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Background: Prognostic models combine several prognostic factors to provide an estimate of the likelihood (or risk) of future events in individual patients, conditional on their prognostic factor values. A fundamental part of evaluating prognostic models is undertaking studies to determine whether their predictive performance, such as calibration and discrimination, is reproduced across settings. Systematic reviews and meta-analyses of studies evaluating prognostic models’ performance are a necessary step for selection of models for clinical practice and for testing the underlying assumption that their use will improve outcomes, including patient's reassurance and optimal future planning. Methods: In this paper, we highlight key concepts in evaluating the certainty of evidence regarding the calibration of prognostic models. Results and Conclusion: Four concepts are key to evaluating the certainty of evidence on prognostic models’ performance regarding calibration. The first concept is that the inference regarding calibration may take one of two forms: deciding whether one is rating certainty that a model's performance is satisfactory or, instead, unsatisfactory, in either case defining the threshold for satisfactory (or unsatisfactory) model performance. Second, inconsistency is the critical GRADE domain to deciding whether we are rating certainty in the model performance being satisfactory or unsatisfactory. Third, depending on whether one is rating certainty in satisfactory or unsatisfactory performance, different patterns of inconsistency of results across studies will inform ratings of certainty of evidence. Fourth, exploring the distribution of point estimates of observed to expected ratio across individual studies, and its determinants, will bear on the need for and direction of future research. © 2021

Human blood is a self-regenerating lipid-rich biological fluid that is routinely collected in hospital settings. The inventory of lipid molecules found in blood plasma (plasma lipidome) offers insights into individual metabolism and physiology in health and disease. Disturbances in the plasma lipidome also occur in conditions that are not directly linked to lipid metabolism; therefore, plasma lipidomics based on MS is an emerging tool in an array of clinical diagnostics and disease management. However, challenges exist in the translation of such lipidomic data to clinical applications. These relate to the reproducibility, accuracy, and precision of lipid quantitation, study design, sample handling, and data sharing. This position paper emerged from a workshop that initiated a community-led process to elaborate and define a set of generally accepted guidelines for quantitative MS-based lipidomics of blood plasma or serum, with harmonization of data acquired on different instrumentation platforms across independent laboratories as an ultimate goal. We hope that other fields may benefit from and follow such a precedent.

Copyright © 2016 Elsevier Inc. All rights reserved. Objectives: Intervention Modeling Experiments (IMEs) are a way of developing and testing behavior change interventions before a trial. We aimed to test this methodology in a Web-based IME that replicated the trial component of an earlier, paper-based IME. Study Design and Setting: Three-arm, Web-based randomized evaluation of two interventions (persuasive communication and action plan) and a "no intervention" comparator. The interventions were designed to reduce the number of antibiotic prescriptions in the management of uncomplicated upper respiratory tract infection. General practitioners (GPs) were invited to complete an online questionnaire and eight clinical scenarios where an antibiotic might be considered. Results: One hundred twenty-nine GPs completed the questionnaire. GPs receiving the persuasive communication did not prescribe an antibiotic in 0.70 more scenarios (95% confidence interval [CI] = 0.17-1.24) than those in the control arm. For the action plan, GPs did not prescribe an antibiotic in 0.63 (95% CI = 0.11-1.15) more scenarios than those in the control arm. Unlike the earlier IME, behavioral intention was unaffected by the interventions; this may be due to a smaller sample size than intended. Conclusions: A Web-based IME largely replicated the findings of an earlier paper-based study, providing some grounds for confidence in the IME methodology. Postprint

In this article, a critique of cancer nursing literature on the issue of sexuality is presented, with particular reference to literature on cancers common to women. The paper begins with an account of two competing perspectives on sexuality. The first is a version of sexuality rooted in sexology, underpinned by biomedical science that makes a claim to having identified 'normal' sexuality. The second is a version of sexuality developed within feminist scholarship that tends to reject biological determinism as a basis for understanding sexuality, instead favouring constructionist perspectives, with the socio-political context of sexual relations problematised. The focus of the article then shifts to cancer nursing literature on sexuality that deals primarily with cancers common to women, to appraise the extent to which either of the above perspectives on sexuality is invoked. Within this body of nursing knowledge, I argue that there has largely been an uncritical endorsement of biomedical constructions of sexuality, rooted in orthodox sexology, with a dominant focus on sexual functioning and on sexual rehabilitation for women with cancer. Moreover, in this knowledge base, phallocentric heterosexuality over and above other forms of sexual expression is privileged, and the socio-political context of unequal gender power relations is largely excluded. References to the social sphere as a dimension of nursing care are focused almost exclusively on maintaining normality, and reflect the emphasis on functional restoration. The largely individualistic, uncritical and biocentric emphasis in this literature may serve inadvertently to reinforce and maintain existing gender inequalities in heterosexual relationships. Finally, I consider the difficulties for oncology nurses in dealing with contradictory truth claims or conventional wisdoms about sexuality from the disparate disciplines of which holism is comprised. kpw13/3/13

Objectives: To determine the prevalence of congenital hypothyroidism in children with filter-paper blood-spot TSH (b-TSH) between 5 and 10 μIU/mL in the neonatal screening. Methods: This was a retrospective study including children screened from 2003 to 2010, with b-TSH levels between 5 and 10 μIU/mL, who were followed-up during the first two years of life when there was no serum TSH normalization. The diagnosis of congenital hypothyroidism was defined as serum TSH ≥10 μIU/mL and start of levothyroxine treatment up to 2 years of age. Results: Of the 380,741 live births, 3713 (1.04%) had filter paper TSH levels between 5 and 10 μIU/mL and, of these, 339 (9.13%) had congenital hypothyroidism. Of these, 76.11% of the cases were diagnosed in the first three months of life and 7.96% between 1 and 2 years of age. Conclusion: The study showed that 9.13% of the children with b-TSH levels between 5 and 10 μIU/mL developed hypothyroidism and that in approximately one-quarter of them, the diagnosis was confirmed only after the third month of life. Based on these findings, the authors suggest the use of a 5 μIU/mL cutoff for b-TSH levels and long-term follow-up of infants whose serum TSH has not normalized to rule out congenital hypothyroidism. Resumo: Objetivos: Determinar a prevalência de hipotireoidismo congênito em crianças com TSH em papel filtro (TSH-f) entre 5 e 10 μUI/mL na triagem neonatal. Métodos: Estudo retrospectivo incluindo crianças triadas de 2003 a 2010, com TSH-f entre 5 e 10 μUI/mL, que foram acompanhadas nos dois primeiros anos de vida quando não houve normalização do TSH sérico. O diagnóstico de hipotireoidismo congênito foi definido como TSH sérico igual ou superior a 10 μUI/mL e início de tratamento com levotiroxina até os dois anos de idade. Resultados: Dos 380.741 nascidos vivos triados, 3.713 (1,04%) apresentaram TSH-f entre 5 e 10 μUI/mL e, destes, 339 (9,13%) tinham hipotireoidismo congênito. Destes, 76,11% dos casos foram diagnosticados nos primeiros três meses de vida e 7,96% entre um e dois anos de idade. Conclusão: O estudo mostra que 9,13% das crianças com TSH-f entre 5 e 10 μUI/mL desenvolveram hipotireoidismo e que em cerca de um quarto delas o diagnóstico só se confirmou após o terceiro mês de vida. Com base nestes achados, sugere-se a utilização do ponto de corte de TSH-f de 5 μUI/mL e o acompanhamento em longo prazo dos lactentes cujo TSH sérico não tenha se normalizado para descartar o hipotireoidismo congênito. Keywords: Congenital hypothyroidism, Neonatal screening, Infant, Newborn, Thyroid diseases, Palavras-chave: Hipotireoidismo congênito, Triagem neonatal, Lactente, Recém-nascido, Doenças da glândula tireoide

Objectives: To determine the seroprevalence rate of toxoplasmosis, HIV, syphilis and rubella in a population of puerperal women. Methods: A prospective, cross-sectional study was performed from February 2007 to April 2008 at Hospital Geral, Universidade de Caxias do Sul in a population of 1,510 puerperal women. Women that gave birth to live born or stillborn infants were included in the study; maternal and perinatal variables were analyzed. Descriptive statistics and Pearson's chi-square with occasional Fisher's correction were used for comparisons. Alpha was set in 5%. Results: A total of 148 cases of congenital infection (9.8%) were identified: 66 cases of syphilis (4.4%), 40 cases of HIV (2.7%), 27 cases of toxoplasmosis (1.8%) and 15 cases of rubella (1.0%). In ten cases there was co-infection (four cases of HIV and syphilis, two cases of HIV and rubella, one case of HIV and toxoplasmosis, two cases of rubella and syphilis, and one case of toxoplasmosis and rubella). In a comparison between puerperal women with and without infection there was no statistical significance in relation to incidence of abortions, small for gestational age, prematurity, live births and stillbirths, and prenatal care. Need of neonatal intensive care unit (NICU), maternal schooling, maternal age higher than 35 years and drug use (alcohol, cocaine and crack) had statistical significance. Conclusion: The prevalence rate of infections was 9.8%. Need of NICU, maternal schooling lower than eight years, maternal age higher than 35 years and drug use were significantly associated with occurrence of congenital infection. Keywords: perinatal infections, congenital infections, clinical diagnosis, seroprevalence

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. As an important technique for the detection of fingermarks on porous surfaces, 1,2-indanedione is widely used due to its excellent detection performance. In order to optimize the effectiveness of 1,2-indanedione, several institutions have modified the original formulation. In this study, four different 1,2-indanedione formulations were used to treat fingermarks deposited on different porous substrates to determine the most suitable formulation and whether Solstice-PF can be an alternative carrier solvent for the currently used HFE7100. It was found that the Solstice-PF-based formulation performed similarly to the HFE7100-based formulation on copy paper, but when treating fingermarks deposited on brown paper and newspaper, Solstice-PF was superior to HFE7100 by developing up to 10% more marks graded 3 and 4 regardless of the ageing period. The results confirm that Solstice-PF can be used as an alternative carrier solvent for 1,2-indanedione formulations with good detection rates and lower costs.

Purpose This study aims to develop a multi-gene assay predictive of the clinical benefits of chemotherapy in non-small cell lung cancer (NSCLC) patients, and substantiate their protein expression as potential therapeutic targets. Patients and methods The mRNA expression of 160 genes identified from microarray was analyzed in qRT-PCR assays of independent 337 snap-frozen NSCLC tumors to develop a predictive signature. A clinical trial JBR.10 was included in the validation. Hazard ratio was used to select genes, and decision-trees were used to construct the predictive model. Protein expression was quantified with AQUA in 500 FFPE NSCLC samples. Results A 7-gene signature was identified from training cohort (n = 83) with accurate patient stratification (P = 0.0043) and was validated in independent patient cohorts (n = 248, P, Highlights • This paper develops a RT-PCR based 7-gene assay to predict the clinical benefits of chemotherapy for precision oncology • This work discovers a prognostic protein biomarker ZNF71 using AQUA technique as a potential companion test and therapeutic target • This research identifies prognostic mRNA and protein biomarker CD27 with implications in immunotherapy The results from this paper will help clinicians to select NSCLC patients who might benefit from chemotherapy. The identified prognostic protein biomarkers might also be new therapeutic targets in chemotherapy and immunotherapy.

Background: The pressure of pursuing an athletic career simultaneously with education may set adolescent student-athletes at risk for sport and school burnout. Although the 2 life domains of student-athletes are strongly intertwined, so far, there has not been an instrument for investigating sport burnout parallel to school burnout. The aim of the present study was to introduce a sport burnout measure for adolescents in a dual career context and investigate its validity and reliability by using confirmatory factor analysis. Methods: The participants were 391 student-athletes (51% females) who filled in a questionnaire of sport burnout and background variables in the beginning of upper secondary school. Results: A 3-factor model or a second-order-factor model described the data better and gave better reliability indices than a 1-factor model. The 3 dimensions of sport burnout were shown to be separate, but closely related constructs. Evidence for convergent and discriminant validity was obtained by correlating the 3 sport burnout dimensions with depressive symptoms, self-esteem, and sport task values. Conclusion: The results suggest that Sport Burnout Inventory-Dual Career Form (SpBI-DC) is a valid and reliable instrument for investigating sport burnout among adolescent student-athletes.

Background The social environment might play an important role in explaining people's physical activity (PA) behavior. However, little is known regarding whether personal networks differ between physically active and physically inactive people. This study aimed to examine the relationship between personal network characteristics and adults' physical (in)activity. Methods An egocentric social network study was conducted in a random sample in Switzerland (n = 529, mean age of 53 years, 54% females). Individual and personal network measures were compared between regular exercisers and non-exercisers. The extent of these factors' association with PA levels was also examined. Results Non-exercisers (n = 183) had 70% non-exercising individuals in their personal networks, indicating homogeneity, whereas regular exercisers (n = 346) had 57% regularly exercising individuals in their networks, meaning more heterogeneous personal networks. Additionally, having more regular exercisers in personal networks was associated with higher PA levels, over and above individual factors. Respondents with an entirely active personal network reported, on average, 1 day of PA more per week than respondents who had a completely inactive personal network. Other personal network characteristics, such as network size or gender composition, were not associated with PA. Conclusion Non-exercisers seem to be clustered in inactive networks that provide fewer opportunities and resources, as well as less social support, for PA. To effectively promote PA, both individuals and personal networks need to be addressed, particularly the networks of inactive people (e.g., by promoting group activities)., Journal of Sport and Health Science, 9 (4), ISSN:2095-2546, ISSN:2213-2961

Background: Ovarian clear cell carcinoma (OCCC) is a histological subtype of epithelial ovarian cancer (EOC) with distinct pathological, biological, and molecular features. OCCCs are more resistant to conventional treatment regimen of EOC and have the worst stage-adjusted prognosis amongst EOC subtypes. As the OCCC incidence rate in Asian populations has significantly increased in recent decades, it is critical to elucidate its molecular features that could lead to OCCC-tailored therapeutic strategies. Methods: Gene expression profiles of 222 OCCC were analyzed by hierarchical clustering and statistical analyses. Findings: We identified two OCCC gene expression subtypes: EpiCC—epithelial-like, which is associated with early-stage disease, with a relatively higher rate of gene mutations in the SWI/SNF complex; and MesCC—mesenchymal-like, associated with late-stage and higher enrichment of immune-related pathway activity. Genetic, copy number and transcriptomic analyses showed that both EpiCC and MesCC carried OCCC-associated aberrations. The EpiCC/MesCC classification was reproducible in validation cohorts and OCCC cell lines. MesCC tumors had a poorer progression-free survival (PFS) than EpiCC tumors (HR: 3·0, p = 0·0006). Functional assays in cell lines showed that the MesCC subtype was more proliferative and more anoikis-resistant than the EpiCC. By applying the EpiCC/MesCC classification to the TCGA renal clear cell carcinoma cohort, our results indicated interoperability of the subtyping scheme, and revealed preferential drug response of MesCC to bevacizumab. Interpretation: The EpiCC/MesCC classification shows promise for prognostic and therapeutic stratification in OCCC patients and warrants further investigation in the context of OCCC gene expression subtype-tailored treatment strategies. Keywords: Ovarian cancer, Clear cell, Microarray gene expression, Molecular subtype

Background: Pediatric high-grade gliomas (pHGG) are the leading cause of cancer-related death during childhood. Due to their diffuse growth characteristics, chemoresistance and location behind the blood-brain barrier (BBB), the prognosis of pHGG has barely improved in the past decades. As such, there is a dire need for new therapies that circumvent those difficulties. Since aberrant expression of DNA damage-response associated Fanconi anemia proteins play a central role in the onset and therapy resistance of many cancers, we here investigated if FANCD2 depletion could sensitize pHGG to additional DNA damage. Methods: We determined the capacity of celastrol, a BBB-penetrable compound that degrades FANCD2, to sensitize glioma cells to the archetypical DNA-crosslinking agent carboplatin in vitro in seven patient-derived pHGG models. In addition, we tested this drug combination in vivo in a patient-derived orthotopic pHGG xenograft model. Underlying mechanisms to drug response were investigated using mRNA expression profiling, western blotting, immunofluorescence, FANCD2 knockdown and DNA fiber assays. Findings: FANCD2 is overexpressed in HGGs and depletion of FANCD2 by celastrol synergises with carboplatin to induce cytotoxicity. Combination therapy prolongs survival of pHGG-bearing mice over monotherapy and control groups in vivo (P

Background: The understanding of the regulation of glucagon secretion by pancreatic islet α-cells remains elusive. We aimed to develop an in vitro model for investigating the function of human α-cells under direct influence of glucose and other potential regulators. Methods: Highly purified human α-cells from islets of deceased donors were re-aggregated in the presence or absence of β-cells in culture, evaluated for glucagon secretion under various treatment conditions, and compared to that of intact human islets and non-sorted islet cell aggregates. Findings: The pure human α-cell aggregates maintained proper glucagon secretion capability at low concentrations of glucose, but failed to respond to changes in ambient glucose concentration. Addition of purified β-cells, but not the secreted factors from β-cells at low or high concentrations of glucose, partly restored the responsiveness of α-cells to glucose with regulated glucagon secretion. The EphA stimulator ephrinA5-fc failed to mimic the inhibitory effect of β-cells on glucagon secretion. Glibenclamide inhibited glucagon secretion from islets and the α- and β-mixed cell-aggregates, but not from the α-cell-only aggregates, at 2.0 mM glucose. Interpretation: This study validated the use of isolated and then re-aggregated human islet cells for investigating α-cell function and paracrine regulation, and demonstrated the importance of cell-to-cell contact between α- and β-cells on glucagon secretion. Loss of proper β- and α-cell physical interaction in islets likely contributes to the dysregulated glucagon secretion in diabetic patients. Re-aggregated select combinations of human islet cells provide unique platforms for studying islet cell function and regulation. Keywords: Human alpha cells glucagon, Insulin inhibition, Somatostatin, Glibenclamide, Paracrine regulation, Cell-to-cell contact

Background: Despite toxic side effects and limited durable response, the current standard-of-care treatment for high grade serous ovarian cancer (HGSOC) remains platinum/taxane-based chemotherapy. Given that the overall prognosis has not improved drastically over the past several decades, there is a critical need to understand the underlying mechanisms that lead to tumour development and progression. Methods: We utilized an integrative proteogenomic analysis of HGSOC tumours applying a poor prognosis gene expression signature (PPS) as a conceptual framework to analyse orthogonal genomic and proteomic data from the TCGA (n = 488) and CPTAC (n = 169) studies. Genes identified through in silico analyses were assessed in vitro studies to demonstrate their impact on proliferation and cell cycle progression. Findings: These analyses identified DNA amplification and overexpression of the transcription factor ADNP (Activity Dependent Neuroprotector Homeobox) in poorly prognostic tumours. Validation studies confirmed the prognostic capacity of ADNP and suggested an oncogenic role for this protein given the association between ADNP expression and pro-proliferative signalling. In vitro studies confirmed ADNP as a novel and essential mediator of cell proliferation through dysregulation of cell cycle checkpoints. Interpretation: We identified ADNP as being amplified and overexpressed in poor prognosis HGSOC in silico analyses and demonstrated that ADNP is a novel and essential oncogene in HGSOC which mediates proliferation through dysregulation of cell cycle checkpoints in vitro. Funding: The National Cancer Institute of the National Institutes of Health, the V Foundation for Cancer Research and the New Jersey Commission for Cancer Research. Keywords: High-grade serous ovarian cancer, HGSOC, Proteogenomic, ADNP, Poor prognostic marker

