The two hemispheres are thought to mutually hamper neuroplastic reorganization of the other hemisphere. The results of preceding degeneration and neuroplastic reorganization of white matter may be reflected by Diffusion Tensor Imaging-derived diffusivity parameters such as fractional anisotropy (FA). In this study, we applied Diffusion Tensor Imaging (DTI) to contrast the white matter status of the contralesional hemisphere of young lesioned brains with and without contralateral influences by comparing patients after hemispherotomy to those who had not undergone neurosurgery. DTI was applied to 43 healthy controls  and two groups of in total 51 epilepsy patients with comparable juvenile brain lesions  either after hemispherotomy (30 of 51 patients) or without neurosurgery (21 of 51 patients), respectively. FA values were compared between these groups using the unbiased tract-based spatial statistics approach. A voxel-wise ANCOVA controlling for age at scan yielded significant group differences in FA. A post hoc t-test between hemispherotomy patients and healthy controls revealed widespread supra-threshold voxels in the contralesional hemisphere of hemispherotomy patients indicating comparatively higher FA values . The non-surgery group, in contrast, showed extensive supra-threshold voxels indicating lower FA values in the contralesional hemisphere as compared to healthy controls . Whereas lower FA values are suggestive of pronounced contralesional degeneration in the non-surgery group, higher FA values in the hemispherotomy group may be interpreted as a result of preceding plastic remodeling. We conclude that, whether juvenile brain lesions are associated with contralesional degeneration or reorganization partly depends on the ipsilesional hemisphere. Contralesional reorganization as observed in hemispherotomy patients was most likely enabled by the complete neurosurgical deafferentation of the ipsilesional hemisphere and, thereby, the disinhibition of the neuroplastic potential of the contralesional hemisphere. The main argument of this study is that hemispherotomy may be seen as a major plastic stimulus and as a prerequisite for contralesional neuroplastic remodeling in patients with juvenile brain lesions.Cerebral lesions may cause degeneration and neuroplastic reorganization in both the ipsi- and the contralesional hemisphere, presumably creating an imbalance of primarily inhibitory interhemispheric influences produced  For the hemispherotomy group, inclusion criteria were the following: (1) treatment at the Department of Epileptology at the University of Bonn Medical Center between 1992 and 2014; (2) transsylvian functional hemispherotomy , a 3D T2-weighted sequence , and a single-shot diffusion-weighted sequence  with 60 diffusion-encoding directions and a b-value of 1,000 s/mm2 as well as six baseline volumes with a b-value of 0 s/mm2. Sequences acquired after the scanner update were run with a 32-channel head-coil equally including a MPRAGE sequence , T2-weighted sequence , and a single-shot diffusion-weighted sequence  with 60 diffusion-encoding directions and a b-value of 1,000 s/mm2 as well as six baseline volumes with a b-value of 0 s/mm2.Magnetic resonance imaging data was acquired using a 3T Magnetom Trio . T1, T2 and DTI data were collected for all patients and healthy controls. As part of a scanner update, a new head coil was implemented in October 2014 leading to minimal changes in sequence parameters: all scans acquired before the scanner update were run with an eight-channel-coil and a scanning routine containing a 3D MPRAGE sequence  to the respective T1-weighted volume.Preprocessing was run using FMRIB’s Software Library 5.0  t-tests between all groups. Group effects in all post hoc t-tests were equally adjusted for scanner update and age at scan. The anatomical location of resulting clusters was identified with the JHU ICBM-DTI-81 white-matter labels atlas  and late  onset of pathology. Scannerupdate and age at surgery were distributed equally in both subgroups and, thus, had not to be adjusted for this model. Lastly, we performed a voxel-wise regression analysis to link the volume of the lesion masks to voxel-wise FA values. All analyses were run using Monte Carlo permutation tests with 10,000 permutations. As we performed voxel-wise testing, as many statistical tests as there are voxels in the white matter skeleton were carried out. To keep the conventional level of significance at 5% and to control for the number of false positives, results were corrected for family-wise error if not indicated otherwise. Threshold-free cluster enhancement was performed to avoid an arbitrary initial cluster-forming threshold for final voxel-wise inference : clusters were widely distributed in the contralesional hemisphere, whereas they only covered frontal areas and cerebral peduncle in the lesional hemisphere. Adjusting for scanner update and age at scan, FA values were subjected to two post hoc t-tests comparing the hemispherotomy group and the healthy control group (A) and the non-surgery group and the healthy control group (B). A: widespread clusters indicating higher FA values in hemispherotomized patients as compared to healthy controls were found primarily in the internal capsule, the external capsule, superior and posterior corona radiata, posterior thalamic radiations, and brainstem regions in the contralesional hemisphere  was used to determine differences between subgroups of hemispherotomy patients with early and late-onset of pathology. Patients with late-onset of pathology (>0.5 years old at onset) exhibited higher FA values in residual ipsilesional brainstem regions , even when omitting a correction for multiple comparisons (p > 0.10).A voxel-wise regression analysis aiming to explain FA values by lesion size did not yield statistically significant results  and a higher antiepileptic drug load, both thought to lower FA values Jehi, . In the via transcallosal pathways. The contrasting result of higher FA values in the contralesional hemisphere in a group of patients with similar pathologies who had undergone hemispherotomy as compared to healthy controls may be interpreted as a correlate of preceding neuroplastic reorganization of the contralesional hemisphere. This reorganization has most likely been enabled by the lacking inhibitory influence of the ipsilesional hemisphere resulting in the disinhibition of the contralesional one. Our study probes models of interhemispheric balance by applying it to two patient groups with early brain lesions after and without hemispherotomy. The pattern of degenerative and plastic structural alterations is most likely a result of interhemispheric inhibition and, therefore, substantiates these models. We conclude, that the procedure of hemispherotomy should be seen as a major plastic stimulus for reorganization in the contralesional hemisphere. Complete disconnection of inhibitory influences from the ipsilesional hemisphere releases the full rehabilitative potential of the contralesional hemisphere for functional recovery in patients with extended unilateral lesions.Lower FA values unexpectedly found in the white matter infrastructure of the contralesional hemisphere of epilepsy patients with juvenile brain lesion when compared to healthy controls most likely reflect secondary degeneration and may constitute the structural correlate of diaschisis. This degeneration may be aggravated and neuroplastic reorganization in this hemisphere may be hindered by the inhibitory influence of the ipsilesional hemisphere produced The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available as they contain information that could compromise the privacy of research participants.The studies involving human participants were reviewed and approved by Ethics Committee of the Medical Faculty of the University of Bonn. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JG, CP, EH, BW, HV, RS, CE, and TR contributed to the conception and design of the study. JG, CP, and LE organized the database. JG, LE, BD, and TR performed the statistical analysis. JG wrote the first draft of the manuscript. CH and TR wrote sections of the manuscript. EH, BW, HV, RS, CE, and TR gave administrative, technical or material support. EH, BW, HV, RS, CE, and TR supervised the study. All authors contributed to manuscript revision, read and approved the submitted version. JG and CP have contributed equally to this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pulmonary infection is a leading cause of hospitalization in world. Lung damage due to infection and host mediated pathology can have life threatening consequences. Factors that limit lung injury and/or promote epithelial barrier function and repair are highly desirable as immunomodulatory therapeutics. Over the last decade, interleukin−22 has been shown to have pulmonary epithelial protective functions at the mucosal immune interface with bacterial and viral pathogens. This article summarizes recent findings in this area and provides perspective regarding the role of IL-22 in mucosal host defense. Protective immune mechanisms of lung injury during infection are an important area of research that could provide new immunomodulatory therapeutic options. Maintenance of lung epithelial structural integrity during bacterial or viral pneumonia is of critical importance to limit lung edema and pathogen dissemination. The function of the cytokine interleukin (IL)−22 in the lung has only recently been elucidated and much is still to be discovered. Work over the last decade has shown that IL-22 likely plays a role in epithelial biology during infectious diseases –4. IL-22Klebsiella pneumoniae model of pneumonia  and (+) bacteria are common causes of both community and hospital acquired pneumonia. The first description of a host defense role for IL-22 in the lung was demonstrated using the neumonia . In thatPseudomonas aeruginosa model of lung infection, antibody blockade of IL-22 was shown to increase infection, lung damage, and neutrophil accumulation  host defense has also been shown. Deletion or antibody neutralization of IL-22 resulted in increased lung bacterial burden and dissemination compared with controls during nfection . These dnfection , 13. IL-K. pneumoniae model, IFNλR–/– mice exhibited better bacterial control and improved epithelial barrier function  has recently emerged. IFNλ is an antiviral cytokine family that has been shown to be protective during viral infections without pro-inflammatory effects, an important consideration in the lung. In the function . These Iell line . This innfection . Howevernfection . These din vitro and in vivo. In this study, IL-22 production was manipulated by deletion of iNKT cells and no effect on viral load was observed. These data suggest an epithelial protective role for IL-22 during viral infections.Influenza virus is the cause of world-wide, annual epidemics and can result in severe lung pathogenesis in people of all ages. Initial study into the role of IL-22 during influenza infection produced limited results. Antibody neutralization of IL-22 was not effective at reducing influenza mediated morbidity or mortality in mice, although a reductive effect on viral load was shown . Shortlyin vitro. Another study found similar effects on lung epithelial repair following influenza infection  on epithelial cells  or middle east respiratory syndrome virus (MERS). A recent report suggests that IL-22 may be systemically elevated in the acute phase of SARS-CoV2 infection and may play a role in cardiovascular injury . Severe in vitro. While data exist regarding the effects of IL-22 on epithelial cells in vitro, there remains much to be discovered. It will be important to assess the effects of IL-22 on epithelial cells in inflammatory settings, perhaps in combination with pathogen associated molecular patterns (PAMPs) or toxins. Evidence for direct impact of IL-22 on immune cells is less abundant and controversial. More work in this area is needed to determine if IL-22 affects lymphocyte function in the absence of signaling through lung stromal cells. Mechanisms by which IL-22 mediates pathogen clearance, in the case of bacterial infection, are mostly unknown. Several studies have shown that lung bacterial burden is altered when IL-22 is manipulated, indicating that IL-22's role is not solely focused on preventing dissemination from the lung. It is likely that IL-22, signaling through the epithelium, impacts host defense in several undiscovered ways. IL-22 has many described functions outside of the lung in the gastrointestinal tract, liver, and thymus. It is possible that IL-22 regulation of immunity in the lung is not restricted to direct effects in lung tissue. Focused use of floxed IL-22Ra1 mice should enable high resolution study of tissue and cellular compartments where IL-22 signaling is required