Thanks to the use of combination antiretroviral treatment (cART) people living with HIV (PLWH) have a similar life expectancy to HIV‐negative people.Table+: 576 (463–701)], with particular attention to the indicators of osteosarcopenia. Percentage of fat mass (FM), fat free mass (FFM) at arms, limbs, and as total body, and bone mineral density (BMD), t‐score and z‐score at spine (L3/L4), femoral neck, and trochanter were measured by DEXA . The appendicular skeletal muscle mass (ASMMI) index was calculated to assess the presence of sarcopenia .t‐score was calculated to assess the presence of osteoporosis (−2.5) and osteopenia (−1 to −2.5). Bone remodelling biomarkers were measured in cryopreserved plasma samples by commercially available enzyme‐linked immunosorbent assays . These included osteoprotegerin (OPG), receptor activator of NF‐kappaB ligand (RANKL), c‐terminal telopeptide (CTX), and bone alkaline phosphatase (BAP). Samples were analysed in batch at the end of the study and blindly with respect to group assignment. Quantitative variables were expressed as median, first, and third quartiles (Q1–Q3). Per cent changes between baseline (BL) and after 12 week of training W12 within each group were assessed by Wilcoxon signed rank test and between‐groups by Mann–Whitney test.We assessed body composition and bone health in a subset of 25 cART‐treated PLWH Table from thiFigure2; men: 6.5 (6.4–6.7) kg/m2; age 52 (48–53)]. At Week 12, six of these eight participants normalized ASMMI, including two women [both in the walk group: 6.1 (6.0–6.1) kg/m2] and four men [two of the walk group and two of the strength‐walk group: 7.2 (7.1–7.2) kg/m2]. Neither significant W12 changes from BL nor significant change differences between groups were observed for BMD, t‐score, z‐score at spine, femoral neck, and trochanter. According to the t‐score, three participants were diagnosed with osteoporosis at BL (12%) and 20 (80%) with osteopenia. At the end of the training protocol, none of the participants improved the t‐score (TableTableAll participants completed the 12 week programme with a median adherence of 64% (Q1–Q3: 59–75%). Among all participants, significant W12 changes in ASMMI from BL were observed in both training groups, and in total, arms and legs FFM in the strength‐walk group, with changes from BL significantly larger in the strength‐walk than in the walk group for all measurements  and Associazione Nazionale Lotta all'AIDS (ANLAIDS), Italy. This study was supported also by the Italian Ministry of Health (Ricerca Corrente).The authors have no conflict of interest to declare.Table S1. Participants' characteristics at baseline.Table Note. Values are either expressed as number of participants (%) or as median (Q1‐Q3). Data were compared between groups by Mann–Whitney and Fisher Exact tests. P values were not significant for all parameters. a: chronic treatment, with no changes during the training period or the 6 weeks before; BMI: Body Mass Index; VACS: Veterans Aging Cohort Study Risk index (this index includes: i) age; ii) laboratory tests: white blood cell count, HIV‐1 RNA, hemoglobin, platelets, AST, ALT, creatinine; iii) liver fibrosis (FIB‐4): composed of AST, ALT, platelets and age; iv) impaired renal function (eGFR): composed of age, gender, race and creatinine; v) HCV status: if the patient ever had a positive antibody test or detectable virus prior the study); NRTI: nucleoside reverse transcriptase inhibitors; NNRTI; non‐nucleoside reverse transcriptase inhibitors; HDL‐C: High density lipoprotein cholesterol; SBP: systolic blood pressure. *Among the inclusion criteria were either objective evidence of lipodystrophy, as established by the visiting physician, or of at least one of the Adult Treatment Panel III definition criteria of the metabolic syndrome.Click here for additional data file.


Iron deficiency anemia (IDA) is common in the pediatric population and often accompanied by mild thrombocytosis, but rarely profound thrombocytopenia is seen. We describe the data of children with IDA and thrombocytopenia in two centers and discuss the published data in the literature.In this retrospective case series, the medical records of patients under the age of 19 years old diagnosed with IDA in two tertiary medical centers over the last 10 years, were reviewed. The data were collected and compared to the data published in the medical literature.All the patients presented with severe IDA and thrombocytopenia improved with iron treatment. Although none of the patients had signs of major bleeding, the thrombocytopenia could mostly be classified as severe (platelet count <50×10E9/L). Due to the severity of the anemia, in about half of the cases, a red blood cell transfusion was given. The peak of the platelet count was seen in the first month after the start of iron treatment. In eight cases of children with IDA, the thrombocytopenia appeared after the supplementation of iron was started.Clinically stable children with severe IDA and thrombocytopenia, where other causes are very unlikely, warrant an empiric monotherapy with iron to prevent unnecessary investigations and treatments. Patients with pre‐existing thrombocytopenia or with a disease explaining the thrombocytopenia at the time of diagnosis were excluded from the study. To compare our data to those from the literature, a PubMed search was conducted in the English‐language literature using the keywords “iron deficiency anemia” and “thrombocytopenia” in the age group 0‐18 years. The papers were reviewed and summarized in Table 2.1Five patients were diagnosed during the study period. The clinical and laboratory characteristics are summarized here and visualized in Figure 2.1.1A 10‐year‐old female was seen by the family physician for paleness, dizziness, and fatigue. Green vegetables and red meat were missing from her diet, while there was no menarche or history of suspected gastrointestinal blood loss. She was the second child of healthy, non‐consanguineous Moroccan parents. The family history was unremarkable. Physical examination revealed blood pressure 105/60 mmHg, pulse 105 per minute, respiratory rate 20 per minute, paleness, systolic murmur, and otherwise normal findings. The blood test showed severe microcytic anemia , severe thrombocytopenia (platelets 38×10E9/L), low ferritin (3 ng/mL), normal coagulations tests, negative parvovirus serology, and normal hemoglobin electrophoresis. Because two cell lines were down, a bone marrow investigation was performed, revealing the absence of iron staining and an increased megakaryocyte count. She received a red blood cell transfusion and oral iron medication was started (5 mg/kg/daily). Her blood cell counts improved rapidly.2.1.2An 8‐year‐old female presented to the clinic because of paleness without complaints of fatigue. Over the last year, she complained of recurrent abdominal pain with suspicion of constipation. There was no weight loss and stools were normal. Her diet consisted mainly of whole milk. She was the first of two children of healthy, non‐consanguineous Dutch Moroccan parents. The family history was unremarkable, besides two nieces with IDA. Physical examination revealed pale skin and conjunctivae, a systolic murmur over the heart with a blood pressure of 115/65 mmHg, a pulse of 95 per minute, and a respiratory rate of 18 per minute. Otherwise, normal physical findings. The blood test showed severe microcytic anemia  and thrombocytopenia (platelets 18x10E9/L) with low ferritin (4 ng/mL). Additional blood work, including folic acid, vitamin B12, coeliac screening, and hemoglobin electrophoresis, was normal. Because two cell lines were down, a bone marrow investigation was performed, revealing the absence of iron staining and some dysplastic erythropoiesis and megakaryopoiesis. Oral iron medication was started (6 mg/kg/daily). The platelet count improved rapidly, while her hemoglobin recuperated more gradually.2.1.3A 15‐year‐old male presented to the clinic because of paleness and fatigue. For a month he had suffered from watery diarrhea, 1‐2× daily, without fever, loss of appetite, or weight loss. No recent travels abroad. He dislikes eating vegetables but otherwise keeps a normal diet. Born at term by uncomplicated vaginal delivery after a normal pregnancy, he was the first of two children of healthy, non‐consanguineous Belgium‐Kenyan parents. The family history was unremarkable. Physical examination revealed a pale adolescent with a systolic heart murmur, mild splenomegaly, and otherwise normal findings . The blood test showed severe microcytic anemia  with low reticulocyte count and low ferritin (<1 ng/mL). Serology for cytomegalovirus, parvovirus, and Epstein‐Barr virus, G6PD level, and hemoglobin electrophoresis was normal. Genotyping of the α‐globin cluster revealed silent carrier status for alpha thalassemia (αα/α‐). Several days after starting oral iron treatment (6 mg/kg/daily), no increase of hemoglobin and reticulocyte count was seen, but thrombocytopenia (platelets 34×10E9/L) appeared. Because two cell lines were down, a bone marrow investigation was performed, revealing the complete absence of iron staining and increased erythropoiesis and megakaryopoiesis. Some dysplastic features (like nuclear segmentation) of the megakaryocytes were seen. The bone marrow puncture was conducted under general anesthesia; therefore, a red blood cell and platelet transfusion were given beforehand. The following day iron was administered intravenously to quickly improve his iron status. Upon discharge oral iron supplementation was restarted. The platelet count improved rapidly, while his hemoglobin recovered more gradually. The gastro‐intestinal evaluation did not reveal a cause of chronic blood loss.2.1.4A 14‐year‐old female presented to the family physician for paleness. She had menorrhagia without complaints of abdominal pain or fatigue. She was the second of three children of healthy, non‐consanguineous Bulgarian‐Belgian parents. Medical history and family history were unremarkable, except that her sister and mother also suffered from menorrhagia. Physical examination revealed normal parameters and besides paleness no clinical abnormalities. The blood test showed severe microcytic anemia  and mild thrombocytopenia (platelets 87×10E9/L) with low ferritin (2 ng/mL). Additional blood work, including folic acid, vitamin B12, coagulation tests, and hemoglobin electrophoresis, was normal. Serology for cytomegalovirus, Epstein‐Barr virus, and parvovirus did not show any active disease. Oral iron medication was started (6 mg/kg/daily) and the platelet count improved quickly. On D27 after the start of oral iron treatment, the hemoglobin did not show any improvement, therefore, a red blood transfusion was given, and oral anti‐conceptive medication was added. Her hemoglobin recuperated to normal levels.2.1.5A 17‐year‐old female was admitted to the hospital for balloon valvuloplasty for congenital aortic valve stenosis. Besides some fatigue, no clear complaints. Her diet was severely iron deficient, and she had menorrhagia. Until a few months ago, she used oral contraceptives for heavy periods. She was the first child of healthy, non‐consanguineous parents from Suriname. The family history was unremarkable. Physical examination revealed normal parameters , and except for paleness and a systolic heart murmur, no abnormalities. The blood test showed severe microcytic anemia  and thrombocytopenia (platelets 16×10E9/L) with low ferritin (7 ng/mL). Additional blood work, including coagulation tests, parvovirus serology, and hemoglobin electrophoresis, was normal. A bone marrow investigation was performed, revealing the absence of iron staining and an increased megakaryocyte count. She received red blood cell and platelet transfusions, and a one‐time gift of IV iron. Oral iron medication was started (5 mg/kg/daily). After the transfusions, her hemoglobin and platelet were raised to, respectively, 7.4 g/dL and 35x10E9/L. The platelet counts rapidly improved to normal value, while the hemoglobin started to increase more gradually.3The cases discussed, supplemented by cases from the literature, are summarized in Table 4IDA is more often associated with mild to moderate thrombocytosis than with thrombocytopenia. The severity of thrombocytosis increases with the severity of anemia. There are conflicting opinions on whether or not cytokines are responsible for this (including interleukin‐6 and thrombopoietin).Thrombocytopenia is much less common in IDA and is often associated with severe iron deficiency.Reports of thrombocytopenia after the start of iron replacement therapy, are extremely rare.5The combination of severe IDA and thrombocytopenia are reported in children. With this rarely occurring entity, the use of red blood cell transfusion and the performance of a bone marrow examination is common. Iron monotherapy shows improvement of the platelet count as well as the hemoglobin. Therefore, if clinically feasible and other causes are unlikely , empiric iron therapy is warranted, and bone marrow examination and red cell or platelet transfusion can be omitted.No funding was provided for this study.The authors have declared that there is no conflict of interest.Conceptualization: Machiel van den AkkerFormal Analysis: Machiel van den AkkerWriting – original draft: Machiel van den Akker, Laura Chielens, Lisa Lopes, Jaques van Heerden, and Mahmoud ZaqoutWriting – review and editing: Machiel van den Akker, Jutte van der Werf Ten BoschAuthors grant Wiley a license to publish the article and identify itself as the original publisher. Authors also grant any third party the right to use the article freely as long as its integrity is maintained and its original authors, citation details and publisher are identified.This manuscript is an honest, accurate, and transparent account of the study being reported and no important aspects of the study have been omitted.


