Project description:Background: DOCK8 deficiency is an autosomal recessive form of hyperimmunoglobulinemia E syndrome (HIES). Severe atopic dermatitis (AD) shares with DOCK8 deficiency some clinical symptoms, including eczema, eosinophilia, and increased serum IgE levels. The deficiency of DOCK8 protein is potentially a life-threatening autosomal recessive HIES and only curable with bone marrow transplantation. Despite identified metabolomics and cytokine biomarkers, novel proteomics biomarkers need to be identified, as the connecting networks are critical to our understanding of this disease. Hence we performed serum proteomics profiling using LC-MSE SynaptG2. Method: Label-free untargeted proteomics analysis was used to identify potentially reliable, sensitive, and specific protein biomarkers in serum collected from DOCK8 (n=10), AD (n=9) patients, which were compared to ctrls (n=5). Results: From a total of 275 quantifiable proteins, binary comparisons between AD vs. Ctrl, DOCK8 vs. Ctrl, and DOCK8 vs. AD revealed 109, 105 and 85 dysregulated proteins, respectively. 24 among 85 proteins were specific potential biomarkers among the DOCK8 and AD groups. The sensitivity and specificity of few proteins like Claspin, Immunoglobulin kappa and heavy, complement components as potential biomarkers to distinguish between DOCK8 and AD patients were evaluated using the receiver operating characteristic curve. DOCK8 deficiency and AD groups' profiling revealed a shared role of ERK1/2 among the commonly dysregulated proteins. Conclusion: In this study, we have identified potential proteomics biomarkers and profile to distinguish between DOCK8 and AD, with possible diagnostic and therapeutic applications to help create effective interventions for managing these diseases. Further studies to confirm these associations in prospective cohorts are warranted.
Project description:In this study we developed metaproteomics based methods for quantifying taxonomic composition of microbiomes (microbial communities). We also compared metaproteomics based quantification to other quantification methods, namely metagenomics and 16S rRNA gene amplicon sequencing. The metagenomic and 16S rRNA data can be found in the European Nucleotide Archive (Study number: PRJEB19901). For the method development and comparison of the methods we analyzed three types of mock communities with all three methods. The communities contain between 28 to 32 species and strains of bacteria, archaea, eukaryotes and bacteriophage. For each community type 4 biological replicate communities were generated. All four replicates were analyzed by 16S rRNA sequencing and metaproteomics. Three replicates of each community type were analyzed with metagenomics. The "C" type communities have same cell/phage particle number for all community members (C1 to C4). The "P" type communities have the same protein content for all community members (P1 to P4). The "U" (UNEVEN) type communities cover a large range of protein amounts and cell numbers (U1 to U4). We also generated proteomic data for four pure cultures to test the specificity of the protein inference method. This data is also included in this submission.
Project description:To examine the effect of DOCK8 deficiency on helper T cell differentiation, we employed microarray expression profiling and found that 850 genes were expressed at higher levels in Dock8–/– AND CD4+ T cells than Dock8+/– controls after antigen stimulation.
Project description:Genome wide DNA methylation profiling of isolated monocyte samples from healthy Kenyan children, the same children during an episode of acute malaria, healthy Kenyan adults, and healthy adults from the United States. The Illumina Infinium MethylationEPIC BeadChip microarray was used to obtain DNA methylation profiles across approximately 860,000 CpGs in negatively selected monocyte samples. Samples included monocytes from 8 children from western Kenya obtained while healthy and matching samples from the same 8 Kenyan children obtained during an episode of acute uncomplicated Plasmodium falciparum malaria, 8 healthy malaria-immune adults from western Kenya, and 8 healthy malaria-naive adults from the US. Abstract -- Background: Age-related changes in adaptive and innate immune cells have been associated with a decline in effective immunity and chