Homozygous whole body Cbs knockout in adult mice features minimal pathology during ageing despite severe homocysteinemia

Deficiencies in Cystathionine-β-synthase (CBS) lead to hyperhomocysteinemia (HHCy), which is considered a risk factor for cardiovascular, bone and neurological disease. Moreover, CBS is important for the production of cysteine, hydrogen sulfide (H2 S) and glutathione. Studying the biological role of CBS in adult mice has been severely hampered by embryological disturbances and perinatal mortality. To overcome these issues and assess the effects of whole-body CBS deficiency in adult mice, we engineered and characterized a Cre-inducible Cbs knockout model during ageing. No perinatal mortality occurred before Cbs-/- induction at 10 weeks of age. Mice were followed until 90 weeks of age and ablation of Cbs was confirmed in liver and kidney but not in brain. Severe HHCy was observed in Cbs-/- (289 ± 58 µM) but not in Cbs+/- or control mice (<10 µM). Cbs-/- showed impaired growth, facial alopecia, endothelial dysfunction in absence of increased mortality, and signs of liver or kidney damage. CBS expression in skin localized to sebaceous glands and epidermis, suggesting local effects of Cbs-/- on alopecia. Cbs-/- showed increased markers of oxidative stress and senescence but expression of other H2 S producing enzymes (CSE and 3-MST) was not affected. CBS deficiency severely impaired H2 S production capacity in liver, but not in brain or kidney. In summary, Cbs-/- mice presented a mild phenotype without mortality despite severe HHCy. The findings demonstrate that HHCy is not directly linked to development of end organ damage

Cholangiopathy and biliary fibrosis in Cyp2c70-deficient mice are fully reversed by ursodeoxycholic acid

BACKGROUND AND AIMS: Bile acids (BAs) aid intestinal fat absorption and exert systemic actions by receptor-mediated signaling. BA receptors have been identified as drug targets for liver diseases. Yet, differences in BA metabolism between humans and mice hamper translation of pre-clinical outcomes. Cyp2c70-ablation in mice prevents synthesis of mouse/rat-specific muricholic acids (MCAs), but potential (patho)physiological consequences of their absence are unknown. We therefore assessed age- and gender-dependent effects of Cyp2c70-deficiency in mice. METHODS: The consequences of Cyp2c70-deficiency were assessed in male and female mice at different ages. RESULTS: Cyp2c70-/- mice were devoid of MCAs and showed high abundances of chenodeoxycholic and lithocholic acids. Cyp2c70-deficiency profoundly impacted microbiome composition. Bile flow and biliary BA secretion were normal in Cyp2c70-/- mice of both sexes. Yet, the pathophysiological consequences of Cyp2c70-deficiency differed considerably between sexes. Three-week old male Cyp2c70-/- mice showed high plasma BAs and transaminases, which spontaneously decreased thereafter to near-normal levels. Only mild ductular reactions were observed in male Cyp2c70-/- mice up to 8 months of age. In female Cyp2c70-/- mice, plasma BAs and transaminases remained substantially elevated with age, gut barrier function was impaired and bridging fibrosis was observed at advanced age. Addition of 0.1% ursodeoxycholic acid to the diet fully normalized hepatic and intestinal functions in female Cyp2c70-/- mice. CONCLUSION: Cyp2c70-/- mice show transient neonatal cholestasis and develop cholangiopathic features that progress to bridging fibrosis in females only. These consequences of Cyp2c70-deficiency are restored by treatment with UDCA, indicating a role of BA hydrophobicity in disease development

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.</p

Relationship between molecular pathogen detection and clinical disease in febrile children across Europe:a multicentre, prospective observational study

Background: The PERFORM study aimed to understand causes of febrile childhood illness by comparing molecular pathogen detection with current clinical practice. Methods: Febrile children and controls were recruited on presentation to hospital in 9 European countries 2016–2020. Each child was assigned a standardized diagnostic category based on retrospective review of local clinical and microbiological data. Subsequently, centralised molecular tests (CMTs) for 19 respiratory and 27 blood pathogens were performed. Findings: Of 4611 febrile children, 643 (14%) were classified as definite bacterial infection (DB), 491 (11%) as definite viral infection (DV), and 3477 (75%) had uncertain aetiology. 1061 controls without infection were recruited. CMTs detected blood bacteria more frequently in DB than DV cases for N. meningitidis (OR: 3.37, 95% CI: 1.92–5.99), S. pneumoniae (OR: 3.89, 95% CI: 2.07–7.59), Group A streptococcus (OR 2.73, 95% CI 1.13–6.09) and E. coli (OR 2.7, 95% CI 1.02–6.71). Respiratory viruses were more common in febrile children than controls, but only influenza A (OR 0.24, 95% CI 0.11–0.46), influenza B (OR 0.12, 95% CI 0.02–0.37) and RSV (OR 0.16, 95% CI: 0.06–0.36) were less common in DB than DV cases. Of 16 blood viruses, enterovirus (OR 0.43, 95% CI 0.23–0.72) and EBV (OR 0.71, 95% CI 0.56–0.90) were detected less often in DB than DV cases. Combined local diagnostics and CMTs respectively detected blood viruses and respiratory viruses in 360 (56%) and 161 (25%) of DB cases, and virus detection ruled-out bacterial infection poorly, with predictive values of 0.64 and 0.68 respectively. Interpretation: Most febrile children cannot be conclusively defined as having bacterial or viral infection when molecular tests supplement conventional approaches. Viruses are detected in most patients with bacterial infections, and the clinical value of individual pathogen detection in determining treatment is low. New approaches are needed to help determine which febrile children require antibiotics. Funding: EU Horizon 2020 grant 668303.</p