during infection. Regardless, pre-clinical animal models suggest that IL-22 has significant therapeutic potential in the context of infectious diseases. Additional study is required to confirm the current reports in the literature and expand the field beyond the few pathogens mentioned herein. Undoubtedly, studies of IL-22 in SARS-CoV2 infection would be of great interest. Beyond pre-clinical animal models, translational studies are now needed to determine if IL-22 can limit lung damage and promote repair in humans. Less is known with regard to human IL-22 production and signaling in human infectious diseases. This area will be critical to evaluate in human pneumonia.The highlighted work and those of others have shown an emerging role for IL-22 in promoting epithelial integrity and repair following infectious pathogen challenge in the lung. Precise mechanisms of IL-22 action have been more elusive, as IL-22 often has limited biologic effects on uninjured cells The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.JA conceived and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Due to the great impact of bariatric surgery on the overweight epidemic, the number of post-bariatric body-contouring procedures is constantly increasing worldwide. The portable incisional negative pressure wound therapy (piNPWT) is a promising medical device for accelerating wounds closure and controlling post-operative complication, which have been shown promising results in post-bariatric population. We aimed to evaluate the role of piNPWT in optimizing wound healing and controlling post-operative complications after a post-bariatric brachioplasty.26 post-bariatric female patients who underwent a brachioplasty followed by either a piNPWT (14 cases) or a standard wound treatment (12 controls) were analyzed. The number of post-operative dressing changes, the rate of local post-operative complications , the time to dry as well as the scar quality and hospitalization length were evaluated.None of the patients prematurely stopped treatment with piNPWT due to intolerance. The piNPWT patient group showed a significant lower healing time as well as a significant reduction of the number of post-operative dressing changes and hospital stay. Despite the scarring process was excellent from the functional point of view in the long term, we noticed a higher rate of hyperchromic scarring at 90 days after surgery.The piNPWT is a cost-effective and user-friendly medical tool that increase and promote wound healing. We suggest the use of this device in post-bariatric patients who undergo a brachioplasty, especially if there is the need to minimize the number of post-operative dressing changes.www.springer.com/00266.This journal requires that authors assign a level of evidence to each article. For a full description of these evidence-based Medicine ratings, please refer to Table of Contents or the online Instructions to Authors  The epidemic of overweight and obesity represents a significant threat for patients’ health and a major challenge for the healthcare system . BariatrDrastical changes in the BMI modify the structure of soft tissues, leading progressively to excess skin in different body districts , 8–10. TAmong different body areas, the postero-medial region of the arms represents a particular region where adipo-cutaneous excess of MWL patient accumulate in the distal half due to the progressive loosening of the support of superficial, longitudinal and axillary fascial systems of the proximal half .In addition, surgical scars of brachioplasty are the most commonly exposed by every day clothing if compared to other procedures, representing the heavier surgical burden.Despite surgery is constantly improving, minor and major complications including seroma, hematoma, infection, lymphocele, numbness, peripheral nerve pain and wound dehiscence are still frequent. Post-operative complications rates are as high as 40% in many studies, especially in associated multiple procedures  , 7.Given the steep learning curve and the high risks, body-contouring procedures should be performed in dedicated centers with managed through defined protocols from experienced surgeons.From the surgical point of view, the deep investing fascia, which envelopes arm musculature, should not be violated during surgery, in order to avoid medial antebrachial cutaneous nerve injuries. An axillary Z-plasty or its sinusoidal variation, can be performed to the proposed skin excision in order to prevent severe contractures and to restore the axillary dome’s appearance. Tension free closure should be obtained. Moreover, intraoperative infiltration of diluted epinephrine and liquid warming have been shown to reduce seroma rate formation and intraoperative bleeding .The preoperative management is of paramount importance in preventing complications with metabolic and nutritional homeostasis achieved before surgery. A stable weight and nutritional evaluation should be granted . FinallyAt present, the piNPWT has been shown effective in improving wound healing in several surgical fields, reducing at the same time risks of complications even in other surgeries: inguinal dissection , mastectAltogether, by assuming that this dressing could act as effective, user-friendly and cost-effective, we compared the effect of piNPWT with a traditional dressing change in MWL brachioplasty patients.The study was conducted in accordance with the World Medical Association Declaration of Helsinki (June 1964) and subsequent amendments.We performed a case-control study with 26 post-bariatric female patients (middle age 45.33) who underwent a brachioplasty at the Division of Plastic Surgery, at our University-Hospital, from June 2018 to March 2020. Fourteen patients (middle age 49.33) were treated post operatively with bilateral piNPWT (piNPWT patients) Fig. Massive weight loss at least of 7 points BMI (post MWL surgery or diet reduction)Stable weight and metabolic/nutritional homeostasis for at least 6 months FemaleInclusion criteria:Unstable weight lossPrevious arm surgeryAllergy to glue and tapeNeurologic, psychiatric or vascular disorders of the upper extremityLymphedema of the armsUnrealistic patient expectationsRaynaud’s disease, connective tissue disorders and advanced rheumatoid arthritis , 23–25Exclusion criteria:Before surgery, a physical examination was performed, evaluating upper limb function, including ROM at shoulder/elbow/hand and grip strength. Authors assessed for excess fat and skin, overall skin quality and tone.A watertight layered and subcuticular tension free suture was obtained, and a drain per arm was placed in all patients. Both groups used compressive sleeve garments for 30 days after treatment . DressinDuring every single dressing, the presence of post-operative complications , the number of post-operative dressing change, the hospitalization length and the eventual impact of the associated surgeries were evaluated. Complications were calculated per arm, defined as either hematoma, wound dehiscence, skin necrosis or infection, and classified through Clavien-Dindo classification .The aspect of the scar aspect was evaluated at a mean time of 6 months after surgery with Vancouver Scar Scale  Figs 2,,2, 3.FigAll procedures have been done in agreement with the declaration of Helsinki.p value < 0.05).Sample size was calculated using Pearson’s chi-square test to detect meaningful differences  between the two groups. Anticipated effect for the outcome (−50%) was based on previous experience and literature on the use of piNPWT in abdominoplasty. Results are expressed as mean ± SD. An unpaired Student’s t Test  was used to determine significant differences between groups  and concomitant blistering of the left arm treated with wet to dry dressing (Clavien-Dindo Grade II). One patient of the piNPWT group developed blistering treated with wet to dry dressing (Clavien-Dindo Grade II). Two patients of the TDD group developed ecchymosis (Clavien-Dindo Grade I). Two hematomas were treated conservatively in the piNPWT and one in the TDD group (Clavien-Dindo Grade I). The patient with hematoma in the TDD group subsequently developed a seroma requiring multiple aspirations (Clavien-Dindo Grade II).No reported discomfort was associated with the use of piNPWT or dry dressing. Three patients of the piNPWT group reported increased itching that did not require dressing change or suspension of therapy.t-Test=4.24×10−9; p<0.05). Furthermore, a significant difference was calculated in the “Time to dry” 17.66 ± 4.79 days in the TDD group vs. 9.36 ± 2.15 days in the piNPWT group . Finally, a significant difference in the hospitalization length was recorded 5.33 ± 1.49 days in the TDD group vs. 3.07 ± 1.14 days in the piNPWT group . Seven patients of the piNPWT group developed a hyperchromic scar at 90 days Fig. The number of post-operative dressing changes was 4.91 ± 0.79 in the TDD group vs. 2 ± 0.77 in the piNPWT group   Figs .2 appeared to be negative influence scarring process. Furthermore, concomitant body-contouring procedures seem to increase post-operative complications [MWL patients are at higher risk for developing complications if compared to standard population. Obesity, smoking history and anemia are known risk factors that commonly impair body-contouring procedures outcomes . In addiications . Greaterications .The present study focused on brachioplasty as one of the main surgeries requested by post-bariatric female patients and more frequently burdened by complications related to wound healing and scar quality.Our outcomes are consistent with those reported for other field of surgery and post-bariatric abdominoplasty as well, confirming the role of piNPWT to manage high risk surgical wounds , 22, 31.The ability of the piNPWT to reduce the lateral tense of closed incision improved wound healing in our patients . The rolThe combination of body-contouring procedures is considered a risk factor for potential increasing of complication. Even if the combination of mastopexy and brachioplasty is a general favorable combination of procedures, the decision should be made evaluating patient’s expectations, BMI loss, comorbidities and smoking status .Among our groups, the higher number of patients undergoing a combination of mastopexy and brachioplasty in piNPWT sample (8 vs. 2) may have negatively influenced the post-operative course of the patients and the wound healing process. On the contrary, the portable incision NPWT may have lessen the risk. Although the scarring was from the functional point of view extremely satisfactory and comparable in both groups, according to our previous experience , the hypVL Young et al.  indicateThe reduced number of post-operative dressing changes and shorter extent of the hospital stay of piNPWT patients together with the reduction of local complications is known to impact favorably the cost management of this group of patients , 35.Nonetheless, the presence of two dressing devices constantly attached to patients’ arms could be annoying for patients. Even though, accordingly to the literature, nobody of piNPWT group complained about their dressing, patients should be accurately informed about the need to carry two devices hung to their arms for 7–12 days.In addition, in the middle of the COVID-19 era, the reduction of the hospital stay, number of outpatient visits and complications allow limiting the exposure of patients to the risk of Sars-Cov2 infection , 38.Our preliminary report, even if retrospectively, confirms the role of piNPWT in improving postoperative management of post-bariatric patient undergoing body-contouring procedures. Other comparative studies are needed to validate this approach in breast lifts and thigh lift. Further research has to be carried out before routine clinical adoption of this technique in particular to find the ideal group of patients that will benefit most .As people heal differently, a prospective comparison of the effect of the application piNPWT in one arm and traditional dressing in the other arm of a single patient could be an option to limit inter-patients variability.Furthermore, a more comprehensive analysis of the cost-effectiveness of the procedure should also be conducted.In conclusion, we demonstrate that piNPWT is an effective medical tool, able to limit minor local complications rates in post-bariatric patients undergoing a brachioplasty.The piNPWT represents a valid alternative to the traditional dry dressing. Given the transiently worrisome aesthetic result, informing the patient about the possible development of a hyperchromic scar is key. We suggest the use of this device in post-bariatric patients who undergo a brachioplasty, especially if there is the need to minimize the number of post-operative dressing changes or outpatients’ visits and when brachioplasty is combined with liposuction and/or mastopexy.