Background: Active immunotherapy is an effective, long-lasting, cheap and safe way to suppress the progression of cancer, however, the key issue is to find the appropriate tumor vaccines. Oncoproteins are up-regulated under various stresses, such as radiation, chemodrugs, targeted drugs, hypoxia, malnutrition and so on to promote the cell growth, suppress apoptosis and alter the micro-environment for survival locally and remotely. Oncoproteins and their immunogenic domain could well serve as tumor vaccines to prime the host active anti-tumor immunity, which might enhance the immune (such as anti-PD-1) modulators given exogenously on effective stage. Methods: Proteomics and bio-information analysis were performed to identify potential tumor associated antigens (TAAs) induced by 8 Gy irradiation (IR). Then, peptides derived from CD151 were designed and synthesized according to MHC I binding and immunogenicity. Cell killing assay, flow cytometer, immunohistochemistry staining, and in vivo bioluminescence imaging were applied to assessed active anti-tumor immunity triggered by CD151 peptides in H22 hepatoma primary tumor and experimental 4T1 breast cancer lung metastasis model. Results: We utilized 8 Gy irradiation as therapeutic stress to up-regulate a panel of oncoproteins, and identified CD151 as a TAA according to proteomics and bio-information analysis. Two CD151 peptides were synthesized as "tumor vaccine" to immunize the mice, which triggered active anti-cancer immunity against both the primary growth of H22 hepatoma and the lung metastasis of 4T1 breast cancer in two mouse model via activation of CD8+IFNγ+ lymphocytes and their targeted cytotoxicity as well as reduction of negative regulator of MDSC cells. The survival of mice with lung metastases in CD151 peptide immunized group was also prolonged. Conclusions: The stress-upregulated oncoproteins defined by proteomics and bio-information on 8 Gy irradiated tumor cells are the good candidates for designing immunogenic peptides as tumor vaccines. Anti-tumor active immunity primed by peptides from tetraspanin oncoprotein CD151 could exert effective, long-lasting, cheap and safe way to suppress the progression of cancer. Funding: The project was funded in part by the grants of Fujian Province Natural Science Foundation (2017J01260); the Key Clinical Specialty Discipline Construction Program of Fujian; the National Clinical Key Specialty Construction Program; Fujian Engineering Research Center of Cancer precision Diagnosis and Immunotherapy; Science&Technology Program of Fujian Province, China (#2018Y2003). Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: Animal experiments were approved by Fujian Medical University Institutional Animal Ethical Committee (FJMU IACUC #2018-075). The applicable institutional guidelines for the care and use of animals were followed.

Background: Although various molecular subtypes of bladder cancer (BC) have been investigated, most of these studies have focused on muscle-invasive BC (MIBC). A few studies have investigated non-muscle-invasive BC (NMIBC) or NMIBC and MIBC together, but none has classified progressive NMIBC or immune checkpoint inhibitor (ICI)-based therapeutic responses in early-stage BC patients. Methods: A total of 1,934 samples from seven patient cohorts were used. We performed unsupervised hierarchical clustering to stratify patients into distinct subgroups and constructed a classifier by applying SAM/PAM algorithms. We then investigated the association between molecular subtypes and immunotherapy responsiveness using various statistical methods. Findings: We explored large-scale genomic datasets encompassing NMIBC and MIBC, redefining four distinct molecular subtypes, including a subgroup containing progressive NMIBC and MIBC with poor prognosis that would benefit from ICI treatment. This subgroup showed poor progression-free survival with the distinct features of high mutation load, activated cell cycle, and inhibited TGFβ signalling. Importantly, we verified that BC patients with this subtype were significantly responsive to an anti-PD-L1 agent in the IMvigor210 cohort. Interpretation: Our results reveal an immunotherapeutic option for ICI treatment of highly progressive NMIBC and MIBC with poor prognosis. Funding: This research was supported by the National Research Foundation of Korea grant funded by the Korean government, a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health and Welfare, Republic of Korea, and a grant from the KRIBB Research Initiative Program. Keywords: Bladder cancer, Disease progression, Subtype, Immunotherapy, Genomic signature

Summary: Background: Bronchiolitis obliterans syndrome (BOS) after allo-HSCT is a devastating complication with limited therapeutic options. We aimed to assess the efficacy and safety of mesenchymal stem cells (MSCs) in BOS after allo-HSCT. Methods: This multicenter prospective cohort study enrolled 81 allo-HSCT recipients whose BOS were diagnosed within 6 months. The choice of prednisone and azithromycin combined with or without MSCs was based on patient preferences (MSC n = 49, non-MSC n = 32). The primary endpoint was response rate at 3 months, defined as the proportion of patients achieving FEV1 improvement or steroid sparing. The trial was registered at ClinicalTrials.gov (NCT02543073). Findings: Response rate was 35/49 patients (71%, 95% CI 59 to 84%) and 14/32 (44%, 27 to 61%) in MSC and non-MSC group, respectively (p = 0.013). The addition of MSCs was associated with a better difference for change in FEV1 rate of decline, compared to non-MSC group (53 mL/months, 2 to 103; p = 0.040). The 3-year overall survival post-diagnosis was 70.6% (55.9 to 85.3%) and 58.2% (36.1 to 78.5%) in MSC and non-MSC group, respectively (p = 0.21). Clinical improvement was accompanied by a significant increase of interleukin (IL)-10-producing CD5+B cells. There was no statistical difference in the rates of infections and leukemia relapse between the two groups. MSCs were well-tolerated with no serious adverse events. Interpretation: MSCs offer an effective and safe therapeutic option for BOS after allo-HSCT. Our study strengthens evidence for clinical use of MSC therapy in BOS. These data also provide novel insight into potential biological mechanisms of MSC treatment and support further investigation in larger randomized controlled trials. Funding: National Key R&D Program of China, National Natural Science Foundation of China, Health Collaborative Innovation Major Projects of Guangzhou City, Science and Technology Planning Project of Guangdong Province. Keywords: Allogeneic hematopoietic stem cell transplantation, Chronic graft-versus host disease, Bronchiolitis obliterans syndrome, Mesenchymal stem cells

Background Cirrhosis-associated immune dysfunction (CAID) contributes to high sepsis risk in patients with chronic liver disease. Various innate and; to a lesser extent; adaptive immune dysfunctions have been described as contributors to CAID leading to immune-paresis and impaired anti-microbial response in cirrhosis. In this study, we examined the phenotype of CD8+ T cells in chronic liver disease with the aim to evaluate changes that might contribute to impaired immune responses. Methods Sixty patients with cirrhosis were prospectively recruited for this study. CD8+ T cells from peripheral blood, ascites and liver explants were characterized using flow cytometry and immunohistochemistry, respectively. The transcriptional signature of flow-sorted HLA-DR+CD8+ T cells was performed using Nanostring™ technology. HLA-DR+CD8+ T cells interactions with PBMCs and myeloid cells were tested in vitro. Findings Peripheral CD8+ T cells from cirrhotic patients displayed an altered phenotype characterized by high HLA-DR and TIM-3 surface expression associated with concomitant infections and disease severity, respectively. Paired peritoneal CD8+ T cells expressed more pronounced levels of HLA-DR and PD-1 compared to peripheral CD8+ T cells. HLA-DR+CD8+ T cells were enriched in cirrhotic livers compared to controls. TIM-3, CTLA-4 and PD-1 levels were highly expressed on HLA-DR+CD8+ T cells and co-expression of HLA-DR and PD1 was higher in patients with poor disease outcomes. Genes involved in cytokines production and intracellular signalling pathways were strongly down-regulated in HLA-DR+CD8+ T cells. In comparison to their HLA-DR− counterparts, HLA-DR+CD8+ T cells promoted less proliferation of PBMCs and induced phenotypic and functional dysfunctions in monocytes and neutrophils in vitro. Interpretation In patients with cirrhosis, CD8+ T cells display a phenotypic, functional and transcriptional profile which may contribute to CAID. Fund This work was supported by Medical Research Council, the Rosetrees Charitable Trust, Robert Tournut 2016 grant (Societe Nationale Francaise de GastroEnterologie), Gilead® sciences, and NIHR Imperial Biomedical Research Centre.