Information regarding changes in renin‐angiotensin‐aldosterone system (RAAS) during cardiac remodeling after mitral valvuloplasty (MVP) in dogs remains lacking.To assess the longitudinal effects of MVP on circulating RAAS activity.Eight client‐owned dogs receiving MVP for myxomatous mitral valve disease (MMVD).This is a cohort study. Plasma renin activity (PRA), angiotensin II (AT2), aldosterone (PAC), blood urea nitrogen (BUN), and creatinine concentrations, were measured in these dogs before (baseline) and at 3 consecutive monthly follow‐ups . Echocardiography was concomitantly used to assess the process of cardiac recovery after MVP.P < .05). There was a significant reduction in the PRA , AT2 , and PAC , respectively at Post‐1M, Post‐2M, Post‐3M compared to the baseline. Additionally, BUN and creatinine concentrations decreased from Post‐1M. The RAAS variables showed significant, weak to moderate, relationship with selected echocardiographic variables.The echocardiography revealed a significant decrease in LVIDDN, LA/Ao, FS, E velocity, E/A, E′ sep, S′ lat, E′ lat, and A′ lat after MVP compared with baseline (Mitral valvuloplasty contributes to decreased RAAS activity in MMVD dogs, which paralleled the process of cardiac reverse remodeling up to Post‐3M. This information facilitates formulating strategies to optimize clinical outcomes for dogs after MVP. AT2angiotensin IIBUNblood urea nitrogenCHFcongestive heart failureLA/Aoratio of the left atrial dimension to the aortic annulus dimensionLVIDDNnormalized left ventricular internal dimension in diastoleMMVDmyxomatous mitral valve diseaseMVPmitral valvuloplastyPACplasma aldosterone concentrationPRAplasma renin activityRAASrenin‐angiotensin‐aldosterone system1Chronic activation of the renin‐angiotensin‐aldosterone system (RAAS) plays an important role in the pathogenesis of congestive heart failure (CHF) both in human and veterinary patients. One aspect of RAAS activation in this setting is evoking potent peripheral vasoconstriction and sodium/water retention, contributing to ventricular hemodynamic loading and subsequent development of left ventricular and left atrial enlargement.Myxomatous mitral valve disease (MMVD) remains the most common acquired heart disease in dogsRecently, a study demonstrated greater RAAS inhibition in American College of Veterinary Internal Medicine (ACVIM) stage D vs stage C, which revealed the effectiveness of RAAS suppressive treatments in dogs with refractory CHF.In this study, we assessed the effect of surgical unloading intervention on RAAS activity by measuring plasma renin activity, angiotensin II and aldosterone concentrations in dogs before and at monthly follow‐ups after MVP. During this midterm postoperative period, alterations in other circulatory markers and echocardiographic variables considered relevant to the cardiac reverse remodeling were also documented and compared with that of the RAAS activity. We hypothesize that decline in RAAS activity will be observed along with the cardiac reverse remodeling process after mitral valvuloplasty in dogs.22.1Client‐owned dogs receiving MVP exclusively for MMVD from August 2019 to April 2020 at a private clinic were enrolled consecutively for this study. Baseline values were obtained within 1 week before the scheduled date of surgery. Postoperatively, all dogs have at least 3 monthly follow‐up evaluations at equal intervals. Clinical diagnosis, staging, and decision for the surgical indication of MMVD were determined through comprehensive evaluation including medical history recoding, physical examination, hematology, biochemistry, diagnostic electrocardiography, radiography, and echocardiography. Specific objects/variables to be evaluated before surgery were as follows: signalment , current cardiac medication, presence of cachexia, renal function , and echocardiographic variables reflecting size and function as delineated in the Echocardiography section below. Inclusion criteria included a diagnosed MMVD, an ACVIM stage of at least B2, and being surgically treated by MVP. The sole criterion for exclusion was a lack of 2 or more measurements during the follow‐up period. Before enrollment, informed client consent was obtained for every dog.2.2Mitral valvuloplasty was performed similarly to an established technique described for small dogs.2.3The echocardiographic assessment was carried out while the dog was maintained in lateral recumbency and examined under standard conditions using a sector probe of 5 MHz . Three consecutive heartbeats were recorded at the end of the expiratory phase. To evaluate structural and functional changes during the course of reverse remodeling of the left atrium and the left ventricle, combined conventional echocardiography protocol including 2‐dimensional, M‐mode, Doppler blood flow, and tissue Doppler imaging techniques from the right and left parasternal long‐ as well as short‐axes views, were obtained.2.4Plasma renin activity (PRA), angiotensin II (AT2), and aldosterone (PAC) concentrations were determined by radioimmunoassay  at each time interval. For this purpose, whole blood samples were withdrawn using disposable EDTA‐treated vacutainers . Clear nonhemolyzed plasma samples were collected after the spinning of blood at 3000 rpm for 10 minutes, frozenly stored, and the temperature strictly kept at −20°C. To minimize the postsampling ex vivo effect on the targeted RAAS activity, all frozen‐stored samples were sent on dry ice within 1 week after collection for measurement, where pretreatment of assay‐specific inhibitors was conducted in accordance with the manufacturer's instructions. The timing of sampling depended on the client's convenience for the visit. Feeding time and diet content were variable among the dogs. For other clinical biochemical tests including measurement of plasma BUN and creatinine, heparinized plasma samples were utilized immediately after centrifugation .2.5P‐value less than .05 was considered to indicate the Gaussian approximation of nonparametric data. Nonparametric data are presented as median and range. All analyses were conducted using GraphPad Prism 8.0 . Spearman's rank correlation and linear regression analysis were used to assess the relationship between RAAS and conventional echocardiographic variables. Coefficient of determination (R2) was computed from the sum of the squares of the distances of the points from the best‐fit curve.Statistical differences between groups were determined by repeated 1‐way analysis of variance for nonparametric data using Friedman test. Post hoc testing was done using Dunn's Multiple Comparison Test. A 33.1A total of 8 dogs met the inclusion criteria and were enrolled in this study. None were excluded during the follow‐up period. Breeds included Chihuahua (n = 3), Cavalier King Charles spaniel (n = 1), Chin (=1), Mongrel (n = 1), Pomeranian (n = 1), and Shih Tzu (n = 1). Six dogs were male (3 intact and the other 3 neutered) while 2 were spayed female. The median [range] age and weight of the dogs were 10.5 [8–14] years and 5.025 [2.4‐7] kilogram, respectively. In accordance with the ACVIM classifications scheme, 1 dog was at stage B2 and 7 at stage C. Before surgery, 3 dogs had been prescribed furosemide, 4 had been prescribed torsemide, 7 had been prescribed pimobendane, 4 had been prescribed an angiotensin‐converting enzyme inhibitor (ACEI), 1 had been prescribed spironolactone, and 6 had been prescribed amlodipine. After surgery, all dogs were discharged within 2 weeks of postoperative care. Complete stop of RAAS‐targeting and other medications affecting hemodynamics  after surgery was achieved at the time of discharge in all cases. Baseline characteristics and individual dosage regime of the study cohort are summarized in Table 3.2P < .05). Moreover, LA/Ao, FS, and S′ lat were significantly reduced at Post‐1M compared with baseline (P < .05). Systolic LV septal and lateral velocities (S′ sep and S′ lat) were significantly decreased at Post‐3M compared with baseline ; meanwhile, no statistically significant change in A′ sep was observed. Collectively, these findings indicated general morphological and functional improvement of the heart after MVP.Echocardiographic data were summarized in Table 3.3P < .05; Figure P < .01), Post‐2M , and Post‐3M , and significantly reduced at Post‐3M (235 [46.4‐960] pg/mL) compared with the baseline .Plasma concentrations of BUN, creatinine and RAAS markers are summarized in Figure 3.4rs = .37, .47, .42, .47, .46, .36, .36, .62; P < .05), respectively. Plasma AT2 concentration displayed significant positive correlation with LVIDDN, E′ sep, A′ sep, E′ lat, and A′ lat , respectively. In addition, PAC exhibited significant positive correlation with E/A ratio and S′ sep , respectively.Table 3.5R2) obtained from linear regression analysis between RAAS and cardiac measurements was illustrated in Table R2 = .15, .32, .19, .43, .43, .18, .27; P < .05), respectively. Angiotensin II exhibited significant effect on LVIDDN, LA/Ao, S′ sep, E′ sep, S′ lat and E′ lat , respectively. Plasma aldosterone concentration demonstrated significant effect on LA/Ao, FS, E velocity, E/A ratio, and S′ lat , respectively.Coefficient of determination  was found to decrease by the first month after MVP in most dogs, even though the extent of this reduction did not reach reference ranges inferred from several other studies involving the same measurements of healthy dogs .Starting from the first month after surgery, a significant difference from the baseline presurgical value was detected in most echocardiographic and RAAS variables. The difference had lasted over the postoperative 3‐month midterm period. Our result implies that in parallel with anatomical/functional normalization of the heart  there exists a corresponding decrease in the RAAS activity after the MVP. This is additionally supported by the discovery of statistically significant, though weak to moderate, correlation and linear regression relationships between the RAAS levels and echocardiographic variables. The moderate‐to‐low values of In humans, there is continued tissue RAAS activation after various surgical interventions,The results of this study might also be relevant to dogs with MMVD that do not receive surgical treatment. RAAS activity at 3 months after MVP where the echocardiographic variables improved to the point that matches B1 criteria was only slightly elevated than the previously reported reference values for dogs. On the other hand, levels of echocardiographic variables and AT2 were not as low at 1 month after the surgery compared with those at 3 months. Our data is unique in that RAAS activity was shown to be associated, at least to some extent, with echocardiographic indicators that reflect the severity of the disease. From this perspective, postoperative echocardiographic changes and RAAS might as well be applicable in reverse chronological order, in other words, natural MMVD progression from mild to severe. Though indirectly, our study supports the relationship between RAAS variables and cardiac enlargement. This information adds to our knowledge about when during the preclinical phase of MMVD the neurohormonal system could become activated. This issue is of clinical significance when determining at what time administration of RAAS blockers should be started for cases and studies dealing with naturally occurring MMVD.To conclude, the present work focused principally on the midterm outcome of MVP for dogs with MMVD, which demonstrated a modest reduction in RAAS activity and sufficient cardiac reverse remodeling such that further RAAS blockade is considered unwarranted.Limitations of the current study include the small number of animals enrolled and consequently inability to perform further assignment to subgroups according to the stage of MMVD, heterogeneity of dogs in various characteristics , time lag between the blood withdrawal and actual inhibitor addition before the assay, inconsistency in the sampling circadian time, and incomprehensive and short‐term documentation of RAAS.One potential bias is the differences in administered medication regimes, especially those affecting hemodynamics (diuretics,Furthermore, only conventional components of RAAS were considered in this relatively short‐term study. Recently, alternative RAAS pathways have been described and investigations regarding their interaction with pharmacological RAAS blockade5The present study demonstrated midterm decrease in RAAS activity after MVP in MMVD dogs, which paralleled the process of reverse remodeling up to the third postoperative month.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.Approved by VCA Japan Shiraishi Animal Hospital IACUC, protocol number R2‐062.Authors declare human ethics approval was not needed for this study.Table S1. Baseline characteristics and individual dosage regime of the study cohort.Click here for additional data file.