Worldwide COVID-19 epidemiology data indicate differences in disease incidence amongst sex and gender demographic groups. Specifically, male patients are at a higher death risk than female patients, and the older population is significantly more affected than young individuals. Whether this difference is a consequence of a pre-existing differential response to the virus, has not been studied in detail. We created DeCovid, an R shiny app that combines gene expression (GE) data of different human tissue from the Genotype-Tissue Expression (GTEx) project along with the COVID-19 Disease Map and COVID-19 related pathways gene collections to explore basal GE differences across healthy demographic groups. We used this app to study differential gene expression of COVID-19 associated genes in different age and sex groups. We identified that healthy women show higher expression-levels of interferon genes. Conversely, healthy men exhibit higher levels of proinflammatory cytokines. Additionally, young people present a stronger complement system and maintain a high level of matrix metalloproteases than older adults. Our data suggest the existence of different basal immunophenotypes amongst different demographic groups, which are relevant to COVID-19 progression and may contribute to explaining sex and age biases in disease severity. The DeCovid app is an effective and easy to use tool for exploring the GE levels relevant to COVID-19 across demographic groups and tissues.  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), is without a doubt the most severe health, economic and social threat of our time. COVID-19 has followed two major infection waves in spring and fall 2020 reaching one million deceased people by September 2020. Our understanding of the disease has evolved over time. While initially COVID-19 was considered to be a severe atypical pneumonia, we have now learned that the virus can infect many different tissues. Moreover, the disease affects blood coagulation and can cause an exacerbated immune response referred to as the “cytokine storm”. Although clinical management of COVID-19 has rapidly evolved over time, no curative treatment is available, and current recommendations support the use of steroids, oxygen and a prophylactic dose of heparin. While accelerated vaccine development efforts have resulted in tremendous advances in a relatively short time, the WHO acknowledges that a vaccine solution for the general population might not be ready before mid to late 2021. At the same time, very recent reinfection cases suggest that immunity may not always persist, and active disease surveillance of past cases may be required. As the second wave of the pandemic steadily progresses in Europe and the US, possibly with new variants of the virus that affect a wider range of the population, novel or improved treatment targets and options are likely to remain important for the management of this disease.One of the most intriguing aspects of COVID-19 is the varying grades of severity in which it affects different people. Although acknowledged risk factors include pre-existing conditions such as cardiovascular disease , diabetehttps://covid.pages.uni.lu/) describing genes related to the COVID-19 disease process, including cytokines and interferon, together with the pathways for the life cycle of SARS-CoV and MERS-CoV. The COVID-19 Disease Map and WikiPathways COVID-19 related pathways are valuable resources to investigate the molecular responses to SARS2-CoV-2 infection and to understand the biological pathways leading to severe manifestations of the disease. These databases also create an opportunity for interrogating existing molecular data on differences associated with COVID-19 risk factors in the general population.The COVID-19 Disease Map initiative was launched in May 2020 with the aim of creating a collection of genes and pathways relevant to SARS-CoV-2 infection derived from the growing COVID-19 literature. This resource contains over 800 COVID-19 genes that have been associated to the disease, and can be used to understand the SARS-CoV-2 virus–host interaction, the SARS-CoV-2 replication cycle and the adaptive and innate immune response pathway [This paper presents the DeCovid, a Shiny app for exploring basal expression level differences between two demographic risk factors, age and sex, for the COVID-19 Disease Map and COVID-19 related pathway genes expressed at several human tissues. DeCovid allows us to interrogate COVID-19 Disease Map and COVID-19 related pathway genes globally or individually. We used data from the GTEx database, which contains RNA-seq profiles for hundreds of demographically diverse healthy individuals in multiple human tissues and cell types. This resource was used to study basal expression differences in COVID-19 associated genes between different demographic groups  to understand the baseline immune patterns they present towards COVID-19. We found that a similar immunological state is prevalent in young people and women, which is different than that found in men and old people. Interestingly, some of these differential pathways have been shown to be relevant for disease severity progression and are characteristic of the sex-biased immune response to the virus, providing ground for hypotheses on the molecular basis of the COVID-19 sex and age bias. We anticipate that the DeCovid app will be a useful tool for researchers to explore the molecular etiology of COVID-19 demographic differences.https://www.gtexportal.org/home/datasets). GTEx data were analyzed for possible biases in relation to the indicated cause of death using the principal component analysis, and blood samples belonging to individuals with a “ventilator case” were discarded  containing data from 44 tissue sources obtained from individuals from both sexes who are deceased but were considered healthy, and that belonged to a wide range of ages . Gene reiscarded . The COVedgeR [https://github.com/ConesaLab/DeCovid.The DeCovid software is a Shiny app written in R with a user-friendly interface. The app can be installed through a Docker image or directly downloaded from GitHub see . DeCovidedgeR  and multedgeR . ResultsedgeR . The tooThe DeCovid shiny app combines GTEx data from a selection of relevant human tissues and cell types with the COVID-19 Disease Map database and COVID-19 related pathways to facilitate the analysis of basal immune differences in the healthy human population for genes described to be important for COVID-19. We included data from whole lungs, heart, kidney, stomach and brain as they were tissues described to be affected by SARS-CoV-2 infection . We alsop-value and a fold-change threshold are provided, the app computes differential expression and provides results as a list of genes and heatmaps with sex and age groups mean values  revealed many specific differentially expressed genes across different tissues/cells see . We founYoung people showed high expression of complement system genes in different immune tissues , women eData showed that females exhibit higher levels of anti-viral type 1 interferons IFNA17, IFNA2, IFIT1, IFIT3 and IFNE in the immune tissues/cells, which serve as the first line of defense against viral and bacterial infections  Figure . MoreoveData showed that young people have a higher expression of matrix metalloproteases (MMPs) genes in all immune tissues/cells . In addiExpression of proinflammatory cytokines such as IL17, IL22 and IL3 were upregulated in older males, when compared to females . In addiHere we present the DeCovid app as a resource to explore sex and age differential expression patterns in the healthy population for genes described to be involved in COVID-19 disease pathways. The GTEx data, used in this work, has been recently mined for sex-specific expression differences concluding that these are tissue-specific and generally small, which was also observed in our analyses . This stWe used DeCovid to investigate gene expression differences between men and women and between old and young people, with the aim of shedding light into possible basal differences in the characteristic immunophenotype of these demographic factors that could influence their distinct susceptibility for COVID-19. We found that four major immune response pathways were differentially expressed across different demographic groups in the healthy population . Women sWe have developed the DeCovid R shiny app to analyze baseline differences between sex an age demographic groups in the expression of COVID-19 relevant genes. Using this resource, we found several immune system related pathways, including interferon, complement system, matrix metalloproteinases, and cytokines that showed differential expression levels between men and women and/or between young and old people. We hypothesize that these population groups are characterized by distinct immunophenotypes that could contribute to their different patterns of progression to severe forms of the disease. Our results show that DeCovid is a useful resource to explore the expression of COVID-19 relevant genes in the healthy population.


