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

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

Isolation von humanen CD4+ dendritischen Zellen aus zirkulierendem Blut und ihr Verhalten bei Stimulation

Dendritische Zellen (DC) sind die wichtigsten antigenpräsentierenden Zellen. Es gibt myeloide und lymphoide DC. In dieser Arbeit konnten mit MACS$\circledR$ CD4+, CD11c- DC isoliert oder im FACS CD4+, CD11c- >> CD11c+ DC identifiziert werden. Mit beiden Methoden werden die gleichen Zellen untersucht. Im Blut sind 0,2-1,2% CD4+ DC, mit lymphozytärer Morphologie, MHC-II+ und CD3-, CD14-, CD16-, CD19-. Nach 48h mit GM-CSF entwickeln sie eine dendritische Zellmorphologie und werden CD83+. Ohne GM-CSF werden die DC in Kultur apoptotisch. CD40-Ligand und TNF-$\alpha$ stimulieren die Expression von CD83, wobei TNF-$\alpha$ der stärkste Reiz ist. LPS hatte wenig Auswirkung. Anhand steigender Größe und Granularität konnten im FACS drei, unterschiedlich CD83+ Populationen abgegrenzt werden. Diese Arbeit zeigt Isolation, Funktion und Reagibilität der mehrheitlich lympoiden CD4+ DC aus Blut. Die Identifikation im FACS ist zeit- und blutsparend (2ml) und eröffnet zukünftige Untersuchungsmöglichkeiten

Vorteilhaftes Langzeitüberleben von Kindern mit rezidivierten Nephroblastomen nach Hochdosis-Chemotherapie und autologem Stammzellrescue

Background: High-dose chemotherapy (HDC) with autologous stem-cell rescue (ASCR) is a treatment option for pediatric patients with relapsed nephroblastoma. We present long term results of 9 patients treated between 1993 and 2013 at our center. Procedure: Reinduction therapy was carried out according to GPOH and SIOP recommendations. The conditioning regimen consisted of carboplatin (1 200 mg/m²), etoposide (800 mg/m² or 40 mg/kg) and melphalan (180 mg/m²). Purging of the grafts with immunomagnetic CD34 positive selection was performed in 5 patients. Results: 8 of 9 Patients (90%) are alive without evidence of disease after a median follow-up of 8.5 years. Leukocyte engraftment occurred after a median of 10 days (range 8-12). Median numbers of 667/µl CD3+, 329/µl CD4+, 369/µl CD8+T cells and 949/µl B cells were reached after 180 days. No negative impact of CD34 selection was observed. No transplantation-related death occurred. Acute toxicity comprised mucositis III°-IV° in all and veno-occlusive disease in one patient. Long term effects probably related to treatment occurred in 3/7 evaluable patients and comprised hearing impairment, reduced renal phosphate reabsorption, mild creatinine elevation and hypothyroidism (n=1, each). Conclusion: Thus, in our experience HDC with ASCR is an effective treatment of recurrent or refractory nephroblastoma with acceptable side effects. However, a randomized trial proving its efficiency with a high level of evidence is needed.Hintergrund: Hochdosischemotherapie mit autologem Stammzellrescue ist eine Therapieoption für paediatrische Patienten mit rezidivierten Nephroblastomen. Wir präsentieren Langzeitergebnisse von 9 Patienten, die in den Jahren 1993–2013 in unserem Zentrum auf diese Weise therapiert wurden. Methods: Reinduktionstherapie wurde nach Empfehlung der SIOPH-GPOH durchgeführt. Das Konditionierungsregime bestand aus Carboplatin (1 200 mg/m²), Etoposid (800 mg/m² oder 40 mg/kg) und Melphalan (180 mg/m²). Immunomagnetische CD-34 positive Selektion wurde bei 5 Patienten durchgeführt. Results: 8 von 9 Patienten sind nach einem medianen Follow-Up von 8,5 Jahren ohne Nachweis der Erkrankung am Leben. Der Leukozyten-Take wurde im Median nach 10 Tagen erreicht (Range 8–12). Nach 180 Tagen wurden hinsichtlich der Immunrekonstitution im Median 667/μl CD3 + Zellen, 329/μl CD4 + Zellen, 369/μl CD8 + Zellen und 949/μl B-Zellen erreicht. Es wurde kein negativer Einfluss auf die Immunrekonstitution durch CD34-Selektion beobachtet. Kein Patient starb an den Nebenwirkungen der Transplantation. Die akuten Nebenwirkungen beinhalten Mukositis °III– °IV bei allen Patienten und veno-occlussive disease (VOD) bei einem Patienten. Langzeitfolgen, die eventuell mit der Therapie in Zusammenhang stehen traten in 3/7 evaluierbaren Patienten auf und beinhalten Hörstörungen, leichte Kreatininerhöhung, Störung der Phosphatrückresorption und Hypothyreodismus (jeweils 1 ×). Schlussfolgerung: Nach unserer Erfahrung ist Hochdosischemotherapie mit autologem Stammzellrescue eine effektive Therapieoption für rezidivierte und refraktäre Nephroblastome mit tolerablen Nebenwirkungen. Jedoch ist eine große randomisierte Studie notwendig um die Effizienz mit hoher Evidenz beweisen zu können

Systematic identification of cancer-specific MHC-binding peptides with RAVEN

Immunotherapy can revolutionize anti-cancer therapy if specific targets are available. Immunogenic peptides encoded by cancer-specific genes (CSGs) may enable targeted immunotherapy, even of oligo-mutated cancers, which lack neo-antigens generated by protein-coding missense mutations. Here, we describe an algorithm and user-friendly software named RAVEN (Rich Analysis of Variable gene Expressions in Numerous tissues) that automatizes the systematic and fast identification of CSG-encoded peptides highly affine to Major Histocompatibility Complexes (MHC) starting from transcriptome data. We applied RAVEN to a dataset assembled from 2,678 simultaneously normalized gene expression microarrays comprising 50 tumor entities, with a focus on oligo-mutated pediatric cancers, and 71 normal tissue types. RAVEN performed a transcriptome-wide scan in each cancer entity for gender-specific CSGs, and identified several established CSGs, but also many novel candidates potentially suitable for targeting multiple cancer types. The specific expression of the most promising CSGs was validated in cancer cell lines and in a comprehensive tissue-microarray. Subsequently, RAVEN identified likely immunogenic CSG-encoded peptides by predicting their affinity to MHCs and excluded sequence identity to abundantly expressed proteins by interrogating the UniProt protein-database. The predicted affinity of selected peptides was validated in T2-cell peptide-binding assays in which many showed binding-kinetics like a very immunogenic influenza control peptide. Collectively, we provide an exquisitely curated catalogue of cancer-specific and highly MHC-affine peptides across 50 cancer types, and a freely available software (https://github.com/JSGerke/RAVENsoftware) to easily apply our algorithm to any gene expression dataset. We anticipate that our peptide libraries and software constitute a rich resource to advance anti-cancer immunotherapy

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

Abstract 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. </jats:p