Successful establishment of a tumour relies on a cascade of interactions between cancer cells and stromal cells within an evolving microenvironment. Both immune and nonimmune cellular components have key roles in this process. Here, we review the major anti‐ and pro‐tumour factors in the early tumour microenvironment and discuss how understanding this process may be exploited in the clinic. Successful establishment of a tumour relies on a cascade of interactions between cancer cells and stromal cells within an evolving microenvironment. Both immune and nonimmune cellular components are key factors in this process, and the individual players may change their role from tumour elimination to tumour promotion as the microenvironment develops. While the tumour–stroma crosstalk present in an established tumour is well‐studied, aspects in the early tumour or premalignant microenvironment have received less attention. This is in part due to the challenges in studying this process in the clinic or in mouse models. Here, we review the key anti‐ and pro‐tumour factors in the early microenvironment and discuss how understanding this process may be exploited in the clinic. Arg1arginase 1ATRAall‐trans retinoic acidCAFcancer‐associated fibroblastCC(L)C‐C motif chemokine (ligand)CDcluster of differentiationChi3L1chitinase‐3‐like protein 1CRCcolorectal cancerCXCLC‐X‐C motif chemokineDAMPsdamage‐associated molecular patternsDCsdendritic cellsECMextracellular matrixEGFepidermal growth factorFAPfibroblast activation proteinFGF(R)fibroblast growth factor (receptor)FSP1fibroblast‐specific protein 1Gas6growth arrest‐specific 6G‐CSFgranulocyte colony‐stimulating factorGDF‐15growth differentiation factor‐15HGFhepatocyte growth factorHLAhuman leucocyte antigenHMGB1high‐mobility group protein 1HPVhuman papillomavirusICAM‐1intercellular adhesion molecule‐1ICPIimmune checkpoint inhibitorsIDOindoleamine dioxygenaseIFNinterferonIGFinsulin‐like growth factorILinterleukinILCsinnate lymphoid cellsJAM2junctional adhesion molecule 2KPCKrasLSL.G12D/+; p53R172H/+; PdxCretg/+LAIR‐1leucocyte‐associated immunoglobulin‐like receptor‐1LIFleukaemia inhibitory factorLOX(L)lysyl oxidase (‐like proteins)MAP(K)mitogen‐activated protein (kinase)M‐CSFmacrophage colony‐stimulating factorMDSCsmyeloid‐derived suppressor cellsMHCmajor histocompatibility complexMICA/BMHC class I‐related proteins A/BmiR‐microRNA‐MMPmatrix metalloproteinaseMMTV‐PyMmouse mammary tumour virus‐polyoma middle tumour antigenMYLmyosin light chainNETneutrophil extracellular trapNF‐kBnuclear factor kappa‐light‐chain‐enhancer of activated B cellsNKG2Dnatural killer group 2, member DNKsnatural killer cellsNKTsnatural killer T cellsNOSnitric oxide synthaseOACoesophageal adenocarcinomaop/oposteoporoticOPNosteopontinP4HA1prolyl 4‐hydroxylase a‐subunit isoform 1PBMCperipheral blood mononuclear cellsPD‐(L)1programmed dead‐(ligand)1PDACpancreatic ductal adenocarcinomaPDGF(R)platelet‐derived growth factor (receptor)PDXpatient‐derived xenograftPGE2prostaglandin E2PLOD‐1, PLOD‐3procollagen‐lysine 2‐oxoglutarate 5‐dioxygenase 1 and 3PSCpancreatic stellate cellsROSreactive oxygen speciesSASPsenescence‐associated secretory phenotypeSHHsonic hedgehogSMAsmooth‐muscle actinSPARCsecreted protein acidic and rich in cysteineSTATsignal transducer and activator of transcriptionTAMtumour‐associated macrophageTANtumour‐associated neutrophilsTAZtranscriptional coactivator with PDZ‐binding motifTCRT‐cell receptorTGF‐βtransforming growth factor betaTHT helperTKItyrosine kinase inhibitorTLRtoll‐like receptorTNFtumour necrosis factorTRAILTNF‐regulated apoptosis‐inducing ligandTRAMPtransgenic adenocarcinoma mouse prostateregTT regulatory cellVEGFvascular endothelial growth factorXCL‐1X‐C motif chemokine ligand 1YAPYes‐associated protein1The stepwise accumulation of genetic changes from healthy tissue through to malignancy, is accompanied by co‐option of neighbouring normal cells to support tumour development and progression. These ‘normal’ cells, which include immune and nonimmune populations, are collectively termed the stroma. Stromal cells and cancer cells collectively form the tumour microenvironment.While much work has focused on the characterisation of oncogenic mutations within epithelial cells from the point of cancer initiation, the acquisition of mutations alone is not always sufficient to give rise to cancer, as the majority of mutagenic cells die or senesce , 2, 3. IWith our increasing appreciation of the complexities of the tumour microenvironment, the concept of exploiting the stroma for therapeutic benefit is becoming more attractive. Thus, whether the characteristics of an evolving microenvironment can be exploited towards early diagnosis and patient stratification approaches in early stages of carcinogenesis remain‐ key questions to be addressed. In this review, we examine the state of our understanding of components within the microenvironment and their contribution to malignant transition. While immune components have been the subject of the most intense research, nonimmune constituents are now emerging as critical players, and here, we attempt to emphasise their role, highlighting emerging strategies to facilitate early detection.2The recognition and elimination of genetically distinct transformed cells by the immune system (immunosurveillance)  represenThe presence of immune cells in tumours capable of recognising mutated cancer cells raises several important questions, which until now remain a significant challenge in oncology: How do cancer cells avoid destruction by the immune system? How is immune control lost? And when during tumorigenesis is this control lost?2.1reg ratios in established tumours are associated with favourable long‐term prognosis [et al. [T lymphocytes are key determinants in tumour fate. Elevated CD8 infiltration and high CD8:Trognosis , 20, 21.rognosis , 25, 26.rognosis , 28. In  [et al.  elegantl [et al. , 32, 33.et al. [reg diversity and numbers of circulating Tregs were indicative of T‐cell dysfunction [regs suppress T‐cell effector functions [regs [However, this is in stark contrast to established tumours which frequently present with a paucity of tumour‐infiltrating lymphocytes exhibiting an impaired cytotoxic capacity. Established tumours escape immune attack through four major mechanisms. First, mutation of constituents of the antigen presentation pathway reduces neoantigen presentation rendering cancer cells ‘invisible’ to T cells. Indeed, 40% of human non‐small‐cell lung cancers exhibit defects in human leucocyte antigens (HLAs), reducing antigen presentation and promoting immune escape . Second,et al.  showed tet al. , 37, whifunction , 38, 39.function , 42. Treunctions , 44, downs [regs , 47, 48.ns [regs , 50, 51.Collectively, these reports add to a growing body of evidence suggesting that while T cells are essential for immune control of premalignant lesions, loss of T‐cell‐mediated immune surveillance may occur prior to malignant transition and that immune involvement actively promotes progression to cancer , 38, 52.2.2Although critical fate determinants, T cells do not operate in isolation, engaging in extensive crosstalk with other immune cells within the developing microenvironment . The inn2.2.1Of all immune cells, macrophages are the most abundant and display arguably the highest plasticity, responding rapidly to a diverse array of environmental stimuli. Activated pro‐inflammatory (M1) and anti‐inflammatory  populations have been described extensively , 54. HowIn very early stages of tumour development, enhanced numbers of macrophages have been observed , 60, andet al. who crossed MMTV‐PyMT mice, which develop breast tumours spontaneously, with osteoporotic (op/op) mice lacking CSF‐1, a cytokine critical for macrophage function. While they observed little impact on the development of premalignant lesions, progression to invasive carcinoma was significantly impaired [However, these antitumour effects of very early lesions are replaced by a tumour‐promoting phenotype at an early stage of carcinogenesis. This requirement for macrophages in malignant progression was elegantly demonstrated by Lin impaired , 78.reg phenotypes [regs and along with CCL5 and CCL17 can inhibit T‐cell proliferation [A key feature of once antitumour, inflammatory macrophages is the acquisition of immune‐suppressive traits to facilitate malignant progression , 79, 80.enotypes , 89. CCLferation , 91. SimKRASG12D upregulated the expression of ICAM‐1 to recruit macrophages, which through matrix metalloproteinase‐9 (MMP9) and TNF‐α production induced metaplasia, the earliest abnormal pancreatic lesions [Macrophages within the premalignant microenvironment may also elicit tumour‐promoting activities beyond influencing immune cells, either via direct effects on epithelial cells or by indirect effects on other nonimmune stromal populations. There are several direct epithelial mechanisms. First, factors such as ROS and NOS also induce DNA damage contributing to genomic instability and mutational burden , 93, 94. lesions . Also in lesions . Macroph lesions . SignallBeyond their direct influence on epithelial cells, as for any organ, an oxygen supply needs to be established to support an expanding epithelial compartment in hyperplastic lesions , 103. HeMacrophages are also involved in remodelling of the underlying tissues. The extracellular matrix (ECM), a 3D network composed of polysaccharides, proteins including collagens and glycoproteins, provides biochemical and biomechanical cues critical for tumour progression , 117. Ma2.2.2Within the early cancer setting, ILCs are extremely efficient at eliminating malignant cells through the expression of perforin and granzyme, as well as death ligands, such as TNF‐regulated apoptosis‐inducing ligand (TRAIL) and Fas ligand , 134 ILCA fluctuating balance of activating and inhibitory receptor–ligand interactions on NK cells controls their activity. The most interesting of these, from a tumour immunology perspective, has been the natural killer group 2, member D (NKG2D) receptor, which can recognise MHC class I‐related proteins A and B (MICA and MICB), ectopically expressed antigens such as Rae1 or H60, as well as DNA damage‐induced cell surface ligands , 140, fa+ T cells – however this is a considerable simplification, and the full nature of these subsets' functions is still under investigation. Analogous to NK cells, ILC1s and ILC3s are also activated by IL‐15, and contribute to immune surveillance through the release of TNF, IL‐8 and IL‐2, promoting leucocyte recruitment and proliferation [The role of the other ILC populations is poorly understood and varies depending on the local microenvironment, with both immune‐protective and tumour‐promoting effects demonstrated in lung, skin and colon cancer , 155. ILferation , 157. Duferation , 149. ILferation .in vitro to the lipid alpha Gal‐Cer. Mice lacking type I NKTs have generally been found to have defects in tumour immune surveillance and there is interest in using alpha Gal‐Cer to stimulate antitumour immunity, though results in patients have been limited. However, in a transgenic model of intestinal polyps (‘APC min’ mouse), type I NKTs were found to promote polyp formation by recruitment of Tregs. ‘Type II’ NKTs  use diverse alpha and beta TCR chains; in contrast to type I, mouse experiments suggest they have an immune‐regulatory, tumour‐promoting function. Like the ILCs, the importance of NKTs in early tumorigenesis is not well‐understood.Another T‐cell population with innate‐like properties are the natural killer T cells (NKTs). These are a unique population of T cells that recognise lipid antigens presented on the class I‐like molecule C1d. ‘Type I’ NKTs use a semi‐invariant TCR made of a specific alpha chain with a small number of beta chains and respond 2.2.32O2, TRAIL and IFN‐γ and expression of costimulatory molecules [Neutrophils operate as vital first responders during infection. Given this critical early role in infection, it follows that