Background: Understanding SARS-CoV-2 dynamics and transmission is a serious issue. Its propagation needs to be modeled and controlled. The Alsace region in the East of France has been among the first French COVID-19 clusters in 2020. Methods: We confront evidence from three independent and retrospective sources: a population-based survey through internet, an analysis of the medical records from hospital emergency care services, and a review of medical biology laboratory data. We also check the role played in virus propagation by a large religious meeting that gathered over 2000 participants from all over France mid-February in Mulhouse. Results: Our results suggest that SARS-CoV-2 was circulating several weeks before the first officially recognized case in Alsace on 26 February 2020 and the sanitary alert on 3 March 2020. The religious gathering seems to have played a role for secondary dissemination of the epidemic in France, but not in creating the local outbreak. Conclusions: Our results illustrate how the integration of data coming from multiple sources could help trigger an early alarm in the context of an emerging disease. Good information data systems, able to produce earlier alerts, could have avoided a general lockdown in France. The coronavirus disease COVID-19 was labelled a pandemic by the World Health Organization (WHO) on 12 March 2020 . The OisTo understand the characteristics of the outbreak, the particular role played by the POC gathering in the virus transmission in Haut-Rhin and to see if the alert could have been triggered earlier, an online population-based survey was launched 22 April 2020 with the support of general physicians and emergency unit health professionals of the Haut-Rhin department . PopulatIn this paper, we compare the results of this survey to those of a retrospective study led by an emergency unit located in Mulhouse in order to check their coherence. Indeed, emergency services have been on the front line of the pandemic, especially during the first weeks of March 2020, when they had to handle a massive increase of COVID-19 cases. Retrospective analysis of medical files in a French hospital North of Paris helped in identifying patients with COVID-19 symptoms as early as December 2019 .Until March 2020, hospital biological laboratories were still performing PCR tests for influenza, while PCR tests for SARS-CoV-2 coronavirus were strictly restricted to persons coming back from regions at risk. In the context of emergency care, influenza PCR tests can be requested for patients displaying clinical symptoms very specific to influenza infection in order to confirm the diagnosis. If COVID-19 was already circulating in the Haut-Rhin population at that time, early COVID-19 cases displaying typical influenza symptoms should have been tested negative for influenza. As a consequence, the rate of negative influenza tests should also provide a useful piece of information to understand the beginning of the COVID-19 epidemic.The objective of our study is to draw a coherent picture of the COVID-19 epidemic history in the Haut-Rhin department from January to April 2020 by confronting data coming from these three sources: an online population-based survey, an emergency care service retrospective study, and influenza PCR laboratory tests performed upon request of emergency care physicians.A population-based survey using an anonymous questionnaire was conducted online to evaluate if people living in the Haut-Rhin department had symptoms commonly experienced in case of COVID-19 infection. In order to achieve a quick deployment to reduce memorization bias, a fast track procedure was followed to build the survey protocol and obtain the needed legal and ethical agreements.After testing the questionnaire on four voluntary families in France and Switzerland, the study was approved by the legal and ethical agreements of Clermont Auvergne University Ethic Committee on 10 April 2020, and Clermont Auvergne University Personal Data Protection Management authorities and National Center for Scientific Research Personal Data Protection Management authorities both on 21 April 2020 (reference number IRB00011540-2020-37). Informed consent was obtained from all participants.All families living in the Haut-Rhin department including POC meeting participants were invited to fill the online questionnaire. Thanks to the support of local media  and to the POC meeting organizers, the survey was well advertised. There was no control nor deadline for participation.The survey considered the family structure and the social relationships and networks of the household members. Respondents were then asked to document the occurrence among family members of the symptoms commonly observed in the case of COVID-19 infection . The intThe second source of information is the emergency care service of the Diaconat-Fonderie Hospital in Mulhouse. This private, not-for-profit hospital is located in the center of Mulhouse and has a capacity of 200 beds. Its emergency care service was created in 2006: the staff includes 7 emergency physicians supported by 17 paramedics. In 2018, the service recorded 28,317 patients, to be compared to 68,552 patients at Mulhouse public hospital emergency service the same year.The detection of COVID-19 patients became simpler in May 2020 thanks to the availability of PCR and serological tests combined with the clinical experience of healthcare professionals. This experience was used to reevaluate retrospectively all the medical records of patients who visited the emergency care service from 30 December 2019 to 18 May 2020 in order to identify potential COVID-19 cases.The patient interview revealed previous contacts with COVID-19 symptomatic persons.The following clinical signs were present: significantly impaired general condition, fever, myalgia, arthralgia, dry cough, significant asthenia, dyspnea, desaturation, sibilant rales at auscultation, chest pain, headaches, unexplained discomfort, anosmia, diarrhea, or abdominal pain. Symptom duration was considered. Productive cough and rhinorrhea were considered symptoms excluding COVID-19.Chest computed tomography (CT) scans suggested COVID-19: the presence of Ground Glass Opacities . AmbiguoBiological indicators were in favor of COVID-19: lymphopenia, a negative influenza PCR test, or thrombocytopenia.For patients admitted before the local sanitary alert on 3 March 2020, the selection process involved several stages. In a first stage, medical records were preselected by the head nurse based on patient reason for emergency consultation. Records of patients presenting symptoms possibly related to a COVID-19 infection were kept, including fever, cough, dyspnea, headache, diarrhea, dizziness, or unexplained discomfort. Files were excluded if medical evaluation recorded at both entrance and exit was not compatible with COVID-19 and if PCR tests for influenza were positive. The preselected patient files were then analyzed by the emergency physicians and classified as probable COVID-19 cases if several of the following clinical conditions were fulfilled:For patients admitted after 3 March 2020, the head nurse reviewed all patient files and performed a first selection of potential COVID-19 cases based on, respectively, the reason for consulting, the nurse’s clinical assessment, and the results of medical examinations, with a particular focus on imaging and biological tests. The selected patients were further evaluated by an emergency physician who established a list of probable COVID-19 patients. This list was then revised by integrating data coming from the radiology service (scanner examinations) and the medical laboratory . COVID-19 infection was retrospectively confirmed for medical records with chest images indicating typical CT features of COVID-19 pneumonia and/or a positive biological test for COVID-19. Patients were classified as confirmed cases only after 3 March 2020, when biological tests were available.At the end of this process, 552 patients were retained as retrospectively diagnosed probable or confirmed cases, out of the 7184 patients admitted to the emergency care service from 29 December 2019 to 18 May 2020.The Diaconat-Fonderie medical biology laboratory is a multi-purpose biological platform open 24 h a day every day to provide continuous support to emergency care services. Its team handles over 600 daily samples coming from more than 20 healthcare centers in the Haut-Rhin department. The laboratory performed influenza PCR tests of the samples sent by the Diaconat-Fonderie emergency care services between 1 January and 15 March 2020 using the Abbott BinaxNOW Influenza A&B Card kit  . After tStatistical analyses were conducted using SAS software v9.4 . Categorical variables were presented as frequencies and percentages, while continuous variables were presented as means and standard deviations (SD).Overall epidemic evolution was compared in the survey data and in the retrospective analysis of emergency care service medical records by computing an epidemic threshold for both data sources.A wide variety of statistical methods are available for defining an epidemic threshold for seasonal epidemics such as influenza ,15,16. IFrom the answers of Haut-Rhin households, we computed every day from 15 December 2019 to 16 May 2020 the number of new possible COVID-19 cases using the first day of illness onset. The non-epidemic level was defined as the highest value of the seven-day moving average of daily incidence over the period from 15 December 2019 to 15 January 2020. The epidemic threshold was then computed as the upper 95% confidence limit of the non-epidemic level.The same approach was adopted for Diaconat-Fonderie medical records. We calculated every day from 30 December 2019 to 16 May 2020 the number of new possible COVID-19 cases using the entrance date in the emergency care service. The non-epidemic level was defined as the highest value of the seven-day moving average of daily COVID-19 cases over the period from 1 January to 15 January 2020.s corresponds to the speed, m to the seven-day moving average of daily incidence, t to the time, and i to the day.In a second step, we checked for both datasets the stability in terms of consecutive days over or under the threshold: if the incidence was rapidly oscillating under and over the threshold, it meant that the threshold did not discriminate between outbreak and background noise. The signal was stable if each period under or over the threshold was larger than seven consecutive days. In order to complete the incidence curve analysis, we also computed the daily speed (Equation (1)) and acceleration (Equation (2)).For the majority of cases documented in the population-based survey, biological proofs of SARS-CoV-2 infection were missing, and there could be confounding epidemic events, as documented for instance in . To confIn order to further discriminate COVID-19 cases from other respiratory syndromes circulating in the population, we particularly focused on anosmia, identified as the symptom with the largest positive predictive value for SARS-CoV-2 infection ,26,27,28From 22 April to 4 June 2020, 1427 households participated to the population-based survey. The 201 households not living in the Haut-Rhin department were excluded from the present analysis, leading to a population of 1226 households, representing 3350 individuals. Among them, 883 households (72.0%), representing 2502 individuals, reported at least one possible case of COVID-19 . A totalBased on the number of cases documented in the reference period (15 December 2019 to 15 January 2020), the epidemic threshold (dashed blue line) was estimated to 5 new cases per day. The curve shape indicates three waves in the epidemic development, which is confirmed by the speed and acceleration analysis (data not shown). From 30 December 2019 to 22 January 2020, a first wave lasted 24 days, with the same one-week increase (1–7 January) and decrease (17–24 January) periods. However, the highest incidence remained under the epidemic threshold. The second wave also lasted 24 days from 23 January to 17 February. It crossed the epidemic threshold on 26 January and remained over this threshold despite a decrease after 10 February. The third wave of the outbreak began on 17 February, with an incidence increase leading to a first maximum on 1 March, 3.6 times higher than the preceding wave. A partial and local lockdown started on 3 March and was extended to the whole country on 17 March [Among households documenting at least one possible COVID-19 case, 47.6% indicated that the onset of the first COVID-19 symptoms occurred before 1 March 2020. On 17 March, the first day of nationwide lockdown, 81.2% of these households had already reported a possible COVID-19 case. On the positive side, the lockdown was very efficient at stopping the epidemic as only 2.9% of the households reported their first case after 31 March, two weeks after entering the lockdown.For the whole period, the secondary attack rate was 37.6%, and the mean generation interval, computed using intra-household data, was 4 days.After analyzing the records of all the patients admitted into the Diaconat-Fonderie emergency care service from 30 December 2019 to 18 May 2020, 552 retrospectively diagnosed probable or confirmed COVID-19 cases were retained.The seven-day moving average of daily incidence according to patient admittance day is documented on In order to check the possible role of emergency attendance, we also computed the fraction of the weekly attendance represented by retrospectively diagnosed