor prognosis vs. good prognosis). Chi‐square test and logistic regression were used to analyze factors associated with prognosis.Here, 78 patients were included. The most common clinical characteristics are cognitive dysfunction (86.0%) and thought disorder (86.0%). Cognitive dysfunction, thought disorder, and seizures tended to appear soon after prodrome symptoms. Logistics analysis results showed that cognitive dysfunction , the score of (GCS ≤ 8) , positive antibodies in serum  and delay immunotherapy  were risk factors of poor clinical outcomes.There are two peaks in the development of autoimmune encephalitis (AE). The first peak is cognitive dysfunction, and the second peak is autonomic dysfunction. Cognitive dysfunction and GCS score ≤8 at admission, antibodies positive in serum, and delay immunotherapy were risk factors for a poor prognosis at discharge. Using electronic medical records (EMRs) of patients between 2013 to 2019 from West China Hospital in China, a retrospective research was conducted to demonstrate the temporary rank of clinical characteristics and disease prognosis of anti‐N‐methyl‐D‐aspartate receptor (NMDAR) encephalitis. We found that the most common clinical characteristics are cognitive dysfunction (86.0%) and thought disorder (86.0%). Logistics analysis results showed that cognitive dysfunction , the score of (GCS≤8), positive antibodies in serum and delay immunotherapy  were risk factors of poor clinical outcomes. They can be associated with T‐cell‐mediated cytotoxicity  or prodrome symptoms . Dalmau's study found that only 23% of patients with anti‐NMDAR encephalitis were initially inspected by a neurologist, while 77% were first seen by a psychiatrist  has been important to decision‐making on the disease  primary discharge diagnosis as anti‐NMDAR encephalitis; (2) first‐episode drug‐naïve anti‐NMDAR encephalitis. Exclusion criteria included were: (1) no lumbar puncture cerebrospinal fluid (CSF) examination ; (2) negative for NMDAR antibodies in CSF examination  is directly extracted from EMRs. Chart review was conducted to gain data regarding incidence trend and clinical characteristics at admission. The incidence trend was calculated by the annual number of diagnosed cases of anti‐NMDAR encephalitis and the time for primary diagnosis  to diagnosis correcting . The clinical characteristics include the score of Glasgow coma scale (GCS), altered behavior, cognitive dysfunction, disturbance of perception, thought disorder, seizure, movement disorder, sleep disturbance, emotional lability, autonomic dysfunction, and viral prodrome. The GCS score was used to assess the level of consciousness of patients at admission. Autonomic dysfunction was detected by sustained atrial tachycardia or bradycardia, orthostatic hypotension , hyperhidrosis, persistently labile blood pressure, ventricular tachycardia, or cardiac asystole , positive (1:32 to 1:100), and strongly positive (1:320), respectively. CSF antibody titers were defined as weakly positive (1:1), positive (1:3.2 to 1:10), and strongly positive (≥1:32)  were directly extracted from the EMRs. We judged whether the patients have tumors according to the results of the chest and abdomen CT.The process of treatment  was reviewed and documented by two authors. Treatment patterns can be divided as first‐line treatment  and second‐line treatment  to divide the anti‐NMDAR encephalitis into good prognosis (mRS scores of 0–2) and poor prognosis (mRS scores of 3–6) at the time of discharge from hospital . Difference between groups (good prognosis vs. poor prognosis) is identified by the Chi‐square test for categorical variables. Estimated coefficients (βs) and odds ratios (ORs) were calculated via logistic regression. 33.1A search of EMRs yielded 1429 patients with a primary discharge diagnosis of encephalitis, of which 78 was considered eligible anti‐NMDAR encephalitis cases. Of the 78 patients, females  outnumbered males . With a median age of 29 at admission, the youngest patient was 14 years old and the oldest was 65 years old.Of the 78 patients, 0 was diagnosed in 2013, accounting for 0% of all patients; 2 in 2014, accounting for 1.7%; 2 in 2015, accounting for 1.7% and 9 in 2016, accounting for 7.4%. There were 16 cases in 2017 and 36 cases in 2018. In 2019, there were 56 cases, accounting for 46.3%. As shown in Figure 3.2The temporal sequence where each clinical characteristic was first reported in EMRs was coded numerically as ranks 1, 2, 3, 4, 5, etc. . Clinical characteristics were recorded in EMRs. If the clinical features were not reported in EMRs, it was scored as “absent.” In turn, it was scored as “present.” If two clinical features occur at the same time, they were coded in the same temporal sequence. If the clinical features were not reported in EMRs, it was scored as “absent.” The frequency of clinical features was presented in Figure Figure 3.3p = 0.03), seizure (p = 0.05) and movement disorder (p = 0.04), antibody‐positive in serum (p = 0.02), utilization of mechanical ventilation (p = 0.04), GCS score ≤8 (p = 0.02) and interval to immunotherapy (p = 0.00) between groups. The details are shown in Table Among 78 anti‐NMDAR encephalitis patients, 33 (42.30%) had a favorable prognosis and 45 (57.70%) patients had a poor prognosis at discharge. There were significant variables including cognitive dysfunction , positive serum antibody , strongly positive serum antibody , score of GCS ≤8  and delay immunotherapy  were risk factors of poor prognosis. There is no significant association in other factors, such as movement disorder and utilization of mechanical ventilation, and a poor prognosis. The details are shown in Table 44.1Previous studies found that nearly 80% of AE are seen in women, of which half of the cases occurred in women above the age of 18  the data is directly extract from the EMRs. Real‐world evidence has the potential to offer useful information about the use and prognosis of a given clinical treatment within a setting most relevant to routine clinical practice in China; (2) we only included initial anti‐NMDAR encephalitis patients so the effect of the medication on anti‐NMDAR encephalitis was ruled out. However, our study had some limitations, including (1) the sample size of the current study was insufficient. (2) Patients with anti‐NMDAR encephalitis coming from only one specific hospital might limit the generalizability of our findings; (3) we only included “definite” anti‐NMDAR encephalitis patients whose anti‐NMDAR was detected in the CSF in this study. Probable anti‐NMDAR encephalitis was not included. (4) We only evaluated the prognosis at discharge, and cannot rule out the patients who were relapsed or dead. There is a need for prospective and multicenter trails in the future. (5) In the retrospective study, for the clinical symptoms that if it did not appear in the EMR was scored as “absent” Gurrera, , ignorin5In summary, we analyzed the temporary rank of clinical characteristics and risk factors for prognosis among anti‐NMDAR encephalitis. Although the spectrum of features at admission and course of anti‐NMDAR encephalitis is wide, new psychiatric symptoms and accompanied prodromal symptoms can be regarded as “red flag” for anti‐NMDAR encephalitis. Cognitive dysfunction, antibodies titers in serum, GCS scores (≤8), and interval to immunotherapy (≥5 days) were risk factors for a poor prognosis.Wei Zhang conceived the study, contributed to the planning, draft, revision of the manuscript. Fenfen Ge, Runnan Yang, and Yue Wang wrote the first draft of this paper. Fenfen Ge and Jingwen Jiang analyzed and interpreted the data. Fenfen Ge and Mengtong Wan revised the draft critically for important intellectual content. All authors contributed to the revision of the manuscript. All authors gave the final approval of the version to be published.https://publons.com/publon/10.1002/brb3.2277.The peer review history for this article is available at