Background: Identifying populations at high risk of HIV transmission is critical for prioritizing treatment and prevention resources and achieving the UNAIDS 90-90-90 Targets. Methods: HIV transmission rates can be estimated from phylogenetic trees as viral lineage-level diversification rates. To identify HIV-1 transmission foci in British Columbia, Canada, we inferred diversification rates from phylogenetic trees of 36 271 HIV-1 sequences from 9630 anonymized individuals. Diversification rates were combined with sociodemographic and clinical data, then aggregated by patients’ area of residence to predict the distribution of new HIV cases between 2008 and 2018. The predictive power of the model was compared with a phylogenetically uninformed model. Findings: Aggregated diversification rate measures were predictive of new HIV cases in the subsequent year after adjusting for prevalent and incident cases in the previous year. For every one-unit increase in the mean of the top five diversification rates, the number of new HIV cases increased by on average 1·38-fold (95% CI, 1·28–1·49). In a blind prediction of 2018 cases, diversification rate improved the model's specificity by 12%, accuracy by 9%, top 20 agreement by 100%, and correlation of predicted and observed values by 162% relative to a model that incorporated epidemiological data alone. Interpretation: By predicting the distribution of future HIV cases, a combined phylogenetic and epidemiological approach identifies hotspots where public health resources are needed most. Funding: Canadian Institutes of Health Research, University of British Columbia, Public Health Agency of Canada, Genome Canada, Genome BC, Michael Smith Foundation for Health Research, and BC Centre for Excellence in HIV/AIDS. Keywords: HIV, Phylogenetics, Epidemiology, Transmission, Predictive model

Background: Non-ischemic dilated cardiomyopathy (NIDCM) responds variably to intramyocardial injection of mesenchymal stem cells (MSCs). We hypothesized that NIDCM genotype may influence responsiveness to MSC therapy and performed genotyping on all patients in the POSEIDON-DCM trial. Methods: POSEIDON-DCM patients (n = 34) underwent genetic sequence analysis and deletion/duplication testing. The results were classified as positive for pathological variants (PV+; n = 8), negative for any variants (V−; n = 6), or as variants of uncertain significance (VUS; n = 20). All outcomes of therapy were analysed for each category of genetic results. Findings: The 3 groups were indistinguishable at baseline with regard to ejection fraction (EF), demographics, medication use, or functional parameters. V− patients had an increase in EF at 12 months: +13.6% (IQR = +7.8%; +20.5%; p = 0.002), compared with VUS (+6.5%; IQR = +0.9%, +11.1%; p = 0.005) and PV+(−5.9%; IQR = −12.7%, +1.0; p = 0.2; p = 0.01 between groups). Six-minute walk distance improved in V- patients, but not in VUS and PV+. V− patients improved MLHFQ, compared to the other 2 groups, which did not improve over time. EPCCFUs increased by 9.7 ± 1.9 in V− (p = 0.009) compared to VUS and PV+ patients. V− patients had one-year survival (100%) compared with VUS (85%) and PV+ (40%; p = 0.015 log-rank). Similarly, MACE rates were lower in V− (0%) than PV+ (61.9%) or VUS (42.2%; p = 0.021 log-rank). Interpretation: Our findings support the concept that the genetic profile of NIDCM patients plays a role in responsiveness to MSC therapy, with V− patients more likely to benefit and the converse for PV+. This observation emphasizes the need for further genetic studies, because of important implications for the management of NIDCM syndromes. Keywords: Precision medicine, Variants of uncertain significance, Positive for pathologic/likely pathologic variant, Dilated cardiomyopathy

Background: End-induction minimal residual disease (MRD) is the strongest predictor of relapse in paediatric acute lymphoblastic leukaemia (ALL), but an understanding of the biological pathways underlying early treatment response remains elusive. We hypothesized that metabolomic profiling of diagnostic bone marrow plasma could provide insights into the underlying biology of early treatment response and inform treatment strategies for high-risk patients. Methods: We performed global metabolomic profiling of samples from discovery (N = 93) and replication (N = 62) cohorts treated at Texas Children's Hospital. Next, we tested the cytotoxicity of drugs targeting central carbon metabolism in cell lines and patient-derived xenograft (PDX) cells. Findings: Metabolite set enrichment analysis identified altered central carbon and amino acid metabolism in MRD-positive patients from both cohorts at a 5% false discovery rate. Metabolites from these pathways were used as inputs for unsupervised hierarchical clustering. Two distinct clusters were identified, which were independently associated with MRD after adjustment for immunophenotype, cytogenetics, and NCI risk group. Three nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, which reduce glycolytic/TCA cycle activities, demonstrated nanomolar-range cytotoxicity in B- and T-ALL cell lines and PDX cells. Interpretation: This study provides new insights into the role of central carbon metabolism in early treatment response and as a potential targetable pathway in high-risk disease. Funding: American Society of Hematology; Baylor College of Medicine Department of Paediatrics; Cancer Prevention and Research Institute of Texas; the Lynch family; St. Baldrick's Foundation with support from the Micaela's Army Foundation; United States National Institutes of Health. Keywords: Acute lymphoblastic leukaemia, Minimal residual disease, Epidemiology, NAMPT, NAMPT inhibitors

Background: To validate a novel method for post-transplant surveillance to detect kidney allograft rejection via a characteristic constellation of the urine metabolites alanine, citrate, lactate, and urea investigated by nuclear magnetic resonance (NMR) spectroscopy a first prospective, observational study was performed. Methods: Within the UMBRELLA study 986 urine specimens were collected from 109 consecutively enrolled renal transplant recipients, and metabolite constellations were analyzed. A metabolite rejection score was calculated and compared to histopathological results of corresponding indication and protocol allograft biopsies (n = 206). Findings: The metabolite constellation was found to be a useful biomarker to non-invasively detect acute allograft rejection (AUC = 0.75; 95% confidence interval (CI) 0.68–0.83; based on 46 cases and 520 control samples). Combined analysis of the metabolite rejection score and the estimated glomerular filtration rate (eGFR) at the time of urine sampling further improved the overall test performance significantly (AUC = 0.84; 95% CI 0.76–0.91; based on 42 cases and 468 controls). Regarding the time course analysis in patients without rejection episodes the test results remained well below a diagnostic threshold associated with high risk of acute rejection. In other cases, a marked increase above this threshold indicated acute allograft rejection already six to ten days before diagnostic renal biopsies were performed. Interpretation: A combination of an NMR-based urine metabolite analysis and eGFR is promising as a non-invasive test for post-transplant surveillance and to support decision making whether renal allografts need histopathological evaluation. Keywords: Kidney transplant rejection, Urinary metabolites, Biomarker, NMR-spectroscopy, Non-invasive test

Background: Distinguishing bacterial and viral respiratory infections is challenging. Novel diagnostics based on differential host gene expression patterns are promising but have not been translated to a clinical platform nor extensively tested. Here, we validate a microarray-derived host response signature and explore performance in microbiology-negative and coinfection cases. Methods: Subjects with acute respiratory illness were enrolled in participating emergency departments. Reference standard was an adjudicated diagnosis of bacterial infection, viral infection, both, or neither. An 87-transcript signature for distinguishing bacterial, viral, and noninfectious illness was measured from peripheral blood using RT-PCR. Performance characteristics were evaluated in subjects with confirmed bacterial, viral, or noninfectious illness. Subjects with bacterial-viral coinfection and microbiologically-negative suspected bacterial infection were also evaluated. Performance was compared to procalcitonin. Findings: 151 subjects with microbiologically confirmed, single-etiology illness were tested, yielding AUROCs 0•85–0•89 for bacterial, viral, and noninfectious illness. Accuracy was similar to procalcitonin (88% vs 83%, p = 0•23) for bacterial vs. non-bacterial infection. Whereas procalcitonin cannot distinguish viral from non-infectious illness, the RT-PCR test had 81% accuracy in making this determination. Bacterial-viral coinfection was subdivided. Among 19 subjects with bacterial superinfection, the RT-PCR test identified 95% as bacterial, compared to 68% with procalcitonin (p = 0•13). Among 12 subjects with bacterial infection superimposed on chronic viral infection, the RT-PCR test identified 83% as bacterial, identical to procalcitonin. 39 subjects had suspected bacterial infection; the RT-PCR test identified bacterial infection more frequently than procalcitonin (82% vs 64%, p = 0•02). Interpretation: The RT-PCR test offered similar diagnostic performance to procalcitonin in some subgroups but offered better discrimination in others such as viral vs. non-infectious illness and bacterial/viral coinfection. Gene expression-based tests could impact decision-making for acute respiratory illness as well as a growing number of other infectious and non-infectious diseases. Keywords: Biomarkers, Gene expression, Respiratory tract infections, Coinfection, Diagnosis, Precision medicine

Summary: Background: Malignant gliomas are rapidly progressive brain tumors with high mortality. Fluorescence guided surgery (FGS) with 5-aminolevulinic acid (5-ALA) provides fluorescent delineation of malignant tissue, which helps achieve maximum safe resection. 5-ALA-based fluorescence is due to preferential accumulation of the fluorophore protoporphyrin-IX (PpIX) in malignant glioma tissue. Additionally, gliomas cells release extracellular vesicles (EVs) which carry biomarkers of disease. Herein, we performed animal and human studies to investigate whether 5-ALA dosed glioma cells, in vitro and in vivo, release PpIX positive EVs in circulation which can be captured and analyzed. Methods: We used imaging flow cytometry (IFC) to characterize PpIX-positive EVs released from 5-ALA-dosed glioma cells, glioma-bearing xenograft models, as well as patients with malignant glioma undergoing FGS. Findings: We first show that glioma cells dosed with 5-ALA release 247-fold higher PpIX positive EVs compared to mock dosed glioma cells. Second, we demonstrate that the plasma of glioma-bearing mice (n = 2) dosed with 5-ALA contain significantly higher levels of circulating PpIX-positive EVs than their pre-dosing background (p = 0.004). Lastly, we also show that the plasma of patients with avidly fluorescent tumors (n = 4) undergoing FGS contain circulating PpIX-positive EVs at levels significantly higher than their pre-dosing background (p = 0.00009) and this rise in signal correlates with enhancing tumor volumes (r 2 = 0.888). Interpretation: Our findings highlight the potential of plasma-derived PpIX-positive EV-based diagnostics for malignant gliomas, offering a novel liquid biopsy platform for confirming and monitoring tumor status. Keywords: Extracellular vesicles, Malignant gliomas, Liquid biopsy, Imaging flow cytometry, 5-ALA, Fluorescence guided surgery