Emergency medical services utilisation among febrile children attending emergency departments across Europe:an observational multicentre study

Children constitute 6–10% of all patients attending the emergency department (ED) by emergency medical services (EMS). However, discordant EMS use in children occurs in 37–61% with fever as an important risk factor. We aimed to describe EMS utilisation among febrile children attending European EDs. This study is part of an observational multicentre study assessing management and outcome in febrile children up to 18 years (MOFICHE) attending twelve EDs in eight European countries. Discordant EMS use was defined as the absence of markers of urgency including intermediate/high triage urgency, advanced diagnostics, treatment, and admission in children transferred by EMS. Multivariable logistic regression analyses were performed for the association between (1) EMS use and markers of urgency, and (2) patient characteristics and discordant EMS use after adjusting all analyses for the covariates age, gender, visiting hours, presenting symptoms, and ED setting. A total of 5464 (15%, range 0.1–42%) children attended the ED by EMS. Markers of urgency were more frequently present in the EMS group compared with the non-EMS group. Discordant EMS use occurred in 1601 children (29%, range 1–59%). Age and gender were not associated with discordant EMS use, whereas neurological symptoms were associated with less discordant EMS use (aOR 0.2, 95%CI 0.1–0.2), and attendance out of office hours was associated with more discordant EMS use (aOR 1.6, 95%CI 1.4–1.9). Settings with higher percentage of self-referrals to the ED had more discordant EMS use (p &lt; 0.05). Conclusion: There is large practice variation in EMS use in febrile children attending European EDs. Markers of urgency were more frequently present in children in the EMS group. However, discordant EMS use occurred in 29%. Further research is needed on non-medical factors influencing discordant EMS use in febrile children across Europe, so that pre-emptive strategies can be implemented. What is Known: •Children constitute around 6–10% of all patients attending the emergency department by emergency medical services. •Discordant EMS use occurs in 37–61% of all children, with fever as most common presenting symptom for discordant EMS use in children. What is New: •There is large practice variation in EMS use among febrile children across Europe with discordance EMS use occurring in 29% (range 1–59%), which was associated with attendance during out of office hours and with settings with higher percentage of self-referrals to the ED. •Future research is needed focusing on non-medical factors (socioeconomic status, parental preferences and past experience, healthcare systems, referral pathways, out of hours services provision) that influence discordant EMS use in febrile children across Europe.</p

Impact of infection on proteome-wide glycosylation revealed by distinct signatures for bacterial and viral pathogens

Mechanisms of infection and pathogenesis have predominantly been studied based on differential gene or protein expression. Less is known about posttranslational modifications, which are essential for protein functional diversity. We applied an innovative glycoproteomics method to study the systemic proteome-wide glycosylation in response to infection. The protein site-specific glycosylation was characterized in plasma derived from well-defined controls and patients. We found 3862 unique features, of which we identified 463 distinct intact glycopeptides, that could be mapped to more than 30 different proteins. Statistical analyses were used to derive a glycopeptide signature that enabled significant differentiation between patients with a bacterial or viral infection. Furthermore, supported by a machine learning algorithm, we demonstrated the ability to identify the causative pathogens based on the distinctive host blood plasma glycopeptide signatures. These results illustrate that glycoproteomics holds enormous potential as an innovative approach to improve the interpretation of relevant biological changes in response to infection

Relationship between molecular pathogen detection and clinical disease in febrile children across Europe: a multicentre, prospective observational study

BackgroundThe PERFORM study aimed to understand causes of febrile childhood illness by comparing molecular pathogen detection with current clinical practice.MethodsFebrile children and controls were recruited on presentation to hospital in 9 European countries 2016-2020. Each child was assigned a standardized diagnostic category based on retrospective review of local clinical and microbiological data. Subsequently, centralised molecular tests (CMTs) for 19 respiratory and 27 blood pathogens were performed.FindingsOf 4611 febrile children, 643 (14%) were classified as definite bacterial infection (DB), 491 (11%) as definite viral infection (DV), and 3477 (75%) had uncertain aetiology. 1061 controls without infection were recruited. CMTs detected blood bacteria more frequently in DB than DV cases for N. meningitidis (OR: 3.37, 95% CI: 1.92-5.99), S. pneumoniae (OR: 3.89, 95% CI: 2.07-7.59), Group A streptococcus (OR 2.73, 95% CI 1.13-6.09) and E. coli (OR 2.7, 95% CI 1.02-6.71). Respiratory viruses were more common in febrile children than controls, but only influenza A (OR 0.24, 95% CI 0.11-0.46), influenza B (OR 0.12, 95% CI 0.02-0.37) and RSV (OR 0.16, 95% CI: 0.06-0.36) were less common in DB than DV cases. Of 16 blood viruses, enterovirus (OR 0.43, 95% CI 0.23-0.72) and EBV (OR 0.71, 95% CI 0.56-0.90) were detected less often in DB than DV cases. Combined local diagnostics and CMTs respectively detected blood viruses and respiratory viruses in 360 (56%) and 161 (25%) of DB cases, and virus detection ruled-out bacterial infection poorly, with predictive values of 0.64 and 0.68 respectively.InterpretationMost febrile children cannot be conclusively defined as having bacterial or viral infection when molecular tests supplement conventional approaches. Viruses are detected in most patients with bacterial infections, and the clinical value of individual pathogen detection in determining treatment is low. New approaches are needed to help determine which febrile children require antibiotics.FundingEU Horizon 2020 grant 668303

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children