After adjustment for age and BMD, pure tone average‐high (p = 0.036), pure tone average‐mid (p = 0.028), and body balance (p = 0.039) were still significantly associated with incident fractures. Bone mineral content, BMD, and treatment at baseline were not associated with subsequent fracture. In conclusion, hearing and body balance at baseline exceeded initial BMD in predicting incident fractures in osteoporotic women regardless of treatment during 25‐year follow‐up. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Hearing and balance deteriorate, and fracture incidence increases with age, especially in women. The aim of the present study was to investigate whether impaired hearing and body balance are stronger predictors of fractures than bone mass. Between 1995 and 1997, 80 women, aged 50 to 70 years, with primary osteoporosis, taking menopausal hormone therapy, mainly for menopausal symptoms, participated in a double‐blind, randomized, placebo‐controlled study of treatment with growth hormone versus placebo. All women received calcium 750 mg and vitamin D 400 U daily. They were then examined yearly until 2007 and followed up by registers until 2020. Hearing was assessed by audiometry. Body balance and fine motor function were tested according to the Bruininks‐Oseretsky test. Bone properties were measured with DXA. Data on fractures were derived from the Gothenburg Hospital register. Over the 25‐year follow‐up, 50 women (63%) sustained 104 fractures, most often related to accidental falls. Thoracic and lumbar spine fractures were most common (36%). Other fractures occurred in the pelvis (14%), humerus (14%), hip (11%), and wrist (10%). Hearing impairment at baseline, measured as pure tone average‐high ( The most common fragility fractures are of the hip and radius. Both are related to falling and are associated with increased mortality in the short and long term. The risk of sustaining a fracture is further greatly increased by the presence of osteoporosis, defined as low BMD or low bone mineral content (BMC).The risk of fractures increases with age, especially in women after menopause. A Canadian study reported a 24% 10‐year absolute risk of fragility fractures in 75‐ to 84‐year‐old women.1 The Impaired balance influences the risk of falling and thereby the risk of fractures. In a previous study, we showed that impaired hearing predicted incident fractures in a general population sample of middle‐aged and elderly Swedish women (up to 98 years of age) followed up for 17 years. In contrast, bone‐regulating hormones, medication, and lifestyle factors did not predict incident fractures.6 However, at the 10‐year follow‐up, BMC had decreased to levels similar to pretreatment levels, and there was no difference in the fracture incidence between women in the GH and placebo groups. The aim of this study was to investigate if body balance and fine motor function, beside impaired hearing, at the start of the study period were stronger predictors of a subsequent first fracture and death than bone mass and treatment in these women with osteoporosis 25 years later.The present study is a follow‐up of 80 women with osteoporosis who participated in a double‐blind, randomized, placebo‐controlled study of treatment with growth hormone (GH) or placebo between 1995 and 1997. At the end of the 3‐year intervention and at follow‐up after 5 years, BMC had increased significantly in a dose‐dependent manner in women treated with GH compared with placebo.7 Howe Osteoporosis was defined according to the World Health Organization (WHO) as BMD lower than −2.5 SD of young women (T‐score) from the Lunar, General Electric Healtcare, Dual energy X‐ray Absorptiometry (DXA), USA Reference Population, measured at the lumbar spine. Exclusion criteria were diabetes mellitus, ischemic heart disease, heart failure, cancer, kidney disease, or other chronic diseases, including chronic disease of the skeleton, as well as treatment with corticosteroids or osteoclast inhibitors. Out of 451 women taking part in the initial screening for osteoporosis, 371 did not meet the inclusion criteria, mainly based on not having osteoporosis. There were 77 women with osteoporosis, according to the WHO criteria, who were included. Because of difficulties in recruiting a sufficient number of participants, another three women with a higher BMD (<−2 SD as T‐score), but with at least one previous osteoporotic fracture were included. All women were menopausal and at least 1 year had passed since their final menstruation. They had been treated with menopausal hormone therapy (MHT) for at least 9 months before inclusion in the study, the majority based on menopausal symptoms.Eighty women aged 50 to 70 years, with primary osteoporosis, were recruited from the Endocrine Outpatient Clinic from 1994 to 1995, from consultants in the city, and by advertisements in the local newspaper. The trial design has been described in detail previously.7 OsteThe women were randomized into a double‐blind, placebo‐controlled study with growth hormone (GH) in two doses, 1.0 U/day (n = 28) or 2.5 U/day (n = 27) versus similar volumes of placebo (n = 25) subcutaneously from 1995 until 1997. The double‐blind phase lasted 18 months. Thereafter, all women who received GH continued the injections for another 18 months, altogether 3 years. Based on ethical considerations, those receiving placebo injections stopped at 18 months. All participants received 750 mg of calcium and 400 U of vitamin D daily during the trial and follow‐up. The study was performed at the Center for Endocrinology and Metabolism at Sahlgrenska University Hospital, Gothenburg, Sweden. The present study reports incidence of the first fracture and mortality among the 80 women initially participating in the GH study up to 2020: a follow‐up of 25 years.The same investigator (K L‐W) examined all women yearly for 7 years after the GH‐treatment was stopped, altogether 10 years. There were no dropouts during this period except for six women who died.8 The At the start of the study in 1995, the participating women answered a questionnaire on past and present health status, previous fractures, medication, smoking habits, and physical activity. Medication was coded according to the Anatomical Therapeutic Chemical (ATC) classification system. Information about the use of bone‐specific treatment during follow‐up after the GH trial was obtained from medical records. Smoking habits were defined as current smokers or nonsmokers . Very few of the participating women were working full‐ or part‐time, so only physical activity during leisure time was analyzed.Physical activity was coded on a four‐grade scale, with: (i) representing sedentary activity; (ii) moderate activity; (iii) regular, strenuous activity; and (iv) regular, very strenuous activity. The two latter categories were combined into one because there were very few women in category iv . The grading was based on a physiological analysis of exercise demands.9 Very2).Body weight was measured barefoot in light underwear to the nearest 0.1 kg. Body height was measured barefoot with a stadiometer to the nearest cm. BMI was calculated as body weight divided by height squared  catching a tossed ball with both hands and thereafter preferred hand—5 trials each, (ii) catching a bounced ball with both hands and preferred hand—5 trials each, (iii) touching the nose with the index finger with closed eyes, (iv) by touching the thumb to the fingertips with closed eyes, and (v) by pivoting the thumb and index finger (90 seconds maximum for items iii–v). The maximum score was 21 points.The Bruininks–Oseretsky test (BOT)10 subIn the manual dexterity subtest, the subjects performed five timed actions: (i) the number of coins placed into a box with preferred hand in 15 seconds, (ii) time to place 12 pairs of coins in two boxes with both hands simultaneously, (iii) the number of shape cards sorted with preferred hand in 15 seconds, (iv) the number of beads strung with preferred hand in 15 seconds, and (v) the number of pegs placed on a pegboard with preferred hand in 15 seconds. The maximum outcome for each item was 8, 10, 10, 7, and 8 points, respectively, giving a maximum score of 43 points. The maximum outcome for the static balance subtests was 29 points and for the dynamic balance tests—8 points, giving a total of 37 points: the better the result, the higher the score. All balance tests were performed by the same physician (M‐LB).Body balance was measured using the BOT subtest running‐speed agility and a balance test using a modification of the BOT subtest balance. Running speed agility was recorded