Background: Despite treatment advances, there remains a significant risk of recurrence in ovarian cancer, at which stage it is usually incurable. Consequently, there is a clear need for improved patient stratification. However, at present clinical prognosticators remain largely unchanged due to the lack of reproducible methods to identify high-risk patients. Methods: In high-grade serous ovarian cancer patients with advanced disease, we spatially define a tumour ecological balance of stromal resource and immune hazard using high-throughput image and spatial analysis of routine histology slides. On this basis an EcoScore is developed to classify tumours by a shift in this balance towards cancer-favouring or inhibiting conditions. Findings: The EcoScore provides prognostic value stronger than, and independent of, known risk factors. Crucially, the clinical relevance of mutational burden and genomic instability differ under different stromal resource conditions, suggesting that the selective advantage of these cancer hallmarks is dependent on the context of stromal spatial structure. Under a high resource condition defined by a high level of geographical intermixing of cancer and stromal cells, selection appears to be driven by point mutations; whereas, in low resource tumours featured with high hypoxia and low cancer-immune co-localization, selection is fuelled by aneuploidy. Interpretation: Our study offers empirical evidence that cancer fitness depends on tumour spatial constraints, and presents a biological basis for developing better assessments of tumour adaptive strategies in overcoming ecological constraints including immune surveillance and hypoxia. Keywords: Tumour ecology, Tumour spatial heterogeneity, Cancer evolution, Ovarian cancer, Histological image analysis

Background: Fundus autofluorescence is a non-invasive imaging technique in ophthalmology. Conventionally, short-wavelength autofluorescence (SW-AF) is used for detection of lipofuscin, a byproduct of the visual cycle which accumulates with age or disease in the retinal pigment epithelium (RPE). Furthermore, near-infrared autofluorescence (NIR-AF) is used as a marker for RPE and choroidal melanin, but contribution of lipofuscin to the NIR-AF signal is unclear. Methods: We employed fluorescence microscopy to investigate NIR-AF properties of melanosomes, lipofuscin and melanolipofuscin granules in histologic sections of wildtype and Abca4−/− mouse eyes, the latter having increased lipofuscin, as well as aged human donor eyes. Differentiation between these pigments was verified by analytical electron microscopy. To investigate the influence of oxidative and photic stress we used an in vitro model with isolated ocular melanosomes and an in vivo phototoxicity mouse model. Findings: We show that NIR-AF is not an intrinsic property of melanin, but rather increases with age and after photic or oxidative stress in mice and isolated melanosomes. Furthermore, when lipofuscin levels are high, lipofuscin granules also show NIR-AF, as confirmed by correlative fluorescence and electron microscopy in human tissue. However, lipofuscin in albino Abca4−/− mice lacks NIR-AF signals. Interpretation: We suggest that NIR-AF is derived from melanin degradation products that accumulate with time in lipofuscin granules. These findings can help to improve the interpretation of patient fundus autofluorescence data. Funding: This work was supported by Bundesministerium für Bildung und Forschung, Deutsche Forschungsgemeinschaft and Chinese Scholarship Council. Major instrumentation used in this work was supported by Deutsche Forschungsgemeinschaft, the European Fund for Regional Development and the state of Baden-Württemberg. Keywords: Melanin, Lipofuscin, Melanolipofuscin, Oxidative stress, Aging

Background: Both idiopathic and familial Parkinson's disease are associated with mitochondrial dysfunction. Mitochondria have their own mitochondrial DNA (mtDNA) and previous studies have reported that the release of mtDNA is a biomarker of Parkinson's disease. Methods: We have now investigated the relationship between mtDNA replication, transcription and release in fibroblasts from patients with idiopathic (iPD) and Leucine-rich repeat kinase 2 -associated Parkinson's disease (LRRK2-PD), using Selfie-digital PCR, a method that allows absolute quantification of mtDNA genomes and transcripts. Findings: In comparison with healthy controls, we found that fibroblasts from patients with iPD or LRRK2-PD had a high amount of mitochondrial 7S DNA along with a low mtDNA replication rate that was associated with a reduction of cf-mtDNA release. Accumulation of 7S DNA in iPD and LRRK2-PD fibroblasts was related with an increase in H-strand mtDNA transcription. Interpretation: These results show that 7S DNA accumulation, low mtDNA replication, high H-strand transcription, and low mtDNA release compose a pattern of mtDNA dysfunction shared by both iPD and LRRK2-PD fibroblasts. Moreover, these results suggest that the deregulation of the genetic switch formed by 7SDNA that alternates between mtDNA replication and transcription is a fundamental pathophysiological mechanism in both idiopathic and monogenic Parkinson's disease., This work was supported by grants to Ramon Trullas and Petar Podlesniy from the Ministerio de Ciencia, Innovación y Universidades of Spain (Grant: SAF2017-89791-R) and from the Instituto Carlos III, Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas, CIBERNED (Grant: PI2016/06-3).

Background: Gene expression profiling (GEP), next-generation sequencing (NGS) and copy number variation (CNV) analysis have led to an increasingly detailed characterization of the genomic profiles of DLBCL. The aim of this study was to perform a fully integrated analysis of mutational, genomic, and expression profiles to refine DLBCL subtypes. A comparison of our model with two recently published integrative DLBCL classifiers was carried out, in order to best reflect the current state of genomic subtypes. Methods: 223 patients with de novo DLBCL from the prospective, multicenter and randomized LNH-03B LYSA clinical trials were included. GEP data was obtained using Affymetrix GeneChip arrays, mutational profiles were established by Lymphopanel NGS targeting 34 key genes, CNV analysis was obtained by array CGH, and FISH and IHC were performed. Unsupervised independent component analysis (ICA) was applied to GEP data and integrated analysis of multi-level molecular data associated with each component (gene signature) was performed. Findings: ICA identified 38 components reflecting transcriptomic variability across our DLBCL cohort. Many of the components were closely related to well-known DLBCL features such as cell-of-origin, stromal and MYC signatures. A component linked to gain of 19q13 locus, among other genomic alterations, was significantly correlated with poor OS and PFS. Through this integrated analysis, a high degree of heterogeneity was highlighted among previously described DLBCL subtypes. Interpretation: The results of this integrated analysis enable a global and multi-level view of DLBCL, as well as improve our understanding of DLBCL subgroups. Keywords: Diffuse large B-cell lymphoma, Independent component analysis, Transcriptomic variability, Gene signatures, prognosis

Background: The immune checkpoint, indoleamine 2,3-dioxygenase 1, is under investigation as target of novel immunotherapies for cancers, including head and neck squamous cell carcinomas (HNSCC). The aim of our study was to analyze DNA methylation of the encoding gene (IDO1) in HNSCC. Methods: Methylation of three CpG sites within the promoter, promoter flank, and gene body was investigated and correlated with mRNA expression, immune cell infiltration, mutational burden, human papillomavirus (HPV)-status, and overall survival in a cohort of N = 528 HNSCC patients obtained from The Cancer Genome Atlas. In addition, IDO1 immunohistochemistry and DNA methylation analysis was performed in an independent cohort of N = 138 HNSCC samples. Findings: Significant inverse correlations of IDO1 methylation and IDO1 mRNA expression were found in the promoter and promoter flank region (Spearman's ρ = −0.163 and ρ = −0.377, respectively) while a positive correlation was present in the gene body (ρ = 0.502; all P

Background: FOXO3a has been widely regarded as a tumor suppressor. It also plays a paradoxical role in regulating the cancer stem cells (CSCs), responsible for tumor-initiation, chemo-resistance, and recurrence in various solid tumors, including oral squamous cell carcinoma (OSCC). This study aims to uncover the role of FOXO3a and its importance for a non-canonical pathway of TGFβ in regulating the OSCC stemness. Methods: We identified FOXO3a expression in OSCC tissues and cell lines using immunohistochemistry and western blot. The correlation between FOXO3a and stemness was evaluated. Stable cell lines with differential expression of FOXO3a were constructed using lentiviruses. The effects of FOXO3a on stem-cell like properties in OSCC was further evaluated in vitro and in vivo. We also explored the effect of TGFβ on FOXO3a with respect to its expression and function. Findings: Our findings suggest that FOXO3a was widely expressed and negatively correlated with the stemness in OSCC. This regulation can be abolished by TGFβ through phosphorylation, nuclear exclusion, and degradation in the non-Smad pathway. We also observed that non-Smad AKT-FOXO3a axis is essential to regulate stemness of CSCs by TGFβ. Interpretation: TGFβ induces stemness through non-canonical AKT-FOXO3a axis in OSCC. Our study provides a foundation to understand the mechanism of CSCs and a possible therapeutic target to eliminate CSCs. Keywords: FOXO3a, TGFβ, Oral squamous cell carcinoma, Stem-cell like properties

Background The heterogeneity in symptomatology and phenotypic profile attributable to COVID-19 is widely unknown. The objective of this manuscript is to conduct a trans-ancestry genome wide association study (GWAS) meta-analysis of COVID-19 severity to improve the understanding of potentially causal targets for SARS-CoV-2. Methods This cross-sectional study recruited 646 participants in the UAE that were divided into two phenotypic groups based on the severity of COVID-19 phenotypes, hospitalized (n=482) and non-hospitalized (n=164) participants. Hospitalized participants were COVID-19 patients that developed acute respiratory distress syndrome (ARDS), pneumonia or progression to respiratory failure that required supplemental oxygen therapy or mechanical ventilation support or had severe complications such as septic shock or multi-organ failure. We conducted a trans-ancestry meta-analysis GWAS of European (n=302), American (n=102), South Asian (n=99), and East Asian (n=107) ancestry populations. We also carried out comprehensive post-GWAS analysis, including enrichment of SNP associations in tissues and cell-types, expression quantitative trait loci and differential expression analysis. Findings Eight genes demonstrated a strong association signal: VWA8 gene in locus 13p14·11 (SNP rs10507497; p=9·54 x10-7), PDE8B gene in locus 5q13·3 (SNP rs7715119; p=2·19 x10-6), CTSC gene in locus 11q14·2 (rs72953026; p=2·38 x10-6), THSD7B gene in locus 2q22·1 (rs7605851; p=3·07x10-6), STK39 gene in locus 2q24·3 (rs7595310; p=4·55 x10-6), FBXO34 gene in locus 14q22·3 (rs10140801; p=8·26 x10-6), RPL6P27 gene in locus 18p11·31 (rs11659676; p=8·88 x10-6), and METTL21C gene in locus 13q33·1 (rs599976; p=8·95 x10-6). The genes are expressed in the lung, associated to tumour progression, emphysema, airway obstruction, and surface tension within the lung, as well as an association to T-cell-mediated inflammation and the production of inflammatory cytokines. Interpretation We have discovered eight highly plausible genetic association with hospitalized cases in COVID-19. Further studies must be conducted on worldwide population genetics to facilitate the development of population specific therapeutics to mitigate this worldwide challenge. Funding This review was commissioned as part of a project to study the host cell receptors of coronaviruses funded by Khalifa University's CPRA grant (Reference number 2020-004).