neutrophils also contribute extensively to early tumorigenesis, with anti‐ and pro‐tumour activities reported. As for macrophages, this may in part be a consequence of their functional plasticity. Indeed, the phenotype of neutrophils in the TME depends on tumour type and stage. In early tumorigenesis, neutrophils are recruited by epithelial‐ and stroma‐derived chemokines such as G‐CSF, CXCL8, CXCL1, CXCL2, CXCL3 and CXCL5 , 161. Heolecules , 164. Atolecules , 166.regs, directly suppressing CD8 T cells via PD‐L1 and indirectly by IL‐8‐driven arginase production [However, directly contradicting this antitumour activity, TANs have also been reported to contribute to carcinogenesis from the point of tumour initiation. Local TGF‐β pushes neutrophils towards a tumour‐promoting phenotype , 167, inoduction , 175. Onoduction , 179. A oduction , metastaoduction  and tumooduction  exists, oduction .2.2.4reg responses [Dendritic cells are professional antigen‐presenting cells that bridge the innate and adaptive immune compartments, stimulating T cells in an antigen‐specific fashion. In the still inflammatory early TME, proficient DCs effectively present tumour antigen to T cells alongside costimulatory molecules CD40, CD80 and CD86, thereby playing a central role in immune surveillance , 186. Heesponses , 193. PDesponses . Cytokinesponses , 199. Toesponses , 200.Thus, although our knowledge is increasing, the role of the immune infiltrate in premalignant tissues and the balance of protection vs. progression remains unclear Fig. . With a The mechanisms underlying the transition from early immune surveillance by innate immune cells to tumour‐promoting phenotypes of macrophages, NK cells, neutrophils and DCs, later during tumorigenesis, are poorly understood. This is in part due to the technical challenges of studying very early lesions in a ‘true’ epithelial setting, which requires transgenic mouse models. Much of the evidence for early innate immune surveillance comes from macroscopic observations of increased cancer rates in immune‐suppressed states, rather than direct observation of the premalignant epithelium. One could hypothesise two key reasons for this shift. According to a cancer cell centric view, premalignant lesions that acquire the ability to co‐opt pro‐tumorigenic innate immunity can grow into tumours , 202. No3While the interactions between malignant cells and the immune system play a key role in establishing the tumour microenvironment during malignant transition, other components of the host tissue play a critical and historically overlooked part. Indeed, the accumulation of stromal components including fibroblasts and vessels is also reported in premalignant lesions including oesophageal, pancreatic and skin cancers , 210. Heet al. [in vitro and xenograft studies in prostate cancer showed that tumour fibroblasts but not normal counterparts could stimulate proliferation and transformation of epithelial cells from benign prostate hyperplasia. Interestingly, CAFs alone were unable to induce growth of normal epithelium, providing early indications of a requirement for reciprocal communication between the two compartments for malignant transformation to occur. Indeed, epithelial cells in precancerous states have been shown to use TGF‐β, PDGF‐D and FGF‐2 to recruit, activate and transform tissue‐resident fibroblasts towards a CAF phenotype [Cancer‐associated fibroblasts exhibit diverse pro‐tumour functions including growth factor secretion, angiogenesis, immunoregulation , 217, ECet al.  depletedet al. , 233. Toet al. , 235. Thet al. , 239. Fiet al. , 242. Eahenotype , 235. Anhenotype , 246. FoIncreasing genomic stress can promote the production of activin and TGF‐β, supporting CAF activation . SimilarCancer‐associated fibroblasts exhibit a capacity to modulate lymphatic vessels, their presence correlating with lymphatic vessel density and metastasis . Here, C3.1Cancer‐associated fibroblasts are emerging as key immune modulators in the tumour context, with increasing evidence to suggest that crosstalk between the nonimmune stroma and leukocytes is essential for escape from early immune control and tumour progression , 266. CAImmune‐modulatory fibroblasts within the developing tumour microenvironment can impact recruitment, retention and polarisation of the innate immune compartment , 269. Weregs contribute to the onset of an immune‐suppressive environment [regs via CXCL12, mediate retention by OX40L, PD‐L2 and JAM2 interactions and support survival [Notably, mounting evidence supports a role for CAFs in modulation of adaptive immune components, shaping T‐cell dysfunction associated with loss of immune surveillance. The production of soluble factors such as IL‐10 and TGF‐β, and metabolic mediators prostaglandin E2 (PGE2), indoleamine 2,3‐dioxygenase (IDO) and arginase can impair activation and cytotoxicity or push responses towards tumour‐promoting TH2 and TH17 responses , 283. FAironment . In humasurvival , 285. CAsurvival , 288. WhAs alluded to previously, a complex interplay exists between CAFs and immune cells, the generation of the tumour microenvironment relies on reciprocal signals. For example, while CAFs impact macrophages and neutrophils, neutrophils also release ROS and NETs to deliver pro‐fibrotic signals , 289. Li3.2Following tissue damage, fibroblasts of healthy tissues rapidly synthesise and deposit ECM as part of the wound‐healing response. In cancer, however, these mechanisms are perturbed to generate a fibrotic, permissive niche , 291. HeThus, within the developing TME, CAFs evolve to alter cellular, structural and chemical aspects of tissues, but the physical changes they afford through deposition and remodelling of the ECM themselves also play a pivotal role in malignant transition.3.3et al. in 2005 [et al dynamically tuned gels from normal  to malignant  stiffness to demonstrate coincident mechanical sensing and loss of mammary epithelial morphology through TGF‐β and YAP signalling. Changes in stiffness together with increasingly aligned fibres promote tumour cell migration, leveraging traction against remodelled matrix [A gradual shift in the rigidity of the ECM from that of normal tissue is implicated in malignant progression due to its far‐reaching impact on cell morphology, migration, alignment, angiogenesis and immune responses , 312. In in 2005 , a conced matrix , 313. Ind matrix , 314.Extracellular matrix tethers and immobilises many soluble mediators, serving as a valuable reservoir for pro‐tumour, CAF‐activating and immune‐suppressive factors such as EGF, FGF, PDGF and critically TGF‐β , 316. MaRemodelling of the ECM modulates immune cell trafficking and functional status , 327. ThThe physical properties of remodelled ECM exert diverse effects on epithelial, stromal and immune cells it surrounds , 343. ThBeyond any direct effects on cell behaviour within the developing TME, an accumulation of collagens, increased cross‐linking and stiffening, and rapidly expanding blood vessels lead to a gradual increase in interstitial fluid pressure , 319. ThA multiplicity of mechanisms exists for CAFs to influence, and be influenced by, the developing TME. With growing evidence and technical precision, this is being ascribed to an increasing number of unique CAF subpopulations, defined by tissue‐specific markers. This overwhelming complexity can be distilled, however, into the recurrent functional observations of these CAFs performing either immune‐modulatory, ECM deposition or mechanical remodelling roles. Whether these specialised subpopulations are present from the outset of malignancy as tissue‐resident fibroblasts or whether they are plastic and interconvert with developing cues in the TME remains an open question in the field.4This review has focussed on the reciprocal microenvironment changes that facilitate early tumour growth. However, as practising oncologists are aware, cancer manifests as a systemic illness. The molecular details of how cancer ‘reaches out’ to influence whole‐body physiology are beginning to be understood , 365. Ag5Historical mouse models of cancer often use injectable human cell lines in immunodeficient mice. While these models have been key to the progress in developing cancer treatments for the past half‐century, they do not model the early microenvironment or the adaptive immune component of the TME. Immune‐based treatments are now central to the practice of oncology, and so, it is essential to use immune‐competent models that reflect the whole TME. Advances over the last decade in genetic engineering have increased the feasibility of spontaneous tumour models, which have a neoantigen profile and local immune infiltrate more representative of human cases than injectable tumour models , 376. Hoin vivo and in vitro toolkit (Table Concerted efforts are now being made to ‘humanise’ murine models as far as reasonably possible. Patient‐derived xenografts (PDX) in combination with an immune system reconstituted from an autologous haematopoietic stem cell are increasingly used in tumour immunology, especially to predict patient response to therapy . Howeverit Table .6For many cancers, early detection and intervention significantly improve survival. In oesophageal cancer, the detection of the premalignant condition Barrett's oesophagus can dramatically increase dismal 5‐year survival rates of less than 20%. However, in the case of Barrett's, monitoring by endoscopic surveillance represents a significant cost and disruption to patients' lives. Thus, identifying the minority that will go on to develop OAC is a priority. Examining the underlying microenvironment may help to define and stratify patients at greater risk. Indeed, molecular imaging has been used to exploit changes in tissue glycosylation for the identification of dysplastic tissues , 388. EaDeveloping our comprehension of how immune and fibroblastic components contribute to the transition from premalignant to cancer affords a range of clinical approaches for early intervention. Understanding kinetics of the host responses in malignant transition, such as loss of immune surveillance, may provide a unique therapeutic window. Preclinical models have explored strategies to prevent or reverse the switch in innate cells to maintain or reinstate an antitumour phenotype. However, while attractive, many pathways disrupted during immune suppression are key to maintain immune tolerance and protection against autoimmunity, thus global approaches to inhibit the innate immune system in early cancer may be counterproductive. Instead, due to their diverse array of functions, targeting the fibroblasts may provide an alternative method to stall malignant transition. While eradication strategies have been investigated, such an approach may in practice be hampered by the lack of specific markers and complexities stemming from heterogeneous pro‐ vs. antitumour subsets , 233. ReBeyond the design of novel therapeutic targets, a thorough understanding of the composition and roles of stroma of the early tumour microenvironment may inform patient prognosis or predict response to treatment , 406. Fo7Tissues surrounding mutated cells play a pivotal role in carcinogenesis, from initial elimination responses through to supporting growth and development of a permissive niche. Recent technological advances have dramatically increased our appreciation of the complex networks at play, the duality of our immune system, the increasing diversity and functional specialisation of fibroblasts, and the impact of changing tissue mechanics. Even so, outstanding challenges remain. We know relatively little about the plasticity, heterogeneity and interdependencies that define the evolving stromal landscape. Nevertheless, it is likely that these complex tumour–stroma interactions compliment the well‐documented tumour heterogeneity and cumulatively contribute to increased robustness of early tumours , and we The authors declare no conflict of interest.JDS conceived the review, and WMM, JOJ and JDS wrote the paper.