by a 25‐m shuttle run in a corridor assessed as time in seconds: the faster the run, the higher the point score. The balance score included measures of both static and dynamic balance. In all the tests, the subject was allowed to use her arms for balance . Static balance was determined as the total score from six items: Romberg's test, the toe‐to‐heel Romberg's test, and standing on the preferred foot with the free (nonpreferred) leg flexed at the standing knee, with eyes open and closed for 30 seconds. The seconds before the subject put the nonpreferred foot down were recorded. Balance was also tested by using item 5 , and item 7  from the BOT protocol. The beam was made up of four parts and the subject scored 1 point if only the first part was passed and 4 points if all four parts were passed. If the subject was unable to reach the maximum time on the first trial of each item, two more trials were permitted, and the best result used. The rough score of the numbers of seconds was then exchanged for a point score .11)11(12), BMC (kg), body fat, and lean body mass were measured with DXA , including total body, lumbar spine (anteroposterior [AP] L2–L4), femoral neck, and distal radius. LUNAR software was used for scanning (version 1.33) and analysis (version 1.33). In‐house precision errors on the scanner used (system 7156), as determined from duplicate examinations in 10 healthy subjects, were 1.46% for total body BMD, 0.81% for AP spine BMD, 1.25% for femoral neck BMD, and 1.66% for forearm BMD. The corresponding variation for total body BMC was 1.94%. The reference database used was the LUNAR USA Reference Population for the region examined. A quality assurance test with a phantom was performed every day of the study. The SD for repeated measures was 0.01 g/cm2 (1%) for L2–L4 and 0.015 g/cm2 (1.5%) for femoral neck during both short‐ and long‐term registrations.BMD  by audiologists. The audiometers  were regularly calibrated in accordance with ISO 389 (1991), using TDH‐39 earphones with MX‐41/AR cushions. Two pure‐tone averages (PTAs) for both ears merged were used as summary statistics: PTA‐High  and PTA‐Mid .Speech audiometry was assessed by performing the speech recognition score in noise, where the percentage of correct answers out of 50 monosyllabic phonemically balanced (PB) words is determined using a speech‐weighted noise and a signal‐to‐noise ratio of +4 dB HL. The test material consisted of six Swedish phonemically balanced word lists, taken from test material commonly used in Swedish speech audiometry practices.13International Classification of Diseases‐10 (ICD‐10):(14) fractures of the vertebrae including thoracic and lumbar spine M48.5, S22.0, and pelvis S32.0, ribs S22.3–4, humerus S42.2–4, forearm S52.0–4, S52.7, wrist S52.5–6, hand S62.0–8, hip S72.0–2, thigh S72.3–9, lower leg S82.1–4, ankle S82.5–6, S82.8, and foot S92.0–9. In addition, a lumbar x‐ray was performed in all subjects at the start of the study, then after 3 and 10 years. If the vertebral height declined >20% (according to the Genant classification) it was considered a new vertebral fracture. X‐ray‐verified fractures were thereafter retrieved until December 31, 2019 by searching the Gothenburg Hospital register. How and when each fracture occurred was asked at every visit. Only the first fracture that was considered osteoporotic was included in the analysis.The fractures were coded according to the (ICD‐10):14 fractuDate of death was retrieved from the Gothenburg Hospital register.2 test was used for ordered categorical variables and the Fisher's nonparametric permutation test was used for continuous variables. The Cox proportional hazard model was used to predict factors of importance for a future first fracture after the study's start. Subsequent fractures were not analyzed. The proportional hazard assumption was tested by including time‐dependent covariates in the Cox model. The Bonferroni correction method was performed to adjust for multiple testing. The results were censored at death. A p < 0.05 was considered significant.Conventional methods were used for the calculation of means and SDs. For comparison between groups, the Mantel–Haenszel χThe University of Gothenburg Ethics Committee approved the study protocol . The study was performed in conformity with the Declaration of Helsinki.2) in the women who had sustained a fracture.Table p < 0.001).All 80 women completed the 3‐year study with GH/placebo. A flow chart including number of women with fracture and type of fracture is given in Fig. Fifty women (63%) sustained a fracture during the 25‐year follow‐up. Twenty‐seven women had one fracture, 11 had two, five had three, and seven women had more than three fractures; one woman sustained seven different fractures on separate occasions. Of the 104 fractures, thoracic and lumbar spine fractures were most common (36%). Other major fracture types were pelvic (14%), humerus (14%), hip (11%), and wrist (10%) fractures. Most fractures were caused by accidental falls.There was no significant difference in body fat, lean body mass, BMC, or BMD among women with and without a subsequent fracture Table .p = 0.040), and there was a trend, although not significant, of higher PTA, indicating a more impaired hearing, in women who fractured during follow‐up. Body balance was significantly worse in women who fractured , PTA‐mid (p = 0.003), and speech recognition score (p = 0.025) at baseline. Moreover, risk of fracture was significantly associated with worse body balance (p = 0.004), higher running‐speed agility (p = 0.012), and worse upper limb coordination (p = 0.044) at baseline. Both impaired hearing and balance were associated with a subsequent fracture even when consideration for multiple testing was made (Bonferroni correction). Impaired hearing, assessed by PTA, and body balance, remained significantly associated with a subsequent fracture also after adjustment for age, BMD or both combined, respectively. However, the association with speech recognition and manual coordination was no longer significant when adjusting for age and BMD. Running‐speed agility was significantly associated with a fracture when adjusted for BMD, but not when adjusted for age, or both age and BMD combined (Table Hazards ratios (HRs) for a fracture during the 25‐year follow‐up are given in Table  p = 0.00, and speFifty‐one (64%) women continued MHT the first years after the GH study was finished, but at follow‐up, 31 (61%) of these women had stopped MHT treatment. Bone‐specific treatment with bisphosphonates and/or teriparatide were given after the GH trial, mainly to those who decreased in BMD and/or fractured during follow‐up. Consequently, the HR for a fracture among women with bone‐specific treatment during the 25‐year follow‐up was nearly doubled, even after adjustment for age and BMD  predicted future fracture.27 It  Similarly, a randomized study of 100 Hungarian women, mean age 69 years, with menopausal osteoporosis who had at least one osteoporotic fracture, showed a significant improvement in postural balance and aerobic capacity after completing a 12‐month training program. Furthermore, a systematic review and meta‐analysis showed that long‐term exercise training reduced the risk of falls and tended to reduce the risk of fractures in older adults.Physical activity might be beneficial to prevent fall‐related fractures. A training program was recently reported to improve muscle strength, balance, and fear of falling in Norwegian women with osteoporosis and a history of vertebral fractures.28 SimA limitation of this study is the small number of participants and no update of variables tested for fracture outcome. Furthermore, the DXA device has been changed through the years, but rigorous calibrations with the phantom have been performed. Besides, all women have been compared with the same device at each occasion to minimize the methodological error. The main outcome of the present long‐term follow‐up study was fracture outcome. The strength is the thorough follow‐up with no dropouts and the long total follow‐up of 25 years by the same observer. Another strength is the objective testing with audiometry, and balance and manual coordination tests, also by a single observer. Furthermore, when and how the women fractured