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

Head of household education level as a factor influencing whether delivery takes place in the presence of a skilled birth attendant in Busia, Uganda: a cross-sectional household study

Abstract Background Assistance during delivery by a skilled attendant is recommended as a means to reduce child and maternal mortality. Globally, higher levels of maternal education have been associated with better health behaviours at delivery. However, given that heads of households tend to be the decision makers regarding accessing healthcare, some educated mothers may find themselves prevented from accessing healthcare at the point of delivery. Methods We examined the association between head of household education level and health seeking behaviours at delivery across a sample of 392 households. Chi-squared analysis and odds ratios were calculated to measure the strength of the relationship between no, some primary, or some secondary or higher education attained by the head of household and the presence or absence of a skilled birth attendant at that child’s birth, and whether the birth took place at a health facility. Results Heads of household (n = 392) were predominantly male (93.4% [(90.9%, 95.8%), a = 0.05]). We found a significant difference in skilled birth attendance between heads of households with some primary education and heads of household with some secondary education or higher (χ 2 (1) = 6.231, p <0.05) whereby those with secondary or higher education were significantly more likely to seek a skilled birth attendant (OR = 1.5,[1.1,2.1]). The difference in health centre delivery between heads of household with a primary education and heads of household with a secondary or higher education was also significant (χ 2 (1) = 7.519, p <0.05). Those with secondary or higher education were significantly more likely to deliver in a health facility (OR = 1.6,[1.2,2.1]). Conclusions The results of our analysis, which identified the vast majority of heads of households as men, suggests that education, or rather limited or a lack of education for the head of household, may be a barrier to women’s use of health care in Uganda and therefore reinforces the need to increase educational access among male heads of households. Improving the rates of health centre deliveries and utilization of services provided by skilled health workers might lie, in part, in increasing overall education levels of heads of households, specifically the education of male heads of households

T Cell Production of IFNγ in Response to TLR7/IL-12 Stimulates Optimal B Cell Responses to Viruses.

Knowledge of the processes that underlie IgG subclass switching could inform strategies designed to counteract infections and autoimmunity. Here we show that TLR7 ligands induce subsets of memory CD4 and CD8 T cells to secrete interferon γ (IFNγ) in the absence of antigen receptor stimulation. In turn, TLR ligation and IFNγ cause B cells to express the transcription factor, T-bet, and to switch immunoglobulin production to IgG2a/c. Absence of TLR7 in T cells leads to the impaired T-bet expression in B cells and subsequent inefficient IgG2a isotype switching both in vitro and during the infection with Friend virus in vivo. Our results reveal a surprising mechanism of antiviral IgG subclass switching through T-cell intrinsic TLR7/IL-12 signaling

IFNγ produced by T cells in response to TLR7/IL-12 simulation is required for T-bet induction in B cells.

<p><b>(A)</b> Sorted memory or naïve CD4 T cells were mixed with purified naïve B cells and incubated in the presence of anti-BCR, anti-BCR and R848, or combination of anti-BCR, R848 and IL-12 for 48h. Cells were stained for surface markers and intracellular T-bet. Bar graph represents gMFI of T-bet expression in B cells (gated as live, B220+, CD19+, CD4-, CD8-). Bars represent the means +/- SEM. <b>(B)</b> WT or TLR7-/- sorted CD4 T cells were mixed with purified naïve WT B cells and incubated for 48h with indicated stimuli. Cells were stained for surface markers and intracellular T-bet. Bar graph represents gMFI of T-bet expression in B cells (gated as live, B220+, CD19+, CD4-, CD8-). Bars represent the means +/- SEM <b>(C)</b> WT or TLR7-/- sorted CD4 T cells were mixed with purified naïve WT B cells and incubated in the presence of indicated stimuli for 7 days. Culture supernatants were assessed for the presence IgG2a by ELISA. <b>(D)</b> Sorted memory or naïve CD4 T cells were mixed with purified naïve B cells and incubated in the presence of indicated stimuli for 7 days. Supernatants were analyzed for the presence of IgG2a by ELISA. Bars represent the means +/- SEM. Data are representative of three or more independent experiments.</p

TLR7 expression in T cells is required for the accumulation of T-bet+ B cells and effective production of anti-viral IgG2a during Friend virus infection.

<p><b>(A)</b> C57BL/6 mice (n = 5 per time point) were infected with 10⁴ SFFU of Friend virus (FV). Spleens were harvested on day 7, 14, 21 or 28 post infection. The presence of T-bet+ cells among B cells was assessed by FACS. Bar graphs represent percentage of T-bet+ cells among B cells. <b>(B, C)</b> Bone marrow chimera were constructed as described in Methods, such as in (TLR7^-/- + TCR^-/-) mice only T cells completely lack TLR7 expression and the rest of the hematopoetic cells were 80% WT and 20% TLR7-/-. Bone marrow chimeras were infected with FV (as in <b>(A)</b>) and spleens and serum were harvested on 14 dpi percentage and numbers of T-bet+ or CD11c+ B cells is shown <b>(B).</b> Serum collected at 14dpi was assessed for the presence of anti-FV IgG2a by ELSIA <b>(C)</b>. <b>(D)</b> T-bet^fl/fl, and T-bet^fl/flxCD19^CRE mice (n = 4 mice per group) were infected with FV as in (A). Serum was collected at 15 dpi and the presence of anti-FV IgG2a was assessed by ELISA. Bars represent the means +/- SEM. Data are representative of three or more independent experiments.</p

Subsets of CD4 and CD8 memory T cells produce IFNγ in response to TLR7 simulation <i>in vitro</i>.

<p>Splenocytes from C57Bl/6 mice were cultured <i>in vitro</i> for 18h in the presence of different TLR agonists (as indicated). Cells were stained for surface markers and intracellular IFNγ. <b>(A)</b> Bars represent percentage of total splenocytes positive for intracellular IFNγ. <b>(B)</b> Gating strategy for IFNγ+ splenocytes upon TLR7 simulation for 18h (Representative FACS plots) and quantification of CD4 and CD8-positive cells among IFNγ+ splenocytes. <b>(C)</b> Bar graphs represent percentages of IFNγ+ among CD4 or CD8 T cells after 18h of splenocytes stimulation with different TLR ligands as indicated. <b>(D, E)</b> Percantages of IFNγ+ CD4 and CD8 T cells in response to different doses <b>(D)</b> or different time <b>(E)</b> of stimulation with R848. Bars represent the means +/- SEM (n = 3). All data are representative of three or more independent experiments. Statistics is shown for each condition over R848 stimulated cultures.</p