N = 38 male, mean age = 74.0 ± 5.7 years, body-mass index = 25.5 ± 4.1 kg/m2) aged 64 years or older participated. Cognitive flexibility and inhibition were assessed by means of a Trail-Making Test (TMT) and a Stroop task. Perceived levels of social burden and perceived stress in general were assessed via standardized questionnaires. Furthermore, diurnal cortisol profiles were assessed. Overall, older participants showed lower cognitive performance than the younger . However, this association did not remain significant when adjusting for multiple comparisons. Social burden was highly correlated with perceived stress, highlighting its importance as a stressor (p < .001). Furthermore, participants who reported higher levels of perceived stress showed lower performance in the TMT as a measure for cognitive flexibility (p < .001). Moreover, social burden was associated with cognitive flexibility, which was assessed by means of the TMT (p = .031). However, this association did not remain significant when using the adjusted α-level. Furthermore, we found that total diurnal cortisol output was a mediator for the relationship between age and inhibition, which was assessed by means of a Stroop task. At last, we found that the mediating role of cortisol interacts with social burden in the way that especially in younger age, high levels of social burden are associated with higher cortisol levels and, therefore, with lower cognitive performance. We conclude that it is important to consider social burden as an additional stressor in older age which is associated with cognitive aging.Stress can be a risk factor for mental and physical health. We investigated the role of social burden as a potential stressor in older age and its associations with cognitive aging . Eighty-three older adults (The online version contains supplementary material available at 10.1007/s10433-021-00616-8. Impairments in cognitive functioning  are one of the most stressful and challenging burdens in older age  and one because of missing data in the social burden questionnaire. The final sample consisted of N = 83 participants. Most of the participants were members in a senior leisure program of the ‘Bavarian Red Cross’ . All participants gave their written and informed consent. Data were anonymized directly after collection to protect participant’s privacy. The study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the local ethics committee of the Friedrich-Alexander University Erlangen-Nürnberg (# 194_18 B).In this study, initially The Mini Mental State Examination , ‘mild to moderate’ (6–10), and ‘severe depression’ (11–15) . Participants were instructed to keep the salivette in their mouth for at least one minute and to move it back and forth, but not to chew on it. Participants were provided with eight salivettes and were instructed to collect four on each of two consecutive days within seven days after the cognitive assessment. The following time points were scheduled: immediately after awakening, before lunch, in the afternoon/before dinner, and at bedtime. Participants were instructed not to eat, drink, or brush their teeth at least 30 min before saliva collection saliva samples were stored in the participants fridge or freezer for approximately 1–2 weeks until they were given back to the experimenters and stored at -30 °C. For analysis, salivettes were thawed at room temperature and were centrifuged at 2000 g and 20 °C for ten minutes immediately before analysis. Salivary cortisol concentrations were determined in duplicate using enzyme-linked immunosorbent assays . Intra- and inter-assay coefficients of variation were below 10%. The total diurnal cortisol output, which was calculated as area under the curve with respect to ground  as well as the diurnal slope, was used as markers for diurnal cortisol profiles. The AUCg can be used as a measure of total diurnal cortisol output, and the diurnal slope can be used as a marker for diurnal cortisol changes. Mean values from both days were calculated and used for further statistical analysis.Seniorennetz,” which is offered by the ‘Bavarian Red Cross’ , is to provide older people access to the "digital world". The members attend age-appropriate courses, which are led by volunteer tutors. Additionally, meetings in groups of interests and one-to-one discussions about anything related to computers, tablets, and smartphones are offered. We recruited most of our participants this way. However, because of time restrictions, we were not able to only focus on this target group and also included other people who take not part in this program.Participants were recruited by means of e-mails, flyers, and posters including information about the study. Furthermore, our study was advertised personally in a local senior leisure program. The aim of this program called “Our study was carried out between May and August 2019. At the day of cognitive testing, the participant and one experimenter (AP or SK) met in a quiet room, usually in the laboratory of the Chair for Health Psychology in Erlangen between 10 a.m. and 5 p.m. First, the declaration of consent was signed by the participants on that day. Subsequently, the experimenter gave instructions about the saliva collection procedure which the participants should do by themselves at their homes within the following week. After the introduction to saliva collection, the cognitive testing and the questionnaire assessment started. This included the Stroop test and the TMT Reitan  as well Y: dependent variable, X: independent variable, M: mediator variable). The mediation analysis includes three steps: first, evaluating the direct path , second, evaluating the indirect paths , and third, investigating whether the direct effect disappears when including the indirect path (X–M–Y) into the model. For the moderated mediation analysis (hypothesis 5), the PROCESS model no. 7 was tested . 10,000 bootstrap samples were used for both models. The data set that has been used for statistical analysis is provided as supplementary material S1. To correct for multiple comparisons, a Bonferroni-adjusted α-level of αadjusted = 0.05/7 = 0.0071 was used for analyses, because seven variables  were compared  was used. Normality of distribution was tested by means of the Kolmogorov–Smirnov test. Because of positive skewness and violation of normality, cortisol-AUCg’s, difference score in the TMT and perceived social burden were transformed by means of the natural logarithm (ln) prior to further statistical analysis. Furthermore, PSS scores were transformed by means of the square root-transformation to achieve a normal distribution. Partial correlations were used for testing the first, second, and third hypotheses. Sex, BMI, and relationship status were included as covariates in the partial correlations as well as in all further statistical analyses. For the mediation and moderation analyses, the SPSS macro PROCESS Hayes  was usedN = 83 participants  = 25.5 ± 4.1 kg/m2). All participants were retired and in most cases without any part-time job. The mean score in the MMSE  differed between participants who took any of these medications and participants who did not report medication intake . Therefore, medication was not included as covariate into the further statistical analyses.The final sample consisted of Mean cortisol profiles of all participants are shown in Fig. t(56.6) = -2.35; p = 0.023; women: 8.0 ± 3.2 s; men: 10.4 ± 5.5). However, this association did not remain significant after adjusting the α-level for multiple comparisons (αadjusted = 0.0071). No further sex differences were found for any of the other variables .Mean processing times in the Stroop task as measure for inhibition were 13.4 ± 3.2 s in the congruent and 22.4 ± 5.9 s in the incongruent condition. Mean Stroop interference scores were 9.1 ± 5.1 s. Because an in-house-developed Stroop task was used, no reference data are available. Mean Stroop interference scores were significantly higher in men than in women  reported being single. Most  were living together with the partner or their family, N = 26 (31.3%) were living alone, and one (1.2%) was living in a rest home.Overall, participants showed relatively low levels of perceived stress (11.7 ± 5.2) and social burden (1.7 ± 0.6). Fifty-nine (71.1%) of the participants reported having an intimate partnership and the others (r(78) = 0.24, p = 0.038; TMT: r(78) = 0.23, p = 0.043; Fig. adjusted = 0.0071).Partial correlations in which sex, BMI, and relationship status were included as covariates, yielded the following results: age was positively correlated with the Stroop and TMT scores, indicating that older participants showed lower performance in the Stroop task as measure for inhibition as well as in the TMT as measure for cognitive flexibility (Stroop: r(78) = 0.38, p < 0.001; Fig. p ≥ 0.732).Our second hypothesis (H2) was confirmed: social burden was positively related with perceived stress (r(78) = 0.44, p < 0.001; Fig. p ≥ 0.351). Furthermore, no associations between any of the cortisol indices and any of the EF domains (cognitive flexibility or inhibition) were found.Our third hypothesis H3) that participants who perceived more stress or show higher cortisol-AUCgs and flatter cortisol slopes will show lower cognitive performance in the domains cognitive flexibility (which was assessed by means of the TMT) and inhibition (which was assessed by means of a Stroop task) was confirmed for the association between TMT and perceived stress (PSS and TMT:  that parr(78) = 0.24, p = 0.031; Fig. adjusted = 0.0071. Moreover, both cortisol indices (AUCg and cortisol slope) and, thus, both measures for diurnal cortisol secretion were associated with age: Older participants showed lower cortisol-AUCgs (r(78) = 0.38, p < 0.001; Fig. r(78) = -0.49, p < 0.001).Further correlation analyses, for which we had no specific hypotheses, showed that social burden was positively associated with performance in the TMT (The whole correlation matrix is provided in Table To test our fourth hypothesis that stress (perceived stress or cortisol measures) mediates the association between age and EF, separate mediation analyses were calculated, for both EF domains inhibition and cognitive flexibility.p = 0.038), the paths between age and cortisol-AUCg (p = 0.0073), as well as between the cortisol-AUCg and Stroop performance (p = 0.010), were significant. However, this association did not pass the correction for multiple comparisons (αadjusted = 0.0071). Furthermore, the whole indirect pathway (age—cortisol-AUCg—Stroop) was significant . This means that the association between age and Stroop performance  is associated with higher cortisol levels in the older participants. However, when including the diurnal cortisol slope or perceived stress into the model, no relevant significant associations were found.For inhibition, which was assessed by means of a Stroop task, the mediation model was confirmed when including the cortisol-AUCg as mediator variable Fig. : The dirp = 0.043) and the indirect path between age and the cortisol-AUCg reached significance (p = 0.007). Note that the association between age and TMT performance did not pass the correction for multiple comparisons. The path between the cortisol-AUCg and the TMT-performance as well as the whole indirect path (age—cortisol-AUCg—TMT) were not significant . Therefore, the hypothesis that the diurnal cortisol output is a mediator for cognitive aging  was not confirmed. For the diurnal cortisol slope, no relevant significant associations were found as well.For cognitive flexibility, which was assessed by means of the TMT, the following significant associations were found for the cortisol-AUCg Fig. : The dirThe aim of our fifth hypothesis was to test whether social burden is an additional stressor that moderates the association between age, EF, and stress that has been investigated in H4. However, because of the small sample size the following findings should be treated with caution and should be considered as preliminary.p = 0.01) as well as with age even when the indirect path was included in the model (p = 0.005) as well as. The cortisol-AUCg was only marginally significantly associated with social burden (p = 0.099). However, this p-value is much higher than our adjusted α-level (αadjusted = 0.0071). The indirect path (age—cortisol-AUCg—Stroop) was significant for participants with high and medium levels of social burden only  = 0.46), but not for participants with low levels of social burden . Perceived stress was considered as psychological stress measure, and diurnal cortisol profiles were considered. Our main findings were that social burden was highly correlated with perceived stress and that the total diurnal cortisol output was a mediator for the relationship between age and inhibition (which was assessed by means of a Stroop task). Furthermore, social burden interacted with the association between age and the cortisol-AUCg in the latter model, in the way that especially in younger older age, high and medium levels of social burden are associated with higher cortisol levels and, thus, with lower cognitive performance.r > 0.2), and thus, our first hypothesis H1 was confirmed. However, this association did not pass the correction for multiple comparisons. Nevertheless, the direction is in line with our hypothesis and a large number of previous studies  with medium effect sizes (p < 0.001 and strong effect size r > 0.3) with perceived stress in general, highlighting its importance as a stressor which has been reported previously (Cranford r > 0.2).Furthermore, we were able to show that social burden was highly correlated , which is in favor of our hypothesis H4. This finding fits well into previous research that also found lower cognitive performances for older adults with higher levels of perceived stress . Differences between subjective stress measures and physiological stress markers have been reported previously , but has not been empirically researched any more than the question of whether this can also be provided by positive support” [Inwieweit positive Ausprägungen [bestimmter] … Variablen … in der Lage sind, Negativunterstützung abzupuffern, kann auf Grund der Ergebnisse der Stressforschung zwar angenommen werden , ist aber empirisch genauso wenig erforscht, wie die Frage, ob dies auch positive Unterstützung vermag]  is considered as a resource or as a protective factor  and not only on the positive ones.S1. Data set that has been used for statistical analysis. (CSV 8 kb)Below is the link to the electronic supplementary material.