is well documented, including the vertebral fractures, which are often underdiagnosed and consequently underreported when assessing fracture risk with the calculator of the fracture risk assessment tool (FRAX).31In conclusion, hearing and body balance at baseline exceeded initial BMD in predicting incident fractures in osteoporotic women regardless of treatment during a 25‐year follow‐up. Including impaired hearing and balance function in the FRAX analysis might contribute to a more reliable calculation of a future risk of fractures, especially those by accidental falls.Annika Dotevall: Conceptualization; datacuration; formal analysis; writing‐original draft; Writing‐review & editing. Emily Krantz: Conceptualization; datacuration; writing‐original draft; writing‐review & editing. Marie‐Louise Barrenäs: Conceptualization;datacuration; investigation; methodology; writing‐original draft; writing‐review & editing. Kerstin Landin‐Wilhelmsen: Conceptualization; principal investigator; datacuration; formal analysis; funding acquisition; investigation; methodology; resources; supervision; validation.The authors have no conflicts of interest to declare.https://publons.com/publon/10.1002/jbm4.10551.The peer review history for this article is available at Supplementary Information S1. Bruininks–Oseretsky Test for Gross Motor FunctionClick here for additional data file.


To harness this observation, we used age‐stratified human transcriptomes to define the age‐related matreotype, which represents the matrisome gene expression pattern associated with age. Using a “youthful” matreotype, we screened in silico for geroprotective drug candidates. To validate drug candidates, we developed a novel tool using prolonged collagen expression as a non‐invasive and in‐vivo surrogate marker for Caenorhabditis elegans longevity. With this reporter, we were able to eliminate false‐positive drug candidates and determine the appropriate dose for extending the lifespan of C. elegans. We improved drug uptake for one of our predicted compounds, genistein, and reconciled previous contradictory reports of its effects on longevity. We identified and validated new compounds, tretinoin, chondroitin sulfate, and hyaluronic acid, for their ability to restore age‐related decline of collagen homeostasis and increase lifespan. Thus, our innovative drug screening approach—employing extracellular matrix homeostasis—facilitates the discovery of pharmacological interventions promoting healthy aging.The identification and validation of drugs that promote health during aging (“geroprotectors”) are key to the retardation or prevention of chronic age‐related diseases. Here, we found that most of the established pro‐longevity compounds shown to extend lifespan in model organisms also alter extracellular matrix gene expression  corresponding to young and old human tissues with drug‐treated expression profiles to predict longevity drugs. Then, we established  To define a young and old human matreotype, we built upon the previous analysis of the GTEx dataset  increased Figure . These fTo extend the matreotype generated from these five tissues, we combined the findings of eight studies that also included transcriptomes across ages from different tissues in order to validate and identify additional multi‐tissue and age‐related matrisome genes Table . By cons2.4i.e., similar gene expression; Figure The ultimate goal of our matreotype signature is its application to identify new geroprotective compounds that modulate the matrisome, and thus, longevity. To first validate this approach, we used known pro‐longevity compounds and identified those causing a youthful matreotype  declined almost linearly within the first 5 days of adulthood , chrysin, and dapsone maintained strong Pcol‐144::GFP expression at old age , enhanced oxidative stress resistance during older age (day 5 adults), and improved lifespan extension compared to empty vector liposomal control treatments  derived from soybeans to have a youthful matreotype profile Figure . Genisten Figure . To optis Figure . A simil Figures . These dC. elegans and mice , parsing healthy versus common aging cohorts  I; rrf‐3(b26) II. LSD2002 was generated by integrating Pcol‐144::GFP transgene (a generous gift from Yelena Budovskaya and Stuart Kim) into N2, outcrossing eight times, and crossing to TJ1060, resulting spe‐9(hc88) I; xchIs001  X genotype.4.2Data analysis was performed utilizing the dplyr . dplyr: A Grammar of Data Manipulation. R package version 1.0.0) and purrr (Lionel Henry and Hadley Wickham (2020). purrr: Functional Programming Tools. R package version 0.3.4). R packages. Data visualization was generated using ggplot2 , ComplexHeatmap (Gu (2016). Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics), and ggpubr (Alboukadel Kassambara (2020). ggpubr: “ggplot2” Based Publication Ready Plots. R package version 0.4.0.999).4.3http://matrisomeproject.mit.edu,  threshold of 0.05 to each dataset if applicable. Tissue‐specific differences were not further investigated due to confounding in the meta‐analysis. Studies analyzing individual tissues were treated as separate sources. To define the aging matrisome, we acquired data from at least three sources implicating the gene in the aging process. Studies that offer directionality were further utilized to determine matrisome genes that were upregulated or downregulated with age using the same thresholds.4.6col‐144::GFP was used to assess age‐related decline in collagen‐144 expression. Animals were age‐synchronized by bleaching and made infertile by culturing at 25℃. NGM culturing plates were prepared with tretinoin (Sigma PHR1187), hyaluronic acid (Sigma H5388), chondroitin sulfate (Sigma 27042), metformin (Sigma D150959), glucosamine (Sigma G4875), and genistein . Distribution scoring is based on intensity observed visual inspection, categorizing to none or very low, low, medium, or high intensity. For some experiments, animals were mounted onto 2% agar pads and pictures were taken with an upright fluorescent microscope . To separate the GFP signal from the autofluorescence of the gut, we used the microscope, settings, and triple‐band filterset as described by Teuscher  alone as control or tretinoin (Sigma PHR1187), hyaluronic acid (Sigma H5388), and chondroitin sulfate (Sigma 27042). Vitamin B12 , chrysin , and dapsone  lifespans were performed in 1.25% DMSO and 50 µM FUdR in 24‐well plates, 4 wells per condition and 30 worms per well, transferred at L4 developmental stage. Animals were classified as dead if they failed to respond to prodding. Exploded, bagged, burrowed, or animals that left the agar were excluded from the statistics. The estimates of survival functions were calculated using the product‐limit (Kaplan–Meier) method. The log‐rank (Mantel‐Cox) method was used to test the null hypothesis and calculate P values (JMP software v.14.1.0).Manual scoring of lifespan as previously described by Ewald et al. . In brieAdditional Information can be found in the Extended Experimental Procedure in the The authors have no competing interests to declare. Correspondence should be addressed to C. Y. E. The authors declare no conflict of interest. FW and FZ are employees of Mibelle Biochemistry.in‐silico analysis. AD and PM translated compound names for list comparison and automated analysis. EJ, CS, AD, and CYE performed lifespan assays. EJ and CYE performed oxidative stress assays. EJ, AD, and SXL performed collagen expression assays. FW and FZ prepared and encapsulated compounds. CYE wrote the manuscript in consultation with the other authors.All authors participated in analyzing and interpreting the data. CYE and CS designed the experiments. CS established and performed Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Figure S6Click here for additional data file.Figure S7Click here for additional data file.Figure S8Click here for additional data file.Figure S9Click here for additional data file.Figure S10Click here for additional data file.Figure S11Click here for additional data file.Figure S12Click here for additional data file.SupFile S1Click here for additional data file.SupFile S2Click here for additional data file.Supplementary MaterialClick here for additional data file.TableS1‐S8Click here for additional data file.