Background: Low-dose aspirin can cause gastric and duodenal ulceration, hereafter called peptic ulcer disease (PUD). Predisposition is thought to be related to clinical and genetic factors; our aim was to identify genetic risk factors associated with aspirin-induced PUD. Methods: Patients (n=1478) were recruited from 15 UK hospitals. Cases (n=505) were defined as patients with endoscopically confirmed PUD within 2 weeks of using aspirin and non-aspirin Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). They were compared to two control groups: patients with endoscopically confirmed PUD without any history of NSAID use within 3 months of diagnosis (n=495), and patients with no PUD on endoscopy (n=478). A genome-wide association study (GWAS) of aspirin-induced cases (n=247) was compared to 476 controls. The results were validated by replication in another 84 cases and 162 controls. Findings: The GWAS identified one variant, rs12678747 (p=1·65×10−7) located in the last intron of EYA1 on chromosome 8. The association was replicated in another sample of 84 PUD patients receiving aspirin (p=0·002). Meta-analysis of discovery and replication cohort data for rs12678747, yielded a genome-wide significant association (p=3·12×10−11; OR=2·03; 95% CI 1·65-2·50). Expression of EYA1 was lower at the gastric ulcer edge when compared with the antrum. Interpretation: Genetic variation in an intron of the EYA1 gene increases the risk of endoscopically confirmed aspirin-induced PUD. Reduced EYA1 expression in the upper gastrointestinal epithelium may modulate risk, but the functional basis of this association will need mechanistic evaluation. Funding: Department of Health Chair in Pharmacogenetics, MRC Centre for Drug Safety Science and the Barts Cardiovascular NIHR Biomedical Research Centre, British Heart Foundation (BHF)

Background Metabolic syndrome (MetS) is a cluster of multiple cardiometabolic risk factors that increase the risk of type 2 diabetes and cardiovascular diseases. Identifying novel biomarkers of MetS and their genetic associations could provide insights into the mechanisms of cardiometabolic diseases. Methods Potential MetS-associated metabolites were screened and internally validated by untargeted metabolomics analyses among 693 patients with MetS and 705 controls. External validation was conducted using two well-established targeted metabolomic methods among 149 patients with MetS and 253 controls. The genetic associations of metabolites were determined by linear regression using our previous genome-wide SNP data. Causal relationships were assessed using a one-sample Mendelian Randomization (MR) approach. Findings Nine metabolites were ultimately found to be associated with MetS or its components. Five metabolites, including LysoPC(14:0), LysoPC(15:0), propionyl carnitine, phenylalanine, and docosapentaenoic acid (DPA) were selected to construct a metabolite risk score (MRS), which was found to have a dose-response relationship with MetS and metabolic abnormalities. Moreover, MRS displayed a good ability to differentiate MetS and metabolic abnormalities. Three SNPs (rs11635491, rs7067822, and rs1952458) were associated with LysoPC(15:0). Two SNPs, rs1952458 and rs11635491 were found to be marginally correlated with several MetS components. MR analyses showed that a higher LysoPC(15:0) level was causally associated with the risk of overweight/obesity, dyslipidaemia, high uric acid, high insulin and high HOMA-IR. Interpretation We identified five metabolite biomarkers of MetS and three SNPs associated with LysoPC(15:0). MR analyses revealed that abnormal LysoPC metabolism may be causally linked the metabolic risk. Funding This work was supported by grants from the National Key Research and Development Program of China (2017YFC0907004).

Background Little is known about chronic cannabis smoking-associated oral microbiome and its effects on central nervous system (CNS) functions. Methods In the current study, we have analyzed the saliva microbiome in individuals who chronically smoked cannabis with cannabis use disorder (n = 16) and in non-smoking controls (n = 27). The saliva microbiome was analyzed using microbial 16S rRNA sequencing. To investigate the function of cannabis use-associated oral microbiome, mice were orally inoculated with live Actinomyces meyeri, Actinomyces odontolyticus, or Neisseria elongata twice per week for six months, which mimicked human conditions. Findings We found that cannabis smoking in humans was associated with oral microbial dysbiosis. The most increased oral bacteria were Streptococcus and Actinomyces genus and the most decreased bacteria were Neisseria genus in chronic cannabis smokers compared to those in non-smokers. Among the distinct species bacteria in cannabis smokers, the enrichment of Actinomyces meyeri was inversely associated with the age of first cannabis smoking. Strikingly, oral exposure of Actinomyces meyeri, an oral pathobiont, but not the other two control bacteria, decreased global activity, increased macrophage infiltration, and increased β-amyloid 42 protein production in the mouse brains. Interpretation This is the first study to reveal that long-term oral cannabis exposure is associated oral enrichment of Actinomyces meyeri and its contributions to CNS abnormalities.

Background The dynamic monitoring of perioperative carcinoembryonic antigen (CEA) is recommended by current colorectal cancer (CRC) guidelines, while the benefits of additional measurements of carbohydrate antigen 19-9 (CA19-9) and carbohydrate antigen 125 (CA125) have remained controversial. Methods This retrospective longitudinal cohort included 3539 CRC patients who underwent curative resection. Distinct trajectory groups were identified by the latent class growth mixed model. Patients were grouped into subgroups jointly by CEA, CA19-9, and CA125 according to preoperative levels and longitudinal trajectories, respectively. The end points were overall survival (OS) and recurrence-free survival (RFS). Findings Three distinct trajectory groups were characterized for serum CEA, CA19-9, and CA125: low-stable, early-rising, and later-rising. Jointly, patients were grouped into six preoperative (trajectory) joint groups. Compared with the three-low group, the adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) associated with death were 1.87 (1.29-2.70), 3.82 (2.37-6.17), 1.87 (0.97-3.61), 2.81 (1.93-4.11), and 4.99 (2.80-8.86) for the CEA-high, CA19-9-high, CA125-high, two-high, and three-high group, respectively. And compared with the three-stable trajectory group, the corresponding HRs (95% CIs) were 1.59 (1.10-2.30), 1.55 (0.77-3.10), 6.25 (4.02-9.70), 4.05 (2.73-6.02), and 12.40 (5.77-26.70) for the five rising trajectory groups, respectively. Similar associations between joint groups and RFS were observed. Notably, the trajectory joint group still had prognostic significance after adjusting for preoperative levels. The CA19-9-high group (HR: 3.82, 95% CI: 2.37-6.17) was associated with higher risk of death than the two-high group (HR: 2.81, 95% CI: 1.93-4.11). Likewise, for the CA125-rising trajectory group and two-rising trajectory group, the HRs (95% CIs) were 6.13 (3.75-10.00) and 3.99 (2.63-6.05) for death, and 3.08 (2.07-4.58) and 2.10 (1.52-2.90) for recurrence. Interpretation In addition to CEA, the dynamic measurements of CA19-9 and CA125 are recommended to monitor the prognosis of CRC patients. Funding National Natural Science Foundation of China [81973147, 82001986, 81960592, 82073569, 81660545].