probable or confirmed COVID-19 cases . The patIn the period preceding the sanitary alert, the circulation of COVID-19 in the population of the Haut-Rhin department was ignored. Emergency care service visitors with specific influenza-like symptoms were given influenza PCR tests from January to mid-March 2020 to confirm diagnosis.The emergency care services sent 59 samples to the Diaconat-Fonderie medical biology laboratory for influenza PCR testing from 1 January to 15 March 2020; 66% of the tests performed up to 28 January 2020 were positive.In the population-based survey, 3.2% of the cases (48 out of 1516) were tested for influenza. Three-quarters of the tests were negative (n = 36). Out of the 12 persons tested positive for influenza, six also tested positive for COVID-19. Cases of coinfection by COVID-19 and influenza have been confirmed and documented . As a coExisting COVID-19 epidemiological data show that children are significantly less affected than adults ,25. AnotTo check whether other respiratory syndromes could explain the pattern observed on A way to discriminate between COVID-19 and other respiratory syndromes is to study symptoms that are more specific to COVID-19. As discussed earlier, one of the most predictive COVID-19 symptoms is anosmia. Out of the 1516 possible cases documented by Haut-Rhin households, 13 out of 158 children less than 11 years old (8.2%), 19 out of 67 children (28.4%) between 12 and 18 years old, and 506 out of 1265 adults (40%) experienced anosmia at different stages.The curves corresponding to partial (orange) and total (red) loss of smell display similar patterns, in particular a little increase at the end of January. The curve corresponding to smell modification (yellow) is less marked.Of particular interest in the case of the Haut-Rhin cluster is the role played by the POC gathering in the outbreak genesis and acceleration in February and beginning of March. The POC gathering began on 17 February and ended 21 February. In the population-based survey answers, 237 out of the 1516 individuals who experienced symptoms commonly attributed to COVID-19 infection come from households in which one member at least participated in the POC gathering.p < 0.0001. Anosmia was observed more frequently (56.5%) in the households involved in the POC meeting than in the other households (32.0%), The results presented in the previous section were obtained from three sources: a population-based survey of the households living the Haut-Rhin department, a retrospective analysis of the Diaconat-Fonderie private, not-for-profit hospital emergency care unit in Mulhouse, and influenza PCR test data from the Diaconat-Fonderie biological laboratory. They all point toward an early circulation of the COVID-19 in the Haut-Rhin population, much earlier than the first official case identified on 26 February 2020 in the Grand Est region and the epidemic alert on 3 March ,22.The converging evidence for a COVID-19 epidemic starting at the end of January 2020 comes from the simultaneous epidemic threshold crossings of three independent observables: visitors to Diaconat-Fonderie emergency care service retrospectively diagnosed as probable and confirmed COVID-19 cases , possiblOur data also provide evidence for the circulation of other respiratory syndromes including influenza, especially during the second wave from 23 January to 17 February 2020. The ratio of the number of retrospectively diagnosed probable and confirmed COVID-19 cases from medical records to the number of possible COVID-19 cases from the population survey was significantly larger in the third wave compared to the second wave . MoreoveThe generation interval computed from intra-household data is close to what is published elsewhere ,23 and tFinally, our results show very different patterns for households with or without participants to the POC gathering. The curves corresponding to the households related to the POC gathering are very typical of a unique epidemic process, centered on one maximum . On the It is important to note that the Diaconat-Fonderie emergency service is not the main line emergency service in Mulhouse . It is more activated when the public hospital is overcrowded: this could be an alternative explanation to the flat epidemic in January and February, before the generalization of the outbreak. The other explanation documented previously is the circulation of other respiratory syndromes in parallel to the start of the COVID-19 epidemic in January and February.Our data favor the minor role of children in the COVID-19 epidemic. Only limited COVID-19 symptoms were observed among children below 15 years old, especially during the third wave after 20 February.The population-based survey was also extremely helpful to receive testimonies of people who experienced frequently observed COVID-19 symptoms before the end of 2019. Evidence exists that SARS-CoV-2 was already spreading in France in late December 2019 , while tOur work has some limitations. Possible biases include, for the population-based survey, the exclusion of people without access to internet, dependency on participant declarations, and voluntary participation. Nevertheless, the population of respondents was diverse and included families without symptoms. The studies were retrospective, leading to a possible memorization bias for the population-based survey, and the bias of missing data for the emergency patient cohort.We had no biological proofs of infection for the population-based survey, and these proofs came very late in the emergency retrospective study, due to the fact that, in France, we were unable to perform PCR tests on a large scale until the end of April 2020. Therefore, respondents to the survey were free to declare symptoms not due to COVID-19. Only 121 (8.0%) of the 1516 possible COVID-19 cases were tested for COVID-19: 34 tests were negative and 87 were positive. The results of serological tests were not available when the datasets were analyzed.Although Diaconat-Fonderie emergency care services documented probable COVID-19 cases in January, the only biological data used to reach our conclusions were the results of influenza PCR tests. As the hospital laboratory observed a significant increase of negative influenza PCR tests at the end of January 2020, we can hypothesize that the growing number of undiagnosed COVID-19-infected patients resulted in a growing rate of negative influenza tests that went unnoticed.Additionally, due to privacy rules, information on patient localization was very limited, consisting only in the department in which the household is located, excluding therefore a spatial chaining of cases.To estimate the epidemic threshold, we could not implement a complex statistical method such as the moving epidemic method (MEM), used to model influenza epidemics based upon historical data from a specific country or region . Indeed,To further document the virus history and propagation, more data are needed, especially from the other emergency services in Mulhouse—particularly the public hospital, which was on the front line during the COVID-19 crisis. Imaging data from radiology units should also help trace the early cases that were unseen during the early days of 2020. Further analysis of the population-based survey should help understand if the early possible COVID-19 cases had common activities or social networks, as households were also invited to document their social contacts.The role of comorbidities has to be explored. It is also necessary to better understand how the attendants to the POC gathering contributed to the local and nationwide epidemic spread, which lead to a complete French lockdown after 17 March, one month after the beginning of the POC gathering and seven weeks after the outbreak started to become significant. This event is of particular interest to explore the stochasticity and heterogeneity in COVID-19 transmission dynamics. In the hypothesis that COVID-19 transmission is stochastic, dominated by a small number of individuals and driven by super-spreading events, models suggest that the epidemic is not monotonically increasing . Such bePopulation-based survey and hospital data are consistent and support the hypothesis that the COVID-19 outbreak started as early as the end of January 2020 in the Haut-Rhin department, while other respiratory syndromes including influenza were still circulating in the population.An efficient sanitary surveillance requires time to collect and analyze data, and to achieve enough convergence to provide an alert. An efficient surveillance system requires about one week ,39, so aOf course, it could be said that it is easy to re-write history when you know how it turns out. Nevertheless, in order to face a second COVID-19 epidemic wave, our work emphasizes the need for population-based surveys crossed with data from emergency care units, to provide alerts at the best possible time. Delayed decisions always lead to degraded health policies.


Background: The induction of donor-specific immunological tolerance could improve outcome after kidney transplantation. However, no tolerance protocol is available for routine clinical use. Chimerism-based regimens hold promise, but their widespread application is impeded in part by unresolved safety issues. This study tests the hypothesis that therapy with polyclonal recipient regulatory T cells (Tregs) and anti-IL6R (tocilizumab) leads to transient chimerism and achieves pro-tolerogenic immunomodulation in kidney transplant recipients also receiving donor bone marrow (BM) without myelosuppressive conditioning of the recipient.Methods/design: A prospective, open-label, controlled, single-center, phase I/IIa academic study is performed in HLA-mismatched living donor kidney transplant recipients.Study group: Recipients of the study group receive in vitro expanded recipient Tregs and a donor bone marrow cell infusion within 3 days after transplantation and tocilizumab for the first 3 weeks post-transplant. In addition they are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Starting 6 months post-transplant, sirolimus and steroids are withdrawn in a step-wise manner in stable patients.Control group: Recipients of the control group are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Co-primary endpoints of safety  and efficacy  are assessed. Secondary endpoints include frequency of biopsy-proven acute rejection episodes and subclinical rejection episodes on surveillance biopsies, assessment of kidney graft function, and the evaluation whether the study protocol leads to detectable changes in the immune system indicative of pro-tolerogenic immune modulation.Discussion: The results of this trial will provide evidence whether treatment with recipient Tregs and donor BM is feasible, safe and efficacious in leading to transient chimerism. If successful, this combination cell therapy has the potential to become a novel treatment option for immunomodulation in organ transplantation without the toxicities associated with myelosuppressive recipient conditioning.Trial registration: European Clinical Trials Database EudraCT Nr 2018-003142-16 and clinicaltrials.gov NCT03867617. Long-term outcome after kidney transplantation has improved little over the last decades and immune-mediated injury remains a leading cause of graft loss despite modern immunosuppressive drug therapy . The estThree pilot trials have recently been conducted that investigated the simultaneous co-transplantation of donor hematopoietic stem cells  for the purpose of tolerance induction in living donor kidney transplant recipients –16. Tolein vitro activated CD4+CD25+ Tregs, TGFβ-induced Tregs and FoxP3-transduced Tregs)  . Levels d Tregs) –23. Reced Tregs) , in whicd Tregs)  and the d Tregs) –30. Preld Tregs) , but thed Tregs) . The fulThe present study investigates combination cell therapy with recipient Tregs and donor BM, together with IL6 pathway blockade, as a potential strategy for inducing transient chimerism and pro-tolerogenic immunomodulation in kidney transplantation  is the trial sponsor. The study sponsor has no role in the design of the study or the analysis and publication of its results.The study has been approved by the ethics committee of the Medical University of Vienna  and the Austrian Federal Office for Safety in Health Care .Prospective, open-label, controlled, single-center, phase I/IIa study.Subjects are recruited from patients scheduled to receive a living donor kidney transplant at the Vienna General Hospital/Medical University of Vienna.The donor and the recipient need to provide written informed consent. The inclusion and exclusion criteria that need to be fulfilled for enrolment into the trial are the same for recipients in the study group and recipients in the control group. Inclusion/exclusion criteria for donors in the study group are distinct from those for donors in the control group. Inclusion and exclusion criteria are listed in in vitro expanded polyclonal recipient Tregs (RegTivex), anti-IL6R (tocilizumab), and donor BM, in addition to belatacept-based immunosuppression (In vitro expanded regulatory T cells (RegTivex) and tocilizumab  are the investigational medicinal products (IMPs) of this study.The study group receives pression . Tregs aThe control group receives the same immunosuppressive drug combination as the study group consisting of thymoglobulin, belatacept, sirolimus, and steroids . No TregExtensive immune monitoring accompanies the study with the aim to yield information about the activity and the mode of action of the treatment protocol . SamplesNo randomization is performed. The complex design which requires recipient leukapheresis plus thymoglobulin treatment 2 weeks prior to transplant and cell therapy with Tregs and BM in the recipient and BM harvesting from the donor in the study group but not the control group, would impede enrolment within a reasonable time frame, if randomization were performed, as it is assumed that more subjects are willing to participate in the control than the study group. Due to the small sample size, randomization would require stratification, as otherwise outcome predictors would likely be unevenly distributed between groups. Due to the number of living donor transplants performed annually at the Medical University of Vienna, stratification would likely make enrollment in this single center study infeasible. Therefore, also no attempt at matching the number of HLA-disparities between the groups is undertaken. Thus, mainly for reasons of feasibility no randomization is performed.in vitro expanded CD45RA+CD4+CD25highCD127low/neg regulatory T cells isolated from a leukapheresis product of the kidney transplant recipient. The Treg product (RegTivex) is generated at the José-Carreras-Centrum for Somatic Cell Therapy  at the University Hospital Regensburg (Germany). Two weeks prior to the scheduled kidney transplant, the recipient undergoes standard leukapheresis at the Medical University of Vienna. The time point of leukapheresis is chosen to allow optimum Treg expansion until the scheduled day of infusion. The leukapheresis product is shipped to the JCC using validated transport conditions, enriched for CD25+ cells using bead selection technologies, then stained for CD4, CD25, CD127, and CD45RA and FACS-sorted for the CD45RA-positive Treg subpopulation (in vitro expanded for ~2 weeks (2–3 log expansion rates) using CD3/28 stimulation beads and high-dose rh-IL-2 (note: Regensburg is a 4-h drive away from Vienna). Kidney transplant recipients of the study group are infused intravenously with the fresh Treg product within 3 days after the kidney transplant. The Treg and BM cell infusions are both given within 3 days post-transplant, but are administered sequentially with a time interval in-between to assess immediate infusion-related side effects individually. At this time, all kidney transplant recipients are routinely treated in-hospital at the transplant center. Treg therapy in this trial constitutes an autologous cell infusion.The IMP consists of pulation . Cells a rh-IL-2 . After b7 cells/kg body weight. This is the dose for which preliminary safety data exist from GVHD studies. The actual dose of available Tregs/kg body weight will depend on several factors, including the cell number and composition of the leukapheresis product, the efficiency of the Treg expansion and the weight of the recipient. In case the target cell dose cannot be reached, doses as low as 0.3 × 107 cells/kg body weight are permissible. This lower limit is set to minimize the risk that Treg cells from the recipient are discarded without clinical use. The maximum dose of Tregs administered is 1.5 × 107 cells/kg body weight. The cells are administered post-operatively as a single intravenous infusion, no later than 3 days after the kidney transplant (no later than D3).The target dose for the Treg cell product is 1.0 × 108 nucleated cells/kg body weight (corresponding to a CD34+ cell dose of 2–3 × 106/kg body weight). Similar doses have been used previously in kidney transplant recipients  and subsequent doses on D5, D15, and D21 post-transplant. This dosing regimen corresponds to the dosing frequency approved for long-term maintenance therapy in rheumatoid arthritis.Thymoglobulin is administered intravenously at a dose of 6 mg/kg body weight. In the control group, thymoglobulin is given at the time of transplant, in four divided doses, as previously reported . In the Belatacept, a CTLA4Ig derivative, is the only clinically approved alternative to calcineurin-inhibitors (CNIs) as primary immunosuppressant. CNIs impeded outcome in pre-clinical costimulation blockade-based chimerism models . BelatacmTOR inhibition through sirolimus promotes Tregs while inhibiting effector T cells. In addition, sirolimus promoted the engraftment of allogeneic BM in preclinical studies , 19, 39.Corticosteroids  are dosed according to local practice at the Medical University of Vienna for 12 months in the control group and 9 months in the study group.Donor BM  is obtained from the iliac crest under general anesthesia during the donor nephrectomy surgery (D0). Intraoperative BM harvesting during living donor nephrectomy was reported to be feasible and safe by several groups –43.Starting 6 months post-transplant, sirolimus and steroids are withdrawn in a step-wise manner in stable patients of the study group. Pre-defined criteria for starting sirolimus and steroid withdrawal are: absence of immunological injury on 6-month surveillance biopsy  and absence of donor-specific antibodies (DSA). Sirolimus weaning starts first, to be followed by weaning of steroids 3 months later.The Investigator must temporarily interrupt or permanently discontinue the study drug if continued administration of the study drug is believed to be contrary to the best interests of the patient. The interruption or premature discontinuation of study drug might be triggered by an Adverse Event (AE), a diagnostic or therapeutic procedure, an abnormal assessment , or for administrative reasons, in particular withdrawal of the patient's consent. The reason for study drug interruption or premature permanent discontinuation must be documented in the CRF.Primary Objective:1) To examine the safety of Treg therapy together with tocilizumab and donor bone marrow in living donor kidney transplant recipients.2) To assess chimerism levels within the first month post-transplant.Secondary Objectives:To demonstrate that the study protocol allows the initiation of a step-wise reduction of immunosuppression up to a point when patients receive drug monotherapy.To gain insight as to whether the study protocol leads to detectable changes in the immune system indicative of pro-tolerogenic immune modulation.To assess the frequency of biopsy-proven acute rejection episodes.To assess the frequency of subclinical rejection episodes on surveillance biopsies.To assess kidney graft function.To assess the area-under-the-curve (AUC) of chimerism.2] or patient death, whichever occurs first, within the first 12 months post-transplantation (co-primary endpoint 1).The composite safety endpoint is defined as the incidence of GVHD, impaired graft function [eGFR <35 mL/min/1.73 mThe efficacy endpoint is defined as individual peak chimerism levels observed in each patient within 4 weeks post-transplant (co-primary endpoint 2). Total leukocyte donor chimerism will be measured by sensitive ddPCR technology at days 5, 10, 15, 21, and 28 post-transplant in both groups .Six patients (recipients) per group eligible for per-protocol analysis, up to 10 patients per group maximum. For each recipient one donor is enrolled in the same group.co-primary endpoint 1) at a one-sided significance level of 2.5% . This margin of 65% is accepted for statistical analysis to preserve feasibility. A more robust safety analysis will only be possible in potential larger subsequent trials. By including also six patients in the control group the study has 86.5% power to detect a difference in mean peak chimerism on the logarithmic-base-2 scale of 2 log-units (a 4-fold difference in geometric means) given an assumed standard deviation of 1 log-unit (co-primary endpoint 2). No data are available from which a meaningful assumption on the standard deviation of mean peak chimerism  could be derived. Our assumption corresponds to a ratio between upper and lower limits of a normal range (97.5th and 2.5th percentiles) of about 16 (four log units), which was considered conservative enough when discussing it among the principal investigators and the study statistician.The sample size is based primarily on feasibility to allow enrollment within a reasonable time frame. Beyond this premise, sample size calculation yielded a number of six for the study group to conclude with a probability  of 0.8 that the incidence of the safety endpoint is not >65% , the BM infusion and at least one dose of tocilizumab (study group), all subjects of the control group who were treated with at least one dose of belatacept, and who completed 4 weeks of follow-up and who did not violate the protocol in a way that might affect the evaluation of the effect of the study drug(s) on the primary objective, i.e., without major protocol violations.This analysis set comprises all subjects who received the kidney transplant, the Treg infusion (at a minimum dose of 0.7 × 10Co-primary Endpoint 1 (Safety). Co-primary endpoint 1 will be evaluated in the study group, using the modified intention-to-treat set. The endpoint will be summarized by computing the proportion of patients reaching co-primary endpoint 1 and the corresponding two-sided 95% exact (Clopper-Pearson) confidence interval.Null hypothesis: The incidence of the composite safety endpoint  in treated patients is 65% or higher.Alternative hypothesis: The incidence of the composite safety endpoint  in treated patients is <65%.2) eGFR values after the first month post-transplant will be considered . eGFR is calculated according to the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) formula. GVHD diagnosis needs to be confirmed by histology.For assessing impaired graft function  if more than one out of the six patients experiences the safety endpoint, as then the upper limit of the one-sided 97.5% confidence interval for the safety endpoint incidence will reach beyond 65%.Co-primary Endpoint 2 (Efficacy). Total leukocyte donor chimerism is measured by sensitive ddPCR technology on D5, D10, D15, D21, and D 28 post-transplant in both study groups. Co-primary endpoint 2 is defined as the individual peak chimerism level, i.e., the maximum value over the five measurements taken in each patient (Cmax). The endpoint will be evaluated in the per-protocol set.t-test assuming unequal variance (Satterthwaite-Welch-test) with a significance level of 0.025.The endpoint will be summarized by computing mean and standard deviation of log-base-2 peak chimerism in both groups. Mean values of the two treatment arms will be compared using a one-sided two-sample Null hypothesis: The expected level of chimerism (log2 Cmax) in patients treated with the experimental treatment is not higher than that of control patients.Alternative hypothesis: The expected level of chimerism (log2 Cmax) in patients treated with the experimental treatment is higher than that of control patients.The two null hypotheses cover different aspects and thus the two co-primary endpoints will be tested independently and both will be reported. Under the global null hypothesis and assuming independence of safety and efficacy, the one-sided probability to reject one or two null hypotheses is 5%.p-values and confidence intervals are only meant to describe the precision of summary statistics, but not to confirm or reject a study hypothesis. Categorical data will be expressed as frequency counts and percentages. Continuous variables will be summarized by their mean, standard deviation, minimum and maximum. For repeatedly measured outcomes, pre- and all post-baseline values will be assessed and graphically presented.All secondary endpoints will be analyzed in a purely descriptive manner. Thus, any inferential measures, such as Monitoring is performed by the Clinical Trials Coordination Centre of the Medical University of Vienna. A Data and Safety Monitoring Board (DSMB) that it is independent from the sponsor and has no competing interests regularly reviews the clinical data of the trial and has the authority to recommend alterations and/or termination of the trial for safety reasons.An Adverse Event (AE) is defined as any untoward adverse change from the subject's baseline condition, i.e., any unfavorable and unintended sign including an abnormal laboratory finding, symptom or disease which is considered to be clinically relevant by the physician that occurs during the course of the study, whether or not considered related to the study drug. A special section is designated to adverse events in the case report form documenting the details of the AE. The severity of clinical AEs is graded on a three-point scale: mild, moderate, severe, and reported on specific AE pages of the CRF.For all AEs, the Investigator assesses the causal relationship between the study drug and the AE using his/her clinical expertise and judgment (not related—unlikely—possibly related—probably related—related).A Serious Adverse Event (SAE) is defined by the International Conference on Harmonization (ICH) guidelines and GCP guidelines as any AE fulfilling at least one of the following criteria:Results in deaths.Life-threatening—defined as an event in which the subject was, in the judgment of the Investigator, at risk of death at the time of the event.Requiring subject's hospitalization or prolongation of existing hospitalization.Resulting in persistent or significant disability or incapacity .Congenital anomaly or birth defect.Is medically significant or requires intervention to prevent at least one of the outcomes listed above.In case of a serious adverse event, the Investigator has to use all supportive measures for best patient (recipient and donor) treatment. A written report is also to be prepared detailing the SAE. If