An 85‐year‐old woman presented with pain and a palpable mass in her left flank. Abdominal computed tomography revealed massive splenomegaly and para‐aortic lymphadenopathies. Bone marrow biopsy showed CD79a, CD20, and bcl‐2‐positive atypical lymphocytes, which led to the diagnosis of splenic marginal zone lymphoma. The causes of massive splenomegaly are hematologic diseases, malignancies, infection, and infiltrative conditions. When seeing massive splenomegaly, hematologic diseases, especially malignant diseases, should be ruled out first. Abdominal contrast‐enhanced computed tomography revealed massive splenomegaly of >20 cm without mass lesion, and para‐aortic lymphadenopathies .Written informed consent to publish this report was obtained from the patient in accordance with the journal's patient consent policy.Video S1Click here for additional data file.Video S2Click here for additional data file.Supplementary MaterialClick here for additional data file.


Diabetic foot ulcerations or infections (DFUs/DFIs) are common complications of patients with diabetes. This study aimed to explore the impact of non‐dialysis and dialysis CKD on hospitalized patients with DFUs/DFIs.A retrospective cohort study was conducted using the National Inpatient Sample database for the years 2017 and 2018. Patients hospitalized for DFUs/DFIs were included in the study. The primary outcome was lower limb amputations. The secondary outcomes were inpatient mortality, sepsis, length of stay (LOS), total hospitalization charges (THC) and disposition.A total of 121,815 hospitalizations were included . There was no significant difference in amputation rates between those on non‐dialysis CKD  and dialysis CKD  when compared to non‐CKD group. Dialysis CKD group had increased odds of undergoing major amputation , in‐hospital mortality (aOR: 3.77 [95% CI: 1.94–7.31]), sepsis (aOR: 1.83 [95% CI: 1.27–2.62]), longer LOS (adjusted mean difference [aMD]: 1.46 [95 CI: 1.12–1.80) and higher THC . Non‐dialysis CKD group had increased odds of sepsis (aOR: 1.36 [95% CI: 1.02–1.82]), less likely to be discharged home (aOR: 0.87 [95% CI: 0.80–0.95]), longer LOS (aMD: 0.91 [95% CI 0.69–1.13]) and higher THC .Patients with CKD on dialysis had higher odds of undergoing major amputation. CKD increased the odds of in‐hospital morbidity and resource utilization, with the most significant is for those on dialysis. The present study demonstrated that the CKD population, particularly the dialysis CKD group, is posed to higher amputation rates, worse clinical outcomes, and more enormous economic impacts on patients with DFUs/DFIs. The lifetime incidence has been predicted to be more than 19% in patients with diabetes.Diabetes is also frequently complicated by kidney disease. Chronic kidney disease (CKD) is a known marker for the generalized vascular status of patients with diabetes. Overall, CKD is associated with an increased level of in‐hospital mortality, postoperative complications, length of stay and hospital costs.To our knowledge, the impact of non‐dialysis and dialysis CKD on patients with DFUs/DFIs has not been explicitly addressed among hospitalized patients at a national level. Therefore, this study aims to explore the impact of CKD on clinical outcomes and resource utilization.22.1th Revision, Clinical Modification/Procedure Coding System (ICD‐10‐CM/PCS) coding system was used to report all medical diagnoses and inpatient procedures recorded in the NIS database. Detailed information is available at http://www.hcup‐us.ahrq.gov. Data were sought from the 2016 and 2017 National Inpatient Sample (NIS) database. The NIS is a nationwide database of hospital inpatient stays across the United States, funded by the Agency for Healthcare Research and Quality (AHRQ). The NIS is part of the Healthcare Cost and Utilization Project (HCUP) and the largest publicly available national all‐payer inpatient healthcare database. The samples were collected from all US short‐term general and other specialty hospitals, excluding Federal hospitals, long‐term acute care hospitals and rehabilitation hospitals. Samples were stratified based on hospital census division, ownership, urban/rural location, teaching status and hospital beds. It collects data for more than 7 million unweighted hospital discharge records annually and approximately more than 35 million weighted hospitalizations annually. From 2017 to 2018, the NIS included over 71 million weighted discharge records from across 48 states. In 2017 and 2018, the NIS provides up to 40 diagnoses and 25 procedures for each hospitalization record. The NIS contains a large sample size; therefore, it is ideal for developing national and regional estimates of multiple conditions. The International Classification of Disease, 10The NIS database was queried for patients 18 years and older who had a principal diagnosis of foot ulcers or foot infections and any diagnosis of diabetes mellitus using ICD‐10 codes. This cohort was further divided based on at least one secondary discharge diagnosis of non‐dialysis CKD and dialysis CKD. Institutional Review Board approval was not needed because the NIS database has completely removed possible patient identifiers, state level and hospital identifiers.2.2The primary outcome was any lower limb amputation. Lower limb amputation was further divided into major and minor amputation. Major amputation was defined as any amputation above the ankle joint. Minor amputation was defined as any amputation limited to the foot. The secondary outcomes included in‐hospital mortality, sepsis, disposition, length of stay and total hospitalization charges. Disposition was defined as either home discharge or all others . ICD‐10‐CM/PCS codes were used to obtain lower limb amputation, major amputation, minor amputation and sepsis from the cohort Table . The oth2.3p‐value of .2. Adjusted confounders included the following: a) patient‐level: gender, race, median income based on patient's zip code, Charlson comorbidity index, peripheral arterial disease (PAD), obesity, hypertension and b) hospital level: bed size, teaching status, region. Multivariate regression analysis was used to adjust for possible confounders while calculating the primary and secondary outcomes. All p‐values were two‐sided, with.05 as the threshold for statistical significance.The data were analysed using STATA 16.0 (StataCorp). The NIS database represents a collection of a complex sampling design that includes weighting, clustering and stratification. All analyses were conducted using weighted samples based on guidelines outlined by HCUP NIS.33.1p < .001). The highest proportion among the three groups was dominated by Caucasians and males. The non‐dialysis CKD and CKD on dialysis groups also had higher Charlson comorbidities than those without CKD . The baseline characteristic of patients is presented in Table This