The high prevalence of atrial fibrillation (AF) in the very elderly population (aged >80 years) might be underestimated. The elderly are at increased risk of both fatal stroke and bleeding. The Optimal Thromboprophylaxis in Elderly Chinese Patients with Atrial Fibrillation (ChiOTEAF) registry provides contemporary management strategies among the elderly Chinese patients in the new era of non‐vitamin K antagonists.To present the 1‐year follow‐up data from the ChiOTEAF registry, focusing on the use of antithrombotic therapy, rate vs. rhythm control strategies, and determinants of mortality and stroke.The ChiOTEAF registry analyzed consecutive AF patients presenting in 44 centers from 20 Chinese provinces from October 2014 to December 2018. Endpoints of interest were mortality, thromboembolism, major bleedings, cardiovascular comorbidities, and hospital re‐admissions.Of the 7077 patients enrolled at baseline, 657 patients (9.3%) were lost to the follow‐up and 435 deaths (6.8%) occurred. The overall use of anticoagulants remains low, approximately 38% of the entire cohort at follow‐up, with similar proportions of vitamin K antagonists (VKA) and non‐vitamin K antagonists (NOACs). Antiplatelet therapy was used in 38% of the entire cohort at follow‐up, and more commonly among high‐risk patients (41%). Among those on a NOAC at baseline, 22.4% switched to antiplatelet therapy alone after one year.Independent predictors of stroke/transient ischemic attack/peripheral embolism and/or mortality were age, heart failure, chronic kidney disease, prior ischemic stroke, dementia, and chronic obstructive pulmonary disease.The ChiOTEAF registry provides contemporary data on AF management, including stroke prevention. The poor adherence of NOACs and common use of antiplatelet in these high‐risk elderly population calls for multiple comorbidities management. General outcomes of the ChiOTEAF registry. AF, atrial fibrillation. AFatrial fibrillationAFFIRMThe Atrial Fibrillation Follow‐up Investigation of Rhythm ManagementCHA2DS2‐VAScCongestive heart failure or left ventricular dysfunction Hypertension, Age ≥75 (doubled), Diabetes, Stroke (doubled)‐Vascular disease, Age 65‐74, Sex categoryCIconfidence intervalECGelectrocardiographyEORP‐AFEURObservational Research Programme Atrial FibrillationESCEuropean Society of CardiologyHAS‐BLEDhypertension, abnormal renal/ liver function, stroke, bleeding history or predisposition, labile international normalized ratio, elderly, drugs/ alcohol concomitantlyHRhazard ratioIQRinter‐quartile rangemAFAmobile AF ApplicationNOACnon‐vitamin K antagonistOACoral anticoagulantRCTrandomized control trialSDstandard deviationTIAtransient ischemic attackVKAvitamin K antagonist: ChiOTEAF: Optimal Thromboprophylaxis in Elderly Chinese Patients with Atrial Fibrillation1Atrial fibrillation (AF) incidence is increasing over the past decadeRecent data demonstrate the beneficial effect of oral anticoagulants (OACs) for stroke prevention in elderly patients with AF.The introduction of the NOACs has led to a major change in the landscape of stroke prevention in AF, but limited contemporary nationwide data are evident from China. Thus, the prospective, nationwide Optimal Thromboprophylaxis in Elderly Chinese Patients with Atrial Fibrillation (ChiOTEAF) registry aimed to explore contemporary regional management strategies, including antithrombotic therapy among the high‐risk AF population of the elderly Chinese patients, in the new era of the NOACs. In this analysis, we present the 1‐year follow‐up data from the ChiOTEAF registry, focusing on the use of antithrombotic therapy, rate vs. rhythm control strategies, and determinants of mortality and stroke.2The protocol of the ChiOTEAF registry has previously been published.The registry was conducted between October 2014 and December 2018. Briefly, the registry population comprises consecutive in‐ and outpatients presenting with AF to cardiologists (mainly), neurologist, and surgeons, enrolled in 44 sites from 20 Chinese provinces. The main inclusions criteria were age ≥65 years  and the qualifying AF event in the 12 months prior to enrolment (recorded by a 12‐lead ECG or 24 hours ECG Holter).Data were collected at the moment of enrolment and during the follow‐up visits (including patient visit and/or chart review and/or telephone follow‐up) by any investigator and reported into an electronic case report form. Follow‐up was performed by the local investigators, initially at 6 and 12 months in the first year and annually for the next 2 years. Endpoints of interest were mortality, thromboembolism, major bleedings, cardiovascular comorbidities, and hospital re‐admissions. For this analysis, we focused on 1‐year outcomes.2DS2‐VASc score.2DS2‐VASc 0 or females with a CHA2DS2‐VASc 1; “moderate risk” was defined as male patients with a CHA2DS2‐VASc score 1 or females with a CHA2DS2‐VASc 2; and “high risk” was defined as CHA2DS2‐VASc score ≥2. Bleeding risk was assessed based on the HAS‐BLED bleeding score.The ChiOTEAF registry has common definitions and protocol for the EURObservational Research Programme Atrial Fibrillation (EORP‐AF) General Registry.2.12 test or Fisher's exact test (if required) was used for among‐group comparisons.Univariate analysis was applied to continuous and categorical variables. Continuous variables were reported as mean+SD and/or as median and inter‐quartile range (IQR). Among‐group comparisons were made using a non‐parametric test . Categorical variables were reported as percentages, and the χP‐value of <.05 was considered statistically significant.All the statistically significant variables at univariate analysis and variables considered of relevant clinical interests were included in the multivariable model to distinguish the independent predictors of all‐cause death and/or stroke/transient ischemic attack (TIA)/peripheral embolism during the 1‐year follow‐up period. A Cox proportional hazard model was performed by adjusting for the following covariates: sex, hypertension, coronary artery disease, liver dysfunction, and prior major bleeding. All Cox regression analyses were reported as hazard ratio (HR) and 95% confidence interval [CI]. A two‐sided 3Available data on patient demography and baseline characteristics in relation to clinical AF subtype are summarized in Table 2DS2‐VASc score ≥2)  in relation to clinical subtype was 75.0 (65.0‐82.0) years, with a vast majority of patients at high risk of stroke (CHA2) Table . Analysi2) Table . Patient3.1Of those patients with reported data, 1098 (20.4%) were symptomatic at 1‐year follow‐up . OACs were used in 27.9%–29.0% of low‐risk patients, in 34.2%–35.8% of moderate‐risk, and in 39.2%–40.1% of high‐risk patients, while the NOACs were used in 14.9%–16.2% of low, 18.1%–19.5% of moderate and 20.9%–21.8% of high‐risk patients, respectively. Antiplatelet therapy was used in 41.2%–41.3% of patients at high risk of stroke.Table 3.3For the analysis of rate and rhythm control strategies, 6022 patients with available data by 1‐year follow‐up were included. Drugs used for rhythm and rate control therapy at follow‐up are summarized in Table Among patients managed with rate control at baseline, only 4.2% continued a rate control strategy, while rhythm control was considered in 43.1% Figure . Of thosTable 3.4After one year, 6.8% 435/6420) of the patients enrolled in the study died between the enrolment and the 1‐year follow‐up visit , 67 for acute coronary syndrome, and 108 for heart failure. In this high‐risk cohort, 102 thromboembolism complications  and 102 major bleedings  occurred. Patients with long‐standing persistent and permanent AF were at the highest risk of both ischemic stroke/TIA and bleeding.3.5P <.001), heart failure , chronic kidney disease , prior ischemic stroke , dementia  and chronic obstructive pulmonary disease .A Cox proportional hazard model was compiled to establish clinical factors associated with the composite outcome of stroke/TIA/peripheral embolism and/or death Table . For str4In this 1‐year follow‐up analysis of high‐risk elderly patients with AF, our principal findings are as follows: (a) patients are frequently asymptomatic, but in a fifth of AF patients, symptoms are present ; (b) the use of OAC remained low, less than 40% of patients, with similar proportions of VKA and NOACs; (c) rhythm control was infrequent, with any rhythm control intervention being performed in 9% of patients (and catheter ablation in only 5.5%); (d) 1‐year mortality was high  and independent predictors of mortality were age, heart failure, chronic kidney disease, chronic obstructive pulmonary disease and dementia; and (e) hospital re‐admissions were common, especially for arrhythmic causes.The ChiOTEAF registry is the first contemporary nationwide prospective survey focused on management practices among Chinese cardiologists, with associated follow‐up data, since the introduction of NOACs. It was designed to have aligned definitions of clinical outcomes and a common protocol with the EORP‐AF registry to compare AF management between European and Chinese populations.While patients are frequently asymptomatic, symptoms at 1‐year follow‐up are common among persistent and long‐term persistent AF patients , particularly palpitations and shortness of breath. Of note, over 50% of patients were treated with beta‐blockers; and rhythm control drugs were used in 12.6% of patients . Consistent with symptom‐based management, any rhythm control intervention was limited and performed only in 9% of the overall cohort (most commonly in persistent and long‐term persistent AF).Given that many elderly AF patients are asymptomatic, opportunistic screening is recommended for early AF detection in those aged ≥65 years.2DS2‐VASc scores ≥2 were anticoagulated.The ChiOTEAF registry showed that the overall use of OACs was relatively low (38% of