applicable, the initial report should be followed by a follow up report, indicating the outcome of the SAE.Suspected unexpected serious adverse reactions (SUSARs) are defined as all serious adverse reactions with suspected causal relationship to the study drug that is unexpected (not previously described in the Summary of Product Characteristics or Investigator's brochure) and serious. The regulatory authorities, and the Institutional Review Board/Independent Ethics Committee (IRB/IEC) must be informed about all SUSAR. Such reports containing all relevant detail shall be made by the sponsor.Recruitment started on August 1, 2019 and is expected to be completed by April 2022.8 nucleated cells/kg body weight) has been used safely (without GVHD) in several clinical transplant trials of “BM augmentation”  (de novo DSA) in patients receiving belatacept-based immunosuppression  . The risk of GVHD is minimal as it is expected that no permanent chimerism is induced. Immunosuppression is reduced to belatacept monotherapy in the study group, but complete withdrawal is not attempted in this first trial of combined Treg and BM cell therapy. Thereby the potential risk of immunological damage to the graft during immunosuppression withdrawal is avoided , 40. Unstioning) , 45, 46.tuzumab) .While a control group was not included in the three pilot trials of chimerism-based tolerance published to date –14, its The protocol is designed to achieve transient chimerism without myelosuppression. Pre-clinical data demonstrate that the administration of polycloncal recipient Tregs at the time of BM infusion can achieve BM engraftment in this setting. The mechanisms how this effect is mediated are not fully delineated yet, but control of donor-reactive NK cells appears to be critical . Blockinin vitro expansion of the recipient Tregs takes 2–3 weeks  and the Austrian Federal Office for Safety in Health Care . The patients/participants provided their written informed consent to participate in this study.RO, ME, GB, PK, NW, GH, MW, TL, and TW designed the study protocol. TW developed the underlying study concept and wrote the draft of the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recent evidence suggests that influenza vaccination may offer protection against COVID-19 severity. Our aim was to quantify the association between influenza vaccination status and risk of hospitalisation or all-cause mortality in people diagnosed with COVID-19.A retrospective cohort study using routinely collected health records from patients registered to a General Practitioner (GP) practice in South West England within the Electronic Care and Health Information Analytics database. The cohort included 6921 people with COVID-19 during the first wave of the pandemic (1 January–31 July 2020). Data on influenza vaccination, hospitalisation and all-cause mortality were ascertained through linked clinical and demographic records. We applied propensity score methods (stabilised inverse probability of treatment weight) to quantify the association between influenza vaccination status and COVID-19 outcomes .2613 (38%) participants received an influenza vaccination between 1 January 2019 and COVID-19 diagnosis. Receipt of influenza vaccination was associated with a significantly lower odds of hospitalisation or all-cause mortality , and 24% reduced odds of all-cause mortality .Influenza vaccination was associated with a 15%–24% lower odds of severe COVID-19 outcomes. The current UK influenza vaccination programme needs urgent expansion as an integral component of the ongoing response plans to the COVID-19 pandemic. We sought to determine the association between influenza vaccination status and risk of hospitalisation or all-cause mortality in people diagnosed with COVID-19.In people with COVID-19 during the first wave of the pandemic, we found that influenza vaccination was associated with a 15%–24% lower odds of hospitalisation or all-cause mortality.Influenza vaccination may have the potential to offer an additional means of mitigating serious adverse complications of COVID-19, and we therefore propose that a rapid expansion of the influenza vaccination programme is urgently needed as an integral component of the ongoing response to the COVID-19 pandemic.COVID-19 caused by the SARS-CoV-2 is a rapidly evolving global public health emergency. Since the outbreak began, over 87 million cases have been reported globally, with over 1.8 million attributed deaths (as of 7 January 2020).5There remain limited effective interventions to treat COVID-19. A number of vaccines against SARS-CoV-2 have recently been approved for use following promising clinical trials18–20This hypothesis has been supported by a limited number of observational studies, including five retrospective cohort studies24Further validation is required from large UK-based cohorts using individual-level data, exploring important outcomes including all-cause mortality and hospitalisation. This may help us better understand the impact of influenza vaccination in COVID-19 disease and inform the current influenza vaccination programme during the second wave of the pandemic. The aim of this study was to quantify the association between influenza vaccination status and COVID-19 outcomes including hospitalisation and all-cause mortality.Retrospective cohort analysis.Data were obtained from the Care and Health Information Analytics (CHIA) database. This is an anonymous National Health Service (NHS) database that includes individual-level data from 1.5 million patients across 160 GP practices in Southern England. Data have been extracted from routine general practice records linked to local secondary care hospitals, including demographic data, primary/secondary care consultations, diagnoses, medications and outcomes of primary/secondary care investigations.A cohort of individuals within the CHIA database diagnosed with COVID-19 during the first wave of the COVID-19 pandemic (1 January–31 July 2020) were identified. We included both confirmed and suspected cases. Confirmed cases had a positive RT-PCR assay for SARS-CoV-2 on nasal/pharyngeal swab. Clinically suspected cases were defined in accordance with Public Health England (PHE) guidance at the time of the first wave of the pandemic in the UK, when testing was not readily available. Further detail on the COVID-19 case definition has been reported in other databases,27Data for the identified cohort were extracted from the CHIA database including age, sex, ethnicity, Index of Multiple Deprivation (IMD), electronic frailty index, comorbidities, smoking status, body mass index (BMI), medications and influenza vaccination status.Receipt of recent influenza vaccination was defined as an electronic recording of vaccination in the CHIA database between 1 January 2019 and the diagnosis of COVID-19.Since death is a competing risk for hospitalisation,Baseline demographics/clinical characteristics of the study cohort were summarised. Categorical variables were summarised as frequencies and percentages, and continuous variables were as means and SD.Since our primary exposure variable, influenza vaccination, is based on observational data , the group of patients that received the vaccine is not directly comparable with the group that did not receive the vaccine. The reasons why some people received the vaccination leads to so-called treatment indication bias. We attempted to address this confounding by treatment indication bias within marginal structural regression framework, because conventional regression adjustment may not be sufficient to address this issue.To check the robustness of our analysis using stabilised IPTW, we estimated the pooled OR by stratifying the propensity score into deciles . Mantel-Haenszel weighted pooled OR was estimated using Woolf approximation for the calculation of SE.There was missing data on ethnicity, BMI, smoking, IMD and age. Ethnicity is frequently missing from routinely collected primary care records. We removed this variable from the analyses for multiple reasons: (1) high amount of missing data on ethnicity (32.8%), (2) very low number of outcome events in some subgroups  and (3) inadequate reduction of bias by traditional missing data imputation methodsCHIA is an anonymous NHS database, and all individuals have consented for collection of their medical records for inclusion in the database. Findings are reported as per Strengthening the Reporting of Observational Studies in Epidemiology and RECORD guidelines for observational studies of routinely collected data.Anonymised individual level data used in this study was extracted from the CHIA database. Direct requests for data sharing can be made to CHIA.Due to the nature of the study, patients or the public were not involved in the design, conduct, reporting or dissemination of the studyA total of 6921 participants were included in the study, of which 774 (11.2%) received a confirmed diagnosis of COVID-19 during the follow-up period, and 6147 (88.8%) were diagnosed clinically. The mean age was 52.4 years (±24.5) and the majority of participants (59.5%) were female. Most participants were white (60.2%); however, ethnicity was not recorded in one-third of cases (32.8%). Approximately half of participants were from the two least deprived socioeconomic status groups . Baseline characteristics of the study cohort are summarised in A total of 2613 (38%) participants in the cohort had received influenza vaccination between 1 January 2019 and COVID-19 diagnosis see . CompareBaselines characteristics of the study cohort, stratified by incidence of the primary study outcome , are displayed in The overall distribution of the propensity score by influenza vaccination status is shown in 10.1136/bmjresp-2020-000857.supp1Supplementary dataIn the stabilised IPTW-weighted regression model, influenza vaccination was associated with 15% reduced odds of hospitalisation or all-cause mortality  and 24% reduced odds of all-cause mortality  see . FurtherIn the sensitivity analysis using pooled OR after stratification by deciles of propensity score, the results were almost identical for the primary outcome  with a slightly stronger effect for all-cause mortality  see . In the In this sample of 6921 participants who received a diagnosis of COVID-19 during the first wave of the pandemic (1 January–31 July 2019), we found that having prior influenza vaccination was associated with a 15%–24% lower odds of hospitalisation or all-cause mortality.2 0.338).Our study is consistent with observational data from outside the UK demonstrating a negative association between influenza vaccination and COVID-19 acquisition, severity and mortality.Possible explanations for these findings include a direct protective effect against COVID-19 from influenza vaccination. Growing evidence, particularly from work studying BCG and measles vaccination,18–20To our knowledge, this is the first study to examine influenza vaccination in relation to all-cause mortality and hospitalisation based on individual-patient data linked to demographic and clinical records in the UK. We applied robust analytic techniques to reduce the treatment indication bias that is inherent to observational studies. However, this does not completely rule out the possibility of residual and unknown confounding. A strength of the study is that it was drawn from a large database of individuals 160 GP practices across South West England; however, the cohort does include low representation of ethnic minority groups, and a relatively large proportion were from high socioeconomic backgrounds. Another limitation is our inability to analyse the effects by ethnicity due to large amount of missing data. Data being from a centralised single-payer healthcare system may limit the generalisability of these findings to other parts of the world.The case definition of COVID-19 disease has also evolved over time, especially as testing has become more readily available in recent months. We included clinically suspected cases of COVID-19 alongside confirmed cases, in accordance with guidance from PHE during the first wave of the pandemic. Recent studies have shown that clinical suspicion of COVID-19 among GPs does closely match with confirmed cases and subsequent clinical outcomes, but it is still plausible that not all those with clinical cases had COVID-19.We used logistic regression for our primary analysis because the date of COVID-19 diagnosis, hospitalisation or deaths was not precise in the database. However, as stated above, additional time-to-event analysis adjusted for the month of COVID-19 diagnosis showed identical results. Furthermore, exact date of death was not always available for patients who died (only months and years of death were reported). Since the follow-up time was small, this is unlikely to have changed the estimates substantially.Our results suggest that influenza vaccination may have the potential to offer an additional means of mitigating serious adverse complications of COVID-19. Further research should aim to validate these findings in larger more diverse cohorts. Additional work is also needed to examine differences between influenza vaccine subtypes and whether the duration from vaccination administration to COVID-19 acquisition is important.In people with COVID-19 during the first wave of the pandemic, we found that influenza vaccination was associated with a 15%–24% lower odds of hospitalisation or all-cause mortality. An expansion of the influenza vaccination programme is needed as an integral component of the ongoing response to the COVID-19 pandemic.