retrospective, nationwide cohort study included 121,815 patients hospitalized for DFUs/DFIs in 2017 and 2018. Of these hospitalizations, there were 31,780 (26.1%) hospitalizations associated with non‐dialysis CKD and 10,285 (8.4%) hospitalizations associated with CKD on dialysis. Patients with non‐dialysis CKD and CKD on dialysis were older than those without CKD . However, the difference in minor amputation rates was not significant when compared between the groups (p = .664).Table p < .001).Results of multivariate logistic regression analysis of the outcomes are shown in Table 3.3p < .001), sepsis , total hospitalization charges  and longer length of stay . Moreover, patients with non‐dialysis CKD and dialysis CKD were less likely to be discharged to home compared to patients without CKD .Table p = .036). In addition, the non‐dialysis CKD group was also significantly less likely to be discharged home , had higher total hospitalization charges  and had longer length of stay .Results of multivariate logistic regression analysis of the outcomes are shown in Table p = .001), sepsis . Moreover, the CKD on dialysis group also significantly had higher total hospitalization charges  and longer length of stay .After adjusting for the potential confounders, the patients with CKD on dialysis were associated with a significantly increased risk of in‐hospital mortality .Several studies showed that CKD was associated with worse mortality and morbidity in patients with diabetic foot.p < .001) and patients on dialysis treatment  when compared to patients with non‐advanced CKD.CKD has consistently been linked to adverse cardiovascular and renal outcomes. This association was not only seen in advanced CKD but also stage 1 or 2 CKD.The present study also demonstrated the financial burden of DFUs/DFIs in the non‐dialysis CKD and dialysis CKD population. Both groups in this study showed an increase in resource utilization, including total hospitalization charges and hospital stays. Some of the differences in expense might be accounted for additional inpatient dialysis and nephrology consult. However, patients with non‐dialysis CKD also faced higher hospitalization charges and more extended hospital stays. Worse clinical outcomes, which were evident in both groups, could have also translated into higher hospitalization costs. These findings were consistent with previous studies investigating the impact of patients with non‐dialysis CKD and CKD on dialysis on other medical conditions, including acute pancreatitis, post‐prostatectomy and heart failure.This study has some important limitations. Firstly, the NIS database uses ICD‐10 codes to characterize diagnoses, procedures and hospitalization events. The database does not provide laboratory or imaging parameters and degrees or extent of DFUs/DFIs. Therefore, there is a possibility of misclassification of the diagnoses. Secondly, CKD might be under‐reported in the NIS database. ICD‐10 codes show a high accuracy for diabetic foot complications and high specificity and low sensitivity for CKD.5The present study demonstrated that the CKD population, particularly the dialysis CKD group, is posed to higher amputation rates, worse clinical outcomes and more enormous economic impacts on patients with DFUs/DFIs. The results from this study highlight the need for more research on ways to prevent diabetic foot complications in this high‐risk population.The author reports no conflicts of interest in this work.MS contributed in the conceptualization, data curation, formal analysis, methodology, software, validation, visualization, draft preparation, review and editing.Table S1Click here for additional data file.


The firmly anchored AgNPs could prevent its entry into the body, thereby eliminating the toxicity of nanomaterials. The outer layer was a polyurethane membrane, which has a nanoporous structure that could maintain free transmission of water vapor. Chitosan/collagen sponge was selected as the inner layer because of its excellent biocompatibility and biodegradability. The presence of AgNPs in the CSNWF was fully characterized, and the high antibacterial activity of CSNWF/AgNPs was confirmed by against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The superior wound healing effect on deep dermal burns of presented composite wound dressing was demonstrated in a porcine model. Our finding suggested that the prepared AgNPs doped sandwich structure composite wound dressing has great potential application in severe wound care.Wounds may remain open for a few weeks in severe burns, which provide an entry point for pathogens and microorganisms invading. Thus, wound dressings with long-term antimicrobial activity are crucial for severe burn wound healing. Here, a sandwich structure composite wound dressing anchored with silver nanoparticles (AgNPs) was developed for severe burn wound healing. AgNPs were  Burn injuries, caused by thermal, electricity, friction, chemical agents, radiation, etc., are a common global health problem and present an enormous medical and financial burden. Burn-initiated pathophysiological events can raise the risk of disfigurement, functional impairment and serious complications by increasing vascular permeability and fibrinolysis, changing in extravasation of plasma proteins and platelet aggregation . AccordiStaphylococcus aureus and vancomycin-resistant Enterococci, has become a major global public health challenge [+) [Antimicrobial wound dressings are developed to prevent wound infection by minimizing bacterial colonization and provide proper coverage for the injured area to aid reepithelialization. Various antimicrobial dressings containing antibiotics or antimicrobial agents have been widely applied for severe and chronic wound healing . Undoubthallenge . Althougenge [+) , cadexomenge [+) , curcumienge [+)  and antienge [+) , 23 haveenge [+) . BesidesRecently, wound dressings with long-term antimicrobial activity have been developed by the incorporation of nanoscale antimicrobial materials into tissue-engineered scaffolds . Among tThe majority of studies on wound dressings were the selection of small animals such as rats and rabbits as clinically relevant wound models to assess wound healing ability. Although dramatic healing performance on these small animal wound models was demonstrated, the effectiveness of presented dressings on humans should be cautioned. For instance, rats are irrelevant to clinical wound healing because they have loose skin and show high resistance to infection. In addition, wound healing in rats is mainly dependent on wound contracture rather than scar tissue formation, whereas the opposite process occurs in humans because the humans’ skin has less elasticity , 37. It +. The AgNPs were in situ synthesized on the fibers of CSNWF and showed strong adhesion capability, which can prevent the detachment and aggregation