patients at follow‐up), with similar uptake of a VKA (18%) and NOACs (20%–21%) among Chinese elderly. In comparison, data from European registries shows over 80% of AF patients being anticoagulated, and NOACs account for 40% of OACs.Despite guideline recommendations, we found that antiplatelet therapy (commonly aspirin) was still used in 23.7% of low‐risk and 41% of high‐risk patients. When a NOAC was discontinued, over a fifth of patients was started on antiplatelet therapy. However, the reasons for this antiplatelet “overuse” in Chinese patients are not evidence‐based; indeed, OACs were found to have superior efficacy with similar safety than aspirin among the elderly with AF.Given that stroke prevention is central to AF management, better education and awareness are needed to improve outcomes in this AF population.Furthermore, our data show high morbidity and mortality rates. Indeed, 1‐year mortality was 6.8% in all cohort, particularly from non‐cardiovascular causes. During the follow‐up, 89 thromboembolism complications occurred. Independent predictors for stroke/TIA/peripheral embolism and/or mortality included age, heart failure, chronic kidney disease, prior ischemic stroke, dementia, and chronic obstructive pulmonary disease. Similarly, the European data of AF patients showed that overall mortality rates remained high (5%) during the 2‐year follow‐up, but mostly due to cardiovascular causes (61.8%).Consistent with other registries, hospital re‐admissions were common in our cohort, especially for cardiac causes . The increasing number of AF‐related hospitalizations is acknowledged as a major healthcare costs.4.1The primary limitation of the study is its observational nature, and given its modest size, it was not powered to detect differences in some endpoints. Patients were enrolled in 44 centers, which implies a potential variability in the therapeutic strategies for AF. Moreover, the enrolment period was relatively long, which may affect the generalizability of the results. There was a moderate proportion of patients lost to follow‐up (9.3%) consistent with large European registries.5The Optimal Thromboprophylaxis in Elderly Chinese Patients with Atrial Fibrillation registry provides contemporary data on AF management, including stroke prevention. The rate of OAC use was <40%, and antiplatelet therapy is still commonly prescribed among high‐risk patients. Given that Chinese patients with AF are increasingly elderly and overburdened with multimorbidity, our large cohort data may help establish best practices to reduce morbidity and mortality.5.1Stroke prevention is central to AF management; better education and awareness are needed to improve outcomes in high‐risk AF populations. Given the substantial clinical impact and healthcare burden associated with AF, the collection of prospective data from local AF cohorts may help establish best practice to reduce AF‐related morbidity and mortality.GYHL: Consultant and speaker for BMS/Pfizer, Boehringer Ingelheim, and Daiichi‐Sankyo. No fees are received personally. Other authors: None declared.Supplementary MaterialClick here for additional data file.


SLC2A1 mutations on chromosome 1p34.2 that impairs glucose transport across the blood–brain barrier resulting in hypoglycorrhachia and decreased fuel for brain metabolism. Neuroglycopenia causes a drug‐resistant metabolic epilepsy due to energy deficiency. Standard treatment for Glut1 deficiency syndrome is the ketogenic diet that decreases the demand for brain glucose by supplying ketones as alternative fuel. Treatment options are limited if patients fail the ketogenic diet. We present a case of successful diazoxide use with continuous glucose monitoring in a patient with Glut1 deficiency syndrome who did not respond to the ketogenic diet.Glut1 deficiency syndrome is caused by  SLC2A1 on chromosome 1p34.2, which impairs transmembrane glucose transport across the blood–brain barrier resulting in hypoglycorrhachia and decreased glucose availability for brain metabolism.Glut1 deficiency syndrome (Glut1DS) is caused by mutations in Continuous glucose monitoring (CGM) was first approved by the FDA in 1999. However, it was not widely used in pediatrics until 2015 after significant technological advances were made. CGM is commonly used by individuals with diabetes mellitus and studies have shown reductions in hemoglobin A1c and hypoglycemia with consistent use.A 14‐year‐old girl with Glut1DS (c.398_399delGCinsTT:p. Lys133Phe) presented with absence seizures at age 2 years. At initial presentation, she had a developmental and epileptic encephalopathy dominated by continual absence seizures and mild cognitive impairment. She also had dysarthria, hyperreflexia, and wobbly legs as a measure of paroxysmal exertional dyskinesia, often precipitated by athletic activities. Laboratory tests revealed a CSF glucose of 36 mg/dL when blood glucose was 93 mg/dL (CSF/blood glucose ratio 0.39). A classic KD was initiated with a 4:1 ratio and transitioned to a modified Atkins diet with a carbohydrate allowance as high as 50 g/day. Nevertheless, she could not tolerate the KD due to severe nausea, vomiting, abdominal pain, and hypertriglyceridemia. Trials of levetiracetam, zonisamide, valproic acid, carbamazepine, lacosamide, alpha lipoic acid, and triheptanoin also failed to control seizures. At age 14 years, diazoxide was started with target blood glucoses of 120–180 mg/dL. She was on no other anti‐seizure medication at that point. Serial EEG seizure counts documented a seizure‐free state after 6 months of diazoxide  and renal insufficiency (peak creatinine 1.11 mg/dL) necessitating transient discontinuation of hydrochlorothiazide. Hydrochlorothiazide was restarted later at a low dose with amiloride (potassium‐sparing diuretic). Daily sodium intake was decreased to <2400 mg/day. Serum electrolytes and renal function were followed closely. Acetazolamide was discontinued after normalization of serum sodium and renal function.At month 8 of diazoxide, the patient developed headache, malaise, and a stiff neck. She did not have papilledema. Lumbar puncture showed opening pressure of 22 cm HTo achieve hyperglycemia in the range of 140–180 mg/dL, cornstarch was supplemented at 7 tbsp/day. Simple carbohydrates were given, when necessary, to raise blood glucose levels. Weight gain (13.7 kg) resulted from this regimen, complicating the fluid retention. Nutrition assessment revealed an average intake of 3193 kcal/day composed of carbohydrates (426 g/day), protein (121 g/day), and fat (108 g/day), which exceeded daily requirements of ˜2000 kcal/day. Her nutrition plan was restructured. The family was counseled regarding daily dietary intake to meet the patient’s energy, sodium, and carbohydrate goals. Daily cornstarch dose was decreased and eventually discontinued. To prevent relative “hypoglycemia”, the patient and family were educated about pairing complex carbohydrates with protein at meals and snacks to help sustain blood glucose levels. If relative “hypoglycemia” did occur, defined as blood glucose <90 mg/dL for more than 3–4 h, the patient and family were instructed to correct with 15 g of simple carbohydrates. Three weeks later, average daily caloric intake had decreased over 400 kcal/day. The patient’s nutritional status continues to be monitored closely as her neurological state continues to improve.This is the first report demonstrating CGM as a tool facilitating safe initiation and real‐time titration of diazoxide in the management of Glut1DS patients who have failed the KD. Diazoxide addresses neuroglycopenia physiologically by raising blood glucose levels. The reasons and benefits for this are twofold. First, the Glut1 transporter facilitates glucose diffusion across tissue barriers including the blood–brain barrier. Reported Km values for the transporter range from 4 to 8 mmol/L (72–144 mg/dL).SLC2A1 adding “insult to injury”. This molecular sequence has been documented in poorly controlled diabetes mellitus.We observed previously that the higher blood glucose levels we achieved with chronic diazoxide administration only correlated with transient benefit (unpublished data). Ultimately, the Glut1DS patients lost the initial clinical benefits and regressed to baseline levels of neurological performance. We speculated chronic hyperglycemia (300–500 mg/dL) caused downregulation of A multidisciplinary team approach has been central to the success of this patient’s care. The specialized team includes neurologists, endocrinologists, genetic counselor, nephrologist, and nutritionist. Diazoxide initiation was coordinated by neurologists and endocrinologists well versed in Glut1DS and diazoxide, respectively. Side effects caused by diazoxide were discussed among the endocrinologists managing the diazoxide, the neurologists following the developmental/epileptic encephalopathy and headache symptoms, a nephrologist managing the diuretics, and the nutritionist managing the dietary therapy. Team meetings were conducted regularly to assess treatment efficacy and re‐evaluate treatment goals. Fortunately, the parents and patient were able to assume increasing responsibility for the CGM and dietary adjustments. The outcome has been beneficial and resulted in optimal control of neurological symptoms and unprecedented quality of life.The authors declare no conflict of interest. Darryl C De Vivo has served as advisor/consultant for AveXis, Biogen, Cytokinetics, Ionis Pharmaceuticals, Inc., Metafora, Roche, Sanofi, Sarepta, and SMA Foundation; received grants from Hope for Children Research Foundation, National Institutes of Health, SMA Foundation, Cure SMA, Glut1 Deficiency Foundation and US Department of Defense; clinical trial funding from Biogen, Mallinckrodt, PTC, Sarepta, Scholar Rock, and Ultragenyx; and as a member of the DSMB for aspa Therapeutics.