Sevoflurane-induced seizures are most often caused by high concentrations of sevoflurane during anesthesia induction. However, in this case, we found a rare case of seizure-like movements caused by residual sevoflurane inside the anesthesia machine. Therefore, we propose that the detection of residual anesthesia-inhaled drugs should be included in pre-anesthesia checkout procedures.An 11-year-old girl with a history of epilepsy was scheduled for emergency appendectomy under general anesthesia. The patient presented with seizure-like movements caused by residual sevoflurane inside the anesthesia machine after pre-oxygenation during rapid sequence induction.Based on the clinical presentation and previous history of seizures, sevoflurane-induced seizures were diagnosed.A washout procedure was performed by turning the oxygen flow up to 10L/min to wash out the residual sevoflurane from the anesthesia machine.The seizures ceased spontaneously, and the vital signs of the patient were stable during the washout procedure. Rapid sequence anesthesia induction and total intravenous anesthesia maintenance were uneventful. Surgery was performed as planned, and there were no postoperative problems. The patient was discharged after 4 days without complications and was well on follow-up.The check-up procedure of residual anesthesia-inhaled drugs inside the anesthesia machine should be included in the checkout design guidelines, or else the washout procedure should be performed in the pre-anesthesia checkout procedures. In most situations, residual anesthesia-inhaled drugs inside anesthesia machines can be neglected, and the check-up of residual anesthesia-inhaled drugs is not recommended in the guidelines.,4 the residual inhaled drugs could reach a high concentration, which could induce clinical complications in special situations, such as patients with a history or family history of malignant hyperthermia (MH).Pre-anesthesia checkout procedures recommended by the Sub-Committee of the American Society of Anesthesiologists Committee on Equipment and Facilities help to detect the problems of the anesthesia machine preoperatively.,7 Here, we report a rare case of seizure-like movements caused by residual sevoflurane inside an anesthesia machine after pre-oxygenation during rapid sequence anesthesia induction in a girl with a family history of epilepsy. We propose that residual anesthesia-inhaled drugs in anesthesia machines could be included in the pre-anesthesia checklist, especially when patients have contraindications for the use of inhalation anesthesia, such as MH and epilepsy.Sevoflurane-induced seizures are most often caused by high concentrations of sevoflurane combined with alveolar hyperventilation during mask induction of anesthesia.This study was approved by the Institutional Review Board of the West China Hospital of Sichuan University. Consent was obtained from the patients’ parents prior to submission of this case for publication.22 increased from 95% in room air to 100%. After 4 minutes of pre-oxygenation, the patient presented with symmetrical seizure-like movements of the upper extremities. We found that the expiratory sevoflurane concentration was as high as 3% on the monitor. Therefore, we checked the breathing circuit, vaporizer, and carbon dioxide absorbent, and found no problems and the vaporizer was closed during the pre-oxygenation procedure. We confirmed that sevoflurane in the breathing circuit was residual sevoflurane inside the anesthesia machine, and the convulsions were caused by residual sevoflurane. We used a new face mask and a bag valve device with a bag reservoir instead of an anesthesia machine for oxygen supply. Immediately, we turn up the oxygen flow to 10L/min in order to wash out the residual sevoflurane. The convulsions ceased spontaneously without treatment. The patient had no obvious discomfort, and his vital signs were stable. When the expiratory sevoflurane concentration decreased to zero on the monitor, a new breathing circuit was used for anesthesia induction. Rapid sequence anesthesia induction and endotracheal intubation were performed smoothly. Considering the contraindication for the use of inhalation anesthesia, total intravenous anesthesia was used during the operation, and anesthesia was uneventful. Surgery was performed as planned, and there were no postoperative problems. The patient was discharged after 4 days without complications and was well on follow-up.An 11-year-old, 135 cm, 30-kg girl presented with pain in the right iliac region with fever and vomiting. Acute appendicitis was diagnosed on physical examination and abdominal ultrasound, and emergency appendectomy was scheduled. The patient had no previous experience of anesthesia, but she had a history of epilepsy. With oral valproate antiepileptic therapy, epilepsy symptoms have not occurred in recent years. Rapid sequence anesthesia induction and endotracheal intubation were selected according to the anesthesia standard for emergency surgery. The anesthesia machine was checked as a pre-anesthesia checklist by an anesthesiologist, and standard monitoring was applied when the patient arrived in the operating room. Pre-oxygenation was performed with a standard face mask attached to the circuit of the Datex-Ohmeda Smart Vent  at a fresh oxygen flow of 6L/min. No drugs were used during the pre-oxygenation process, and the SpO3,9 In these cases, the end-tidal concentration of sevoflurane ranged from 0.75% to 2.5%. Similar to our case, most previous reports of seizure-like activity during sevoflurane were characterized by symmetrical tonic-clonic motor activity, and no serious neurological adverse effects were found in the postoperative period. The mechanisms responsible for seizure-like activity following sevoflurane anesthesia remain to be defined. The action of sevoflurane at γ-aminobutyric acid-mediated receptors could induce interictal epileptiform discharges or seizures in children.–12 Gibert et al demonstrated that the occurrence of major epileptiform signs (MES) is highly dependent on the sevoflurane concentration. The minimal alveolar concentration (MAC) of sevoflurane causing MES, calculated in 100% oxygen, corresponded to 1.75 surgical MAC. When the residual sevoflurane concentration approached 1.75, surgical MAC during pre-oxygenation, the occurrence of MES seems to be highly probable.Despite the frequent use of sevoflurane in anesthesia without adverse reactions, there have been several reports of sevoflurane-induced seizures in patients anesthetized with sevoflurane. However, in special situations, the residual halogenated anesthetics could reach a high concentration, which could induce clinical complications. Therefore, we propose that the check-up of residual anesthesia-inhaled drugs inside the anesthesia machine could be included in the pre-anesthesia checklist.The monitoring of residual anesthesia-inhaled drugs inside the anesthesia machine is not included in the pre-anesthesia checkout procedures, only when the patients have contraindications for the use of inhalation anesthesia, such as those susceptible to MH. In patients susceptible to MH, residual halogenated anesthetics are required to wash out completely. However, in other situations, such as patients with epilepsy or a family history of epilepsy, the residual inhaled drugs were not asked to wash out completely. Recommendations of the washout time for preparing different anesthesia machines for MH-susceptible patients ranges from 15 to 70 minutes. Instructions of the washout time were at least 20 minutes with a fresh gas flow rate of more than 10 L/min.,17 So, we can do this wash out procedure during the pre-anesthesia checkout procedures routinely.In MH-susceptible patients, less than 5 parts per million (0.0005%) concentration of the anesthetic agent are known to be well tolerated.4Although clinical complications caused by residual anesthesia, inhaled drugs inside the anesthesia machine are rare, and no comprehensive clinical studies have been conducted to date. More attention should be focused on the pre-anesthesia checkout procedures of residual anesthesia-inhaled drugs, especially in patients who have contraindications for the use of inhalation anesthesia. We wish to propose that the check-up of residual anesthesia-inhaled drugs inside anesthesia machines should be included in the checkout design guidelines, and that the washout procedure should be routinely performed in the pre-anesthesia checkout procedures.Data curation: Jianqiao Zheng, Li Du.Supervision: Jianqiao Zheng, Lu Zhang.Writing – original draft: Jianqiao Zheng, Li Du.Writing – review & editing: Jianqiao Zheng, Lu Zhang.