of AgNPs. Meanwhile, the CSNWF/AgNPs as interlayer could prevent the AgNPs direct contact with wound tissue and thereby avoiding the toxicity of AgNPs to the human body. The wound healing effect on deep dermal burns of presented composite wound dressing was validated in a porcine model.In this work, an AgNPs anchored sandwich structure composite wound dressing with high antimicrobial activity was prepared for severe burn wound healing in a porcine model. As illustrated in 2) was purchased from Hismer Bio-Technology Co., Ltd. . CS  was purchased from Nantong Xingcheng Biological Industry Limited Co. . COL (Mw = 2 and 100 kDa) was purchased from Haishen Bio-Technology Co., Ltd. . Medical PU membrane with glue on one side was purchased from Sanrui Medical Device Co., Ltd . Silver sulfadiazine (SSD) cream was obtained from Kunming Huarun Shenghuo Pharmaceutical Co., Ltd. . Ketamine, factor VIII kit, vascular endothelial growth factor assay (VEGF) kit, nitric oxide (NO) kit and endothelin kit were provided by the Union Hospital of Fujian Medical University (China). AgNO3, HNO3 and ammonia were obtained from Sinopharm Chemical Reagent Co., Ltd. .CSNWF (120 g/mEscherichia coli (ATCC-8739), S.aureus (ATCC-14458) and Pseudomonasaeruginosa (CMCC B 10104) were purchased from Luwei microbial Sci. & Tech Co., Ltd. . Fetal bovine serum (FBS) was purchased from Gibco Laboratories . Dulbecco's modified Eagle’s medium (DMEM), L-glutamine, penicillin, streptomycin, trypsin/EDTA and dimethyl sulfoxide were purchased from Sigma-Aldrich .The human skin fibroblast (HSF) cell line was obtained from Dingguo Biotechnology Co., Ltd. . Cellstain live/dead double staining kit was purchased from BestBio Co., Ltd. . The WST-1 reagent was obtained from Roche Molecular Biochemicals . in situ growth of AgNPs on the CSNWF, CS/COL sponge preparation and fabrication. The process of composite wound dressing preparation was illustrated in in situ grow AgNPs on the commercial CSNWF. First, CSNWF with a size of 10 cm × 10 cm was quickly washed (1 min) with 5 mM HNO3, followed by thorough rinsing with water several times. Afterward, the pretreated CSNWF was soaked into 50 ml of AgNO3 aqueous solution for 1 h with the Ag+ concentration of 5, 10 and 20 mM, respectively. Subsequently, the CSNWF was exposed to UV irradiation . After UV irradiation, the AgNPs loaded CSNWF was washed with water and then dried under vacuum at room temperature.UV-assisted synthesis method was employed to w = 50 kDa) was dissolved in 100 ml of 0.5% acetic acid solution with constant stirring. Subsequently, 0.1 g CS (Mw = 5 kDa), 0.9 g COL (Mw = 100 kDa) and 0.1 g COL (Mw = 2 kDa) were dissolved into the mixture. After thoroughly mixing, the CS/COL mixture was placed overnight under vacuum to eliminate any air bubbles.Two different molecular weights of CS (5 and 50 kDa) and COL (2 and 100 kDa) were used to prepare the CS/COL sponges without any crosslinking reaction. The mass fraction of high molecular weight CS and COL accounted for 90%. Here, the high molecular weight polymers can enhance the mechanical properties and stability of CS/COL sponges. The incorporated low molecular weight polymers were more beneficial to release and absorption, thereby promoting wound healing. A mount of 0.9 g of CS  was immediately sprayed on the side of CSNWF/AgNPs which was sprayed with water and then frozen 1 h under −80°C. After lyophilization, the CS/COL sponge combined CSNWF/AgNPs matrix was obtained. Finally, the sandwich structure composite wound dressing was prepared by adhering to the CSNWF/AgNPs-CS/COL matrix to a medical PU membrane.8 CFU/ml bacterial suspension  on LB agar plate, the sterile CSNWF/AgNPs disks were placed onto the surface of the agar. After culture 24 h at 37°C, the diameter of the inhibition zone was recorded.The CSNWF/AgNPs was cut into 1.0 cm diameter disks and sterilized by 70% aqueous ethanol solution for 40 min, followed by thorough washing with PBS. After spreading 100 μl of 1 × 105 CFU/ml bacterial suspension  was added and mixed thoroughly. Subsequently, the mixture was placed in a shaker under 25°C with constant shaking at 150 rpm. After 24 h incubation, 1 ml of supernatant was taken to determine the number of bacteria. The supernatant was serially diluted by PBS and 100 μl of the solution was placed on agar plates, on which the grown colonies were counted for analysis. The mixture of PBS and bacterial suspension without any AgNPs/CSNWF as the control group. The tested CSNWF/AgNPs was cut into 1.0 cm × 1.0 cm pieces and sterilized as the method mentioned above. After CSNWF/AgNPs piece immersed into 100 ml of PBS, 5 ml of 1 × 10in vitro cytocompatibility and wound healing of prepared wound dressing were evaluated by culture the HSF cells in the leaching solution of the composite wound dressing according to our previous study [The us study . The lea5 cells/mL, and then cultured at 37°C under a humidified atmosphere containing 5% CO2. After being cultured 12 h, the culture medium was replaced by 100 μl of the leaching solution with 10% of FBS. After incubated at different times, the leaching solution was decanted, and cells were washed with PBS. The viability and proliferation of HSF cells were measured by the live/dead staining and WST-1 assay. As for live/dead staining, 100 μl of live/dead staining stock solution was added to each well and then incubated for 15 min at 4°C. Afterward, the fluorescent images of cells were viewed using a fluorescence microscope . For WST-1 assay, 100 µl of culture medium includes 10 μl of WST-1 reagent was added to each well. After incubation for 4 h, the optical intensity in each well was measured using a microplate reader  at 450 nm.A volume of 100 μl HSF cells suspension in culture medium was seeded into a 96-well plate at a density of 1 × 10in vitro wound healing ability of prepared wound dressing was analyzed by scratch test. HSF cells were seeded into a six-well plate at a density of 1 × 105 cells per well and cultured with culture medium for 24 h at 37°C under a humidified atmosphere containing 5% CO2. After the scratch was made, the culture medium was removed, and the cells were washed with PBS. Subsequently, the PBS was decanted, and 2 ml of leaching solution was added into each well. Microscopy images were taken using the bright-field channel every 6 h and the area of the void between cells was measured using ImageJ software.The All animals were treated humanely throughout all experiments. All protocols were reviewed and approved by the Institutional Ethics Committee for Animal Research of Fujian Medical University. A number of eight ∼12-week-old male pigs (∼20 kg) provided by the Laboratory Animal Center of Fujian Medical University were used in this study. All animals were delivered to the animal house and housed in an individual cage for 5 days with a 12-h light/dark cycle for acclimatization. The