Background: Preoperative determination of breast cancer molecular subtypes facilitates individualized treatment plan-making and improves patient prognosis. We aimed to develop an assembled convolutional neural network (ACNN) model for the preoperative prediction of molecular subtypes using multimodal ultrasound (US) images. Methods: This multicentre study prospectively evaluated a dataset of greyscale US, colour Doppler flow imaging (CDFI), and shear-wave elastography (SWE) images in 807 patients with 818 breast cancers from November 2016 to February 2021. The St. Gallen molecular subtypes of breast cancer were confirmed by postoperative immunohistochemical examination. The monomodal ACNN model based on greyscale US images, the dual-modal ACNN model based on greyscale US and CDFI images, and the multimodal ACNN model based on greyscale US and CDFI as well as SWE images were constructed in the training cohort. The performances of three ACNN models in predicting four- and five-classification molecular subtypes and identifying triple negative from non-triple negative subtypes were assessed and compared. The performance of the multimodal ACNN was also compared with preoperative core needle biopsy (CNB). Finding: The performance of the multimodal ACNN model (macroaverage area under the curve [AUC]: 0.89–0.96) was superior to that of the dual-modal ACNN model (macroaverage AUC: 0.81–0.84) and the monomodal ACNN model (macroaverage AUC: 0.73–0.75) in predicting four-classification breast cancer molecular subtypes, which was also better than that of preoperative CNB (AUC: 0.89–0.99 vs. 0.67–0.82, p

Background: Atherosclerosis-related cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. Cholesterol crystals (CCs) induce inflammation in atherosclerosis and are associated with unstable plaques and poor prognosis, but no drug can remove CCs in the clinic currently. Methods: We generated a phospholipid-based and high-density lipoprotein (HDL)-like nanoparticle, miNano, and determined CC-dissolving capacity, cholesterol efflux property, and anti-inflammation effects of miNano in vitro. Both normal C57BL/6J and Apoe-deficient mice were used to explore the accumulation of miNano in atherosclerotic plaques. The efficacy and safety of miNano administration to treat atherosclerosis were evaluated in the Ldlr-deficient atherosclerosis model. The CC-dissolving capacity of miNano was also detected using human atherosclerotic plaques ex vivo. Findings: We found that miNano bound to and dissolved CCs efficiently in vitro, and miNano accumulated in atherosclerotic plaques, co-localized with CCs and macrophages in vivo. Administration of miNano inhibited atherosclerosis and improved plaque stability by reducing CCs and macrophages in Ldlr-deficient mice with favorable safety profiles. In macrophages, miNano prevented foam cell formation by enhancing cholesterol efflux and suppressed inflammatory responses via inhibiting TLR4-NF-κB pathway. Finally, in an ex vivo experiment, miNano effectively dissolved CCs in human aortic atherosclerotic plaques. Interpretation: Together, our work finds that phospholipid-based and HDL-like nanoparticle, miNano, has the potential to treat atherosclerosis by targeting CCs and stabilizing plaques. Funding: This work was supported by the National Institutes of Health HL134569, HL109916, HL136231, and HL137214 to Y.E.C, HL138139 to J.Z., R21NS111191 to A.S., by the American Heart Association 15SDG24470155, Grant Awards (U068144 from Bio-interfaces and G024404 from M-BRISC) at the University of Michigan to Y.G., by the American Heart Association 19PRE34400017 and Rackham Helen Wu award to M.Y., NIH T32 GM07767 to K. H., Barbour Fellowship to D.L.

International audience; Background: Familial Hypercholesterolemia (FH) is an underdiagnosed condition with an increased cardiovascular risk. It is unknown whether lipid accumulation plays a role in structural myocardial changes. Cardiovascular Magnetic Resonance (CMR) is the reference technique for the morpho-functional evaluation of heart chambers through cine sequences and for myocardial tissue characterization through late gadolinium enhancement (LGE) and T1 mapping images. We aimed to assess the prevalence of myocardial fibrosis in FH patients.Methods: Seventy-two asymptomatic subjects with genetically confirmed FH (mean age 49·24, range 40 to 60 years) were prospectively recruited along with 31 controls without dyslipidaemia matched for age, sex, BMI, and other cardiovascular risk factors. All underwent CMR including cine, LGE, pre- and post-contrast T1 mapping. Extracellular volume (ECV) and enhancement rate of the myocardium (ERM = difference between pre- and post-contrast myocardial T1, normalized by pre-contrast myocardial T1) were calculated.Findings: Five FH patients and none of the controls had intramyocardial LGE (p= 0·188). While no changes in Native T1 and ECV were found, post-contrast T1 was significantly lower (430·6 ± 55ms vs. 476·1 ± 43ms, p

s: Background: During pregnancy a feto-maternal exchange of cells through the placenta conducts to maternal microchimerism (Mc) in the child and fetal Mc in the mother. Because of this bidirectional traffic of cells, pregnant women have also acquired maternal cells in utero from their mother and could transfer grandmaternal (GdM) cells to their child through the maternal bloodstream during pregnancy. Thus, cord blood (CB) samples could theoretically carry GdMMc. Nevertheless this has never been demonstrated. Methods: Using Human Leukocyte Antigen (HLA)-specific quantitative PCR assays on three-generation families, we were able to test 28 CB samples from healthy primigravid women for GdMMc in whole blood (WB) and isolated cells (PBMC, T, B, granulocytes, stem cells). Findings: Five CB samples (18%) had GdMMc which could not be confounded with maternal source, with quantities 100 fold lower than maternal Mc in WB and PBMC. Risk of aneuploidies and/or related invasive prenatal procedures significantly correlated with the presence of GdMMc in CB (p=0.024). Significantly decreased HLA compatibility was observed in three-generation families from CB samples carrying GdMMc (p=0.019). Interpretation: Transgenerational transfer of cells could have implications in immunology and evolution. Further analyses will be necessary to evaluate whether GdMMc in CB is a passive or immunologically active transfer and whether invasive prenatal procedures could trigger GdMMc. Funding: Provence-Alpes-Côte d'Azur APEX grant # 2012_06549E, 2012_11786F and 2014_03978) and the Foundation for Medical Research (FRM Grant #ING20140129045).

Background: The survival time of amyotrophic lateral sclerosis (ALS) is greatly variable and protective or risk effects of the potential survival predictors are controversial. Thus, we aim to undertake a comprehensive meta-analysis of studies investigating non-genetic prognostic and survival factors in patients with ALS. Methods: A search of relevant literature from PubMed, Embase, Cochrane library and other citations from 1st January 1966 to 1st December 020 was conducted. Random-effects models were conducted to pool the multivariable or adjusted hazard ratios (HR) by Stata MP 16.0. PROSPERO registration number: CRD42021256923. Findings: A total of 5717 reports were identified, with 115 studies meeting pre-designed inclusion criteria involving 55,169 ALS patients. Five dimensions, including demographic, environmental or lifestyle, clinical manifestations, biochemical index, therapeutic factors or comorbidities were investigated. Twenty-five prediction factors, including twenty non-intervenable and five intervenable factors, were associated with ALS survival. Among them, NFL (HR:3.70, 6.80, in serum and CSF, respectively), FTD (HR:2.98), ALSFRS-R change (HR:2.37), respiratory subtype (HR:2.20), executive dysfunction (HR:2.10) and age of onset (HR:1.03) were superior predictors for poor prognosis, but pLMN or pUMN (HR:0.32), baseline ALSFRS-R score (HR:0.95), duration (HR:0.96), diagnostic delay (HR:0.97) were superior predictors for a good prognosis. Our results did not support the involvement of gender, education level, diabetes, hypertension, NIV, gastrostomy, and statins in ALS survival. Interpretation: Our study provided a comprehensive and quantitative index for assessing the prognosis for ALS patients, and the identified non-intervenable or intervenable factors will facilitate the development of treatment strategies for ALS. Funding: This study was supported by the National Natural Science Fund of China (Grant No. 81971188), the 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (Grant No. 2019HXFH046), and the Science and Technology Bureau Fund of Sichuan Province (No. 2019YFS0216).

Highlights • In a sample of recurrently depressed women, 12.8% had elevated ADHD symptoms. • 3.4% met ADHD diagnostic criteria; none had a diagnosis from a medical professional. • ADHD was associated with earlier-onset, more impairing and recurrent depression. • Recurrently depressed women with ADHD symptoms were more likely to be hospitalised. • ADHD symptoms were associated with taking non-first-line antidepressant medication., Background Depression is highly heterogeneous in its clinical presentation. Those with attention deficit/hyperactivity disorder (ADHD) may be at risk of a more chronic and impairing depression compared to those with depression alone according to studies of young people. However, no studies to date have examined ADHD in recurrently depressed adults in mid-life. Method In a sample of women in mid-life (n=148) taken from a UK based prospective cohort of adults with a history of recurrent depression, we investigated the prevalence of ADHD and the association of ADHD with clinical features of depression. Results 12.8% of the recurrently depressed women had elevated ADHD symptoms and 3.4% met DSM-5 diagnostic criteria for ADHD. None of the women reported having a diagnosis of ADHD from a medical professional. ADHD symptoms were associated with earlier age of depression onset, higher depression associated impairment, a greater recurrence of depressive episodes and increased persistence of subthreshold depression symptoms over the study period, higher levels of irritability and increased risk of self-harm or suicide attempt. ADHD symptoms were associated with increased risk of hospitalisation and receiving non-first-line antidepressant medication. Limitations ADHD was measured using a questionnaire measure. We focussed on mothers in a longitudinal study of recurrent depression, so the findings may not apply to males or other groups. Conclusions Higher ADHD symptoms appear to index a worse clinical presentation for depression. Clinical implications include that in women with early onset, impairing and recurrent depression, the possibility of underlying ADHD masked by depression needs to be considered.

Background White blood cells (WBC) are commonly measured to investigate suspected infection and inflammation in pregnant women, but the pregnancy-specific reference interval is variably reported, increasing diagnostic uncertainty in this high-risk population. It is essential that clinicians can interpret WBC results in the context of normal pregnant physiology, given the huge global burden of infection on maternal mortality. Methods We performed a longitudinal, repeated measures population study of 24,318 pregnant women in Oxford, UK, to map the trajectory of WBC between 8-40 weeks of gestation. We defined 95% reference intervals (RI) for total WBC, neutrophils, lymphocytes, eosinophils, basophils, and monocytes for the antenatal and postnatal periods. Findings WBC were measured 80,637 times over five years. The upper reference limit for total WBC was elevated by 36% in pregnancy (RI 5.7-15.0×109/L), driven by a 55% increase in neutrophils (3.7-11.6×109/L) and 38% increase in monocytes (0.3-1.1×109/L), which remained stable between 8-40 weeks. Lymphocytes were reduced by 36% (1.0-2.9×109/L), while eosinophils and basophils were unchanged. Total WBC was elevated significantly further from the first day after birth (similar regardless of the mode of delivery), which resolved to pre-delivery levels by an average of seven days, and to pre-pregnancy levels by day 21. Interpretation There are marked changes in WBC in pregnancy, with substantial differences between cell subtypes. WBC are measured frequently in pregnant women in obstetric and non-obstetric settings, and results should be interpreted using a pregnancy-specific RI until delivery, and between days 7-21 after childbirth. Funding None.