Choroidal neovascularization (CNV) is a key characteristic of wet age‐related macular degeneration (AMD) that can lead to severe vision loss in the elderly. Anti‐VEGF therapy is currently the premier strategy for wet AMD, but it has limited efficacy. Previous studies have shown that chitinase‐3‐like‐1 (YKL‐40) can promote microangiogenesis and inflammation, but its effect on CNV formation has not yet been studied. Here, we investigated the potential role of YKL‐40 in wet AMD and the underlying mechanism(s). We report that the serum expression of YKL‐40 in wet AMD patients was significantly higher than that in control patients and was positively correlated with VEGF expression, indicating that YKL‐40 may participate in the development of wet AMD. In addition, YKL‐40 and VEGF expression levels were observed to be increased and the ERK1/2 pathway activated in the neuroretinal (NR) and RPE/choroid tissues of mice with laser‐induced CNV. The YKL‐40 and phosphorylated protein levels of the ERK1/2 pathway were decreased after intravitreal injection with an anti‐YKL‐40 antibody, suggesting that anti‐YKL‐40 could inhibit the activation of the ERK1/2 pathway. These results indicate that YKL‐40 may serve as a novel target for the diagnosis and treatment of wet AMD. We demonstrate that serum expression of YKL‐40 in wet AMD patients was significantly higher than that in control patients. YKL‐40 and phosphorylated protein levels of the ERK1/2 pathway in mice with laser induction were decreased after intravitreal injection with an anti‐YKL‐40 antibody, indicating that YKL‐40 may serve as a novel target for the diagnosis and treatment of wet AMD. AMDage‐related macular degenerationCNVchoroidal neovascularizationERK1/2extracellular regulated protein kinasesRPEretinal pigment epitheliumVEGFvascular endothelial growth factorYKL‐40Chitinase‐3‐like‐1Age‐related macular degeneration (AMD) is the leading cause of irreversible vision impairment in aging populations , 2, 3. CThe chitinase‐3‐like‐1 (CHI3L1) protein, also called YKL‐40, a mammalian member of the 18‐glycosyl hydrolase family , 7, can In this study, we analyzed the correlation between YKL‐40 and VEGF, which was previously reported to be closely related to wet AMD. As neovascularization is the hallmark of wet AMD, we aimed to verify whether the serum concentrations of YKL‐40 in patients with wet AMD were elevated and the diagnostic accuracy of serum YKL‐40 to predict wet AMD. Additionally, we investigated the possible mechanism of YKL‐40 in mice with laser‐induced CNV formation.n = 20) and AMD (n = 45) groups. Clinical parameters of all the subjects such as gender, age, smoking history, low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), total cholesterol (TC), and high sensitivity C‐reactive protein (hs‐CRP) were recorded. All participants signed written informed consent, and the study protocol was approved by the Research Ethics Committee of the First Affiliated Hospital of Chongqing Medical University. All procedures were conducted in accordance with the Helsinki declaration.Subjects from the First Affiliated Hospital of Chongqing Medical University were enrolled. AMD was diagnosed by detailed medical history, slit‐lamp microscopy, fundus photography, optical coherence tomography (OCT), and fluorescein fundus angiography (FFA). The inclusion criteria for male and female participants of both genders were as follows: ≥ 50 years old, nondiabetic, nonhypertensive, and no other ocular complication(s) except AMD. Subjects suffering from arthritis, cancer, and/or use of anti‐inflammatory or anti‐VEGF therapeutics were excluded from the study. Control subjects were required to meet the same inclusion criteria and underwent detailed examinations for confirmation of healthy ocular conditions. Consequently, 65 subjects who fulfilled the inclusion criteria were divided into the control  were gathered and centrifuged at 3000 × The serum concentrations of YKL‐40 and VEGF were quantified by corresponding ELISA kits  according to the protocols. The absorbance at the wavelength of 450 nm was determined with the use of microplate reader  in the dark.A total of 60 male C57BL/6J mice aged 6 to 8 weeks were obtained from the Laboratory Animal Center of Chongqing Medical University and raised in the center under a 12‐/12‐h light‐dark cycle. All procedures involving animals were approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.After undergoing anesthesia with pentobarbital sodium (1% w/v) and pupil dilation with tropicamide, the mice were subjected to laser‐induced photocoagulation  by a krypton‐argon laser . Five laser spots were made in each eye to induce CNV, with white bubbles indicating Bruch’s membrane rupture at each spot. After laser injury, intravitreal injection was performed immediately through a 35‐gauge needle attached to a 10 μL nanofilm syringe. Then, 2 μL of neutralizing monoclonal anti‐YKL‐40 antibody  or PBS solution was gently inserted into the vitreous cavity.After mice were euthanized, the eyeballs were dissected and fixed with 4% formaldehyde solution for 24 h, followed by dehydration with a sucrose gradient before they were embedded in paraffin wax. Sagittal sections at the CNV lesion were selected, and 6.0‐μm‐thick slices were prepared for staining with hematoxylin‐eosin (HE) before they were photographed under a light microscope.YKL‐40 and VEGF mRNA expression at 7 and 14 days after laser induction was examined by real‐time PCR. After the mice were sacrificed, the eyeballs were dissected using an anatomical microscope, and the peripheral fascia of the mice was cut off. The eyeballs were cut along the limbus of the cornea, and retinal tissue could be observed behind the crystal and vitreous separation. The neuroretina (NR) and retinal pigment epithelium (RPE)/choroid were dissected from the eyeballs of the mice . These tg for 15 min, the supernatants were gathered and diluted in loading buffer . The proteins were separated by SDS/PAGE and electrophoretically transferred to PVDF membranes . After being blocked, primary antibodies against YKL‐40 , VEGF , ERK1/2 , p‐ERK1/2 , and GAPDH  were used to probe protein. The membranes were incubated with secondary antibodies and analyzed by enhanced chemiluminescence .On days 7 and 14 after laser induction, NR and RPE/choroid tissues were dissected from mice, homogenized, and lysed in RIPA buffer mixed with 1% protease inhibitor . After centrifugation of the lysates at 15 984 spss Statistics 20.0 software . Student’s t test was used to compare serum levels of YKL‐40 and VEGF. The Pearson correlation coefficient was computed for YKL‐40 and VEGF expression levels. The predictive capabilities  of YKL‐40 and VEGF were investigated by construction of the area under the receiver operating characteristic (ROC) curve (AUC). One‐way ANOVA with Bonferroni correction was used to analyze comparisons of three groups. P values less than 0.05 were considered statistically significant.Data are presented as the means ± SEM and analyzed using The study population comprised 45 patients  and 20 healthy controls . The AMD patients were slightly older than the control patients, with average ages of 71.36 ± 10.19 years and 69.65 ± 9.06 years, respectively. No significant difference was observed in age nor sex between the two groups. Moreover, no significant differences were found in LDL‐C, HDL‐C, TC, hs‐CRP, and smoking between the AMD group and the control group (Table −1) was markedly higher than that in the healthy subjects   was obviously higher than that in the healthy participants   Fig. . The sam18) Fig. .r = 0.866, P < 0.001)   Fig. . Additio01) Fig. .−1, with 66.70% sensitivity and 75.00% specificity (P = 0.001). Similarly, VEGF had an area under the ROC curve of 0.740 (95% CI 0.614–0.866) and a cutoff value of 393.66 pg·mL−1, with 55.60% sensitivity and 80.00% specificity (P = 0.002)  Fig. .We used laser photocoagulation to establish a CNV mouse model and then determined whether YKL‐40 and VEGF expression was elevated in the NR and RPE/choroid tissues. Histological analysis of retina stained with HE confirmed CNV formation on days 7 and 14 after the ruptured Bruch′s membrane induced by laser, with a disordered retinal structure, abnormal cell proliferation, and loss of the outer nuclear layer at the laser‐damaged area Fig. .P < 0.05)    Fig. . Consist01) Fig. . Further01) Fig. .P < 0.05)   Fig. . To furt05) Fig. .P < 0.05)  Fig. .YKL‐40 is classified into the chitinase‐like protein (CLP) family, the members of which do not provide enzymatic activity to degrade chitin directly. The association with the biological activity of YKL‐40 and the regulation of cell proliferation, activation, migration, and adhesion was previously investigated . AlthougP = 0.001) were found when a cutoff level of 835.95 pg·mL−1 was used, which suggested that YKL‐40 could be considered a predictor of wet AMD. Currently, several studies have demonstrated increased serum YKL‐40 in patients with inflammatory diseases and different types of solid tumors, indicating the important role of YKL‐40 in inflammation and tumor development. Moreover, YKL‐40 is deemed a new biomarker used to estimate disease activity in inflammation. Studies have reported elevated serum YKL‐40 and a positive correlation of serum YKL‐40 and disease activity in rheumatoid arthritis [Herein, serum YKL‐40 levels were significantly elevated in patients with wet AMD and positively correlated with VEGF expression, implying the involvement of YKL‐40 in the pathological process of wet AMD. Additionally, moderate sensitivity and specificity values  and the U87 human glioma cell line showed that YKL‐40 and VEGF had a synergistic effect on vascular structure formation [In vivo experiments involving mouse models of human breast cancer, colon cancer and glioma as well as material from patients with glioma highlighted the role of YKL‐40 in promoting angiogenesis [Furthermore, in line with the human serum results, both the mRNA and protein expression levels of YKL‐40 and VEGF were significantly increased in mice after laser induction compared to control mice. These results are consistent with a published report describing that YKL‐40 expression was upregulated in both pathological human and experimental CNV samples , which mormation , 27. In ormation , 27. In ogenesis . In addiogenesis .et al. [in vitro studies have reported that YKL‐40 participates in angiogenesis by activating the MAPK/ERK pathway [MAPK, a common intracellular pathway, plays a crucial role in carcinogenesis, promoting cancer cell proliferation and migration, inhibiting apoptosis and stimulating angiogenesis , 31, 32.et al.  showed t pathway , 27, 34. pathway . In glioIn this research, we utilized a single‐center clinical study with a small sample size and inevitable bias. In future studies, we plan to expand the sample size and consider multicenter cooperation, extend patient follow‐up time, compare serum YKL‐40 levels before and after anti‐VEGF treatment, and provide more reliable evidence for YKL‐40 in the diagnosis and treatment of wet AMD.In conclusion, compared with healthy controls, patients with wet AMD exhibited a higher serum YKL‐40 expression and a positive correlation between YKL‐40 and VEGF expression, which indicates the possible involvement of YKL‐40 in wet AMD. The YKL‐40 and phosphorylated protein levels of the ERK1/2 pathway were decreased after intravitreal injection of the anti‐YKL‐40 antibody, suggesting that anti‐YKL‐40 could inhibit the activation of the ERK1/2 pathway. These results are a prelude to further studying the functional and in‐depth mechanisms of YKL‐40 in wet AMD.The authors declare no conflict of interest.HP conceived and designed the study. YB, YL, and SJ. carried out the experiments. YB and YL analyzed the data and edited the figure. YB wrote the article. HP revised the article. All authors read and agreed on the final version of the article.