room temperature was maintained at 25°C with a humidifying of 50%. Animals were given free access to water and fed a standard grower feed pellet diet throughout the experimental periods.via intramuscular injection. The dorsal surface of the pig was shaved and sterilized with 0.1% benzalkonium bromide solution. Afterward, serve square scald wounds were created using a scald device by placing it on the dorsal skin of pigs for 25 s at 100°C. Burns with a size of 2.0 cm × 2.0 cm  were located on the dorsal of the pig, with ten burns on each side, giving a total of 20 burns per pig , and then viewed and photographed with an optical microscope (Nikon Eclipse CI) equipped with an image-forming system (Nikon DS-U3).Immunohistochemical staining of paraffinic skin sections was performed with factor VIII according to the manufacturer's protocol. Subsequently, skin sections were examined by a microscope, and the microvascular sections were counted. The microvascular density was expressed as the average of 10 fields examined.After removing the dried eschar and bloodstain, 100 mg of collected skin tissue was homogenized in 1 ml sterile saline. For endothelin assay, the specimens were centrifuged at 3000 rpm for 15 min under 4°C, and the supernatant was stored at −20°C. For NO and VEGF assay, the specimens were centrifuged at 10 000 rpm for 15 min under 4°C, and the supernatant was stored at −20°C.P < 0.05), ** (P < 0.01) and *** (P < 0.001) were considered to be statistically significant.All the analytical determinations were carried out at least in triplicate, and the results were reported as means ± standard deviation. All statistical computations were performed using the SPSS 19.0 software. Statistical comparisons were determined by using one-way ANOVA statistical analysis. Value of *  characterization results indicated that the Ag presented on the fibers of CSNWF after AgNO3 impregnation and UV irradiation treatment  nucleation starts to take place after UV irradiation. UV irradiation can reduce Ag+ to Ag° [+ may decrease the potential of Ag+/Ag (EAg+/Ag) and thus promote the reduction of Ag+ [The formation of AgNPs on the fibers of CSNWF was thought to start with the interaction of the substrate with Ag+ to Ag° . It has + to Ag° . In addin of Ag+ . Consequ+ was 5, 10 and 20 mM, respectively , 96.23 ± 3.08% (CSNWF/AgNPs10) and 90.20 ± 3.98% (CSNWF/AgNPs20) of Ag+ were absorbed within 1 h , 93.89 ± 6.10% (CSNWF/AgNPs10) and 87.31 ± 4.56% (CSNWF/AgNPs20) compared with the initial amount of Ag+. The results indicated that the UV irradiation method is highly efficient, which can reduce 96–98% of adsorbed Ag+ into AgNPs.The physical and chemical properties of AgNPs on CSNWF were investigated. The CSNWF showed excellent adsorption capacity for Agthin 1 h . From th+, and CS acts as a stabilizer during the synthesis of AgNPs [+ and sustain the release of Ag+, thereby achieving a long-term antibacterial effect. The results of the Ag+ release test indicated that the Ag+ was released in a manner of sustain and slow . No significant change in AgNPs amount was observed during 90 days of storing , which iof AgNPs . Furtherand slow . All res+ and thiol groups present in the respiratory enzymes in the bacterial cell [Escherichiacoli, P.aeruginosa and S.aureus were selected as representing bacteria that prevalent pathogenic bacterial strains in the wound site. Clear-cut inhibition zones were observed around all CSNWF/AgNPs groups for all bacteria  same as the cells in the normal culture medium  were used in our work. The wound healing effect of the presented wound dressing PU-CSNWF/AgNPs-CS/COL was compared with various control groups, including PU-gauze, PU-gauze-SSD, PU-CSNWF, PU-CSNWF/AgNPs and PU-CS/COL sponge. Herein, commercial available antibacterial agent SSD is widely used in wound treatment as a positive control group, whereas the medical cotton gauze as the negative control group. A 4-week period of the wound healing process was recorded, and the wound closure was investigated at first.No significant changes in wound area were observed during the initial 2 weeks, and the wound closure in all groups was below 5% . Compare+ inhibiting bacterial infections, thereby reducing the inflammatory response. Moreover, the CS and COL, especially the low molecule weight compositions, in composite wound dressings could stimulate the migration of fibroblasts and promote fibroblasts proliferation, respectively.The wound healing process was further evaluated by H&E staining of the tissue section on wound sites. A necrotic epidermal layer observed in all groups on Day 7, and the pore structures under the epidermis indicated blisters occurred after scalding . A clear2. It was significantly higher than the PU-Gauze (11.7 ± 4.2/mm2) and slightly superior to the PU-Gauze-SSD (22.5 ± 5.0/mm2)  . The resVEGF can promote angiogenesis by inducing and stimulating the chemotaxis and proliferation of endothelial cells . Therefoin vivo. The content of NO is low in the physiological state, and it increases when tissue damage occurs [NO and endothelin are playing an essential role in the process of wound deterioration and necrosis after burn injury. NO is a vasodilator substance e occurs . High coe occurs . Moreovee occurs . NormallThe results showed that the content of NO in the wound site was higher than in the healthy tissues on Day 7 . Subsequin situ synthesized on the fiber of CSNWF. The strong adhesion between AgNPs and CS could eliminate AgNPs aggregation and shedding, and thus maintain high antimicrobial activity and minimal toxicity. Meanwhile, the sustained released Ag+ could inhibit bacterial infections, thereby reducing the inflammatory response of the wound site. Moreover, the composition of CS and COL in composite wound dressings can accelerate the wound healing process. The in vivo wound healing effect of the presented wound dressing on deep dermal burn was demonstrated in a porcine model. The results indicated that the prepared wound dressing has excellent reepithelialization and angiogenesis properties through the promotion of the VEGF and endothelin expression as well as inhibition of NO production. Therefore, our finding suggested that the prepared AgNPs anchored sandwich structure composite wound dressing can be represented as a promising antimicrobial wound dressing for severe wound healing.A sandwich structure composite wound dressing with high biocompatibility and antimicrobial activity was developed for severe burn wound healing. The antibacterial agent AgNPs were REGBIO online.This work was supported by the National Natural Science Foundation of China (31800796), the Nature Sciences Funding of Fujian Province (2019J01238) and the Fuzhou University Testing Fund of Precious Apparatus (2021T018).Conflict of interest statement. None declared.rbab037_Supplementary_DataClick here for additional data file.