Background SARS-CoV-2 has been responsible for considerable mortality worldwide, owing in particular to pulmonary failures such as ARDS, but also to other visceral failures and secondary infections. Recent progress in the characterization of the immunological mechanisms that result in severe organ injury led to the emergence of two successive hypotheses simultaneously tested here: hyperinflammation with cytokine storm syndrome or dysregulation of protective immunity resulting in immunosuppression and unrestrained viral dissemination. Methods In a prospective observational monocentric study of 134 patients, we analysed a panel of plasma inflammatory and anti-inflammatory cytokines and measured monocyte dysregulation via their membrane expression of HLA-DR. We first compared the results of patients with moderate forms hospitalized in an infectious disease unit with those of patients with severe forms hospitalized in an intensive care unit. In the latter group of patients, we then analysed the differences between the surviving and non-surviving groups and between the groups with or without secondary infections. Findings Higher blood IL-6 levels, lower quantitative expression of HLA-DR on blood monocytes and higher IL-6/mHLA-DR ratios were statistically associated with the risk of severe forms of the disease and among the latter with death and the early onset of secondary infections. Interpretation The unique immunological profile in patients with severe COVID-19 corresponds to a moderate cytokine inflammation associated with severe monocyte dysregulation. Individuals with major CSS were rare in our cohort of hospitalized patients, especially since the use of corticosteroids, but formed a very severe subgroup of the disease. Funding None.

Objective and hypothesis Klotho is an aging-suppressor gene. Mutation of Klotho gene causes hyperphosphatemia and acute heart failure. However, the relationship of hyperphosphatemia and acute heart failure is unclear. We hypothesize that hyperphosphatemia mediates Klotho deficiency-induced acute heart failure and further that therapeutic reduction of hyperphosphatemia prevents acute heart failure in Klotho mutant (KL(−/−)) mice. Methods and results A significant elevation of serum phosphorus levels and a large reduction of heart function were found in KL(−/−) mice by six weeks of age. Normalization of serum phosphorus levels by low phosphate diet (LPD) rescued Klotho deficiency-induced heart failure and extended lifespan in male mice. Klotho deficiency impaired cardiac mitochondrial respiratory enzyme function and increased superoxide production, oxidative stress, and cardiac cell apoptosis in male KL(−/−) mice which can be eliminated by LPD. LPD, however, did not rescue hyperphosphatemia or heart failure in female KL(−/−) mice. LPD did not affect estrogen depletion in female KL(−/−) mice. Normalization of serum estrogen levels by treatment with 17β-estradiol prevented hyperphosphatemia and heart failure in female KL(−/−) mice. Mechanistically, treatment with 17β-estradiol rescued hyperphosphatemia via inhibiting renal Na-Pi co-transporter expression. Normalization of serum phosphorus levels by treatment with 17β-estradiol also abolished cardiac mitochondrial respiratory enzyme dysfunction, ROS overproduction, oxidative stress and cardiac cell apoptosis in female KL(−/−) mice. Conclusion Klotho deficiency causes acute heart failure via hyperphosphatemia in male mice which can be prevented by LPD. 17β-estradiol prevents Klotho deficiency-induced hyperphosphatemia and heart failure by eliminating upregulation of renal Na-Pi co-transporter expression in female mice., Graphical abstract Image 1

Background: To assess potential roles for tumor-associated autoantibodies (TAAs) in esophageal squamous cell carcinoma (ESCC) screening: detecting early-stage malignancy, and predicting future cancer risk. Methods: Thirteen candidate autoantibodies identified in previous literatures were measured using multiplex serological assays in sera from cases and matched controls nested in two population-level screening cohorts in China. To evaluate the role of TAAs in detecting prevalent esophageal malignant lesions, an identification set (150 cases vs. 560 controls) and an external validation set (34 cases vs. 121 controls) were established with pre-screening sera collected ≤ 12 months prior to screening-related diagnosis. To explore the role of TAAs in predicting future ESCC risk, an exploration set (105 cases vs. 416 controls) with pre-diagnostic sera collected > 12 months before clinical diagnosis was established. Two models, the questionnaire-based model and full model additionally incorporating TAA markers, were constructed. Area under the receiver operating characteristic curve (AUC) and net reclassification improvement (NRI) were calculated to compare the performance of the two models. Findings: In the identification set, NY-ESO-1 (OR=2·12, 95% CI=1·02-4·40) and STIP1 (OR=1·83, 95% CI=1·10-3·05) were positively associated with higher risk of esophageal malignancy. Elevated MMP-7 was associated with higher risk of malignancy in females (ORfemale=5·07, 95% CI=1·30-19·71). The estimates in validation set were consistent with these results, but were close to null in exploration set. Integration of selected TAAs improved the performance of questionnaire-based models in detecting prevalent esophageal malignancy (female: AUCfull model=0·745, 95% CI=0·675-0·814, AUCquestionnaire-based model=0·658, 95% CI=0·585-0·732, NRI=0·604, P

Background: Alzheimer's disease (AD) is generally thought to spare primary sensory function; however, such interpretations have drawn from a literature that has rarely taken into account the variable cognitive declines seen in patients with AD. As these cognitive domains are now known to modulate cortical somatosensory processing, it remains possible that abnormalities in somatosensory function in patients with AD have been suppressed by neuropsychological variability in previous research. Methods: In this study, we combine magnetoencephalographic (MEG) brain imaging during a paired-pulse somatosensory gating task with an extensive battery of neuropsychological tests to investigate the influence of cognitive variability on estimated differences in somatosensory function between biomarker-confirmed patients on the AD spectrum and cognitively-normal older adults. Findings: We show that patients on the AD spectrum exhibit largely non-significant differences in somatosensory function when cognitive variability is not considered (p-value range: .020–.842). However, once attention and processing speed abilities are considered, robust differences in gamma-frequency somatosensory response amplitude (p

Background: If a pregnant woman is overweight, this can evoke metabolic alterations that may have health consequences for both mother and child. Methods: Pregnant women with overweight/obesity (n = 358) received fish oil+placebo, probiotics+placebo, fish oil+probiotics or placebo+placebo from early pregnancy onwards. The serum metabolome was analysed from fasting samples with a targeted NMR-approach in early and late pregnancy. GDM was diagnosed by OGTT. Findings: The intervention changed the metabolic profile of the women, but the effect was influenced by their GDM status. In women without GDM, the changes in nine lipids (FDR

Background Nicaragua experienced a large Zika epidemic in 2016, with up to 50% of the population in Managua infected. With the domesticated Aedes aegypti mosquito as its vector, it is widely assumed that Zika virus transmission occurs within the household and/or via human mobility. We investigated these assumptions by using viral genomes to trace Zika transmission spatially. Methods We analysed serum samples from 119 paediatric Zika cases participating in the long-standing Paediatric Dengue Cohort Study in Managua, which was expanded to include Zika in 2015. An optimal spanning directed tree was constructed by minimizing the differences in viral sequence diversity composition between patient nodes, where low-frequency variants were used to increase the resolution of the inferred Zika outbreak dynamics. Findings Out of the 18 houses where pairwise difference in sample collection dates among all the household members was within 30 days, we only found two where viruses from individuals within the same household were up to 10th-most closely linked to each other genetically. We also identified a substantial number of transmission events involving long geographical distances (n=30), as well as potential super-spreading events in the estimated transmission tree. Interpretation Our finding highlights that community transmission, often involving long geographical distances, played a much more important role in epidemic spread than within-household transmission. Funding This study was supported by an NUS startup grant (OMS) and grants R01 AI099631 (AB), P01 AI106695 (EH), P01 AI106695-03S1 (FB), and U19 AI118610 (EH) from the US National Institutes of Health.

Background Persistent carriage of pneumococcal vaccine serotypes has occurred after introduction of PCV13 vaccination in Africa but the mechanisms are unclear. We tested the feasibility of using a human pneumococcal challenge model in Malawi to understand immune correlates of protection against carriage and to trial alternative vaccine candidates. We aimed to identify a dose of Streptococcus pneumoniae serotype 6B sufficient to establish nasopharyngeal carriage in 40% of those nasally inoculated and evaluate nasal mucosal immunity before and after experimental inoculation. Methods Healthy student volunteers were recruited and inoculated with saline, 20,000 CFU/naris or 80,000 CFU/naris of Streptococcus pneumoniae serotype 6B Post inoculation carriage was determined by nasal sampling for bacterial culture and lytA PCR. Immunological responses were measured in serum and nasal mucosal biopsies before and after bacterial inoculation. Findings Twenty-four subjects completed the feasibility protocol with minimal side effects. pneumococcal carriage was established in 0/6, 3/9 and 4/9 subjects in the saline, 20,000 CFU/naris and 80,000 CFU/naris groups, respectively. Incidental (natural) serotype carriage was common (7/24 participants carried non-6B strains, 29.2%. Experimentally induced type 6B pneumococcal carriage was associated with pro-inflammatory nasal mucosal responses prior to inoculation and altered mucosal recruitment of immune cells post bacterial challenge. There was no association with serum anti-capsular antibody. Interpretation The serotype 6B experimental human pneumococcal carriage model is feasible in Malawi and can now be used to determine the immunological correlates of protection against carriage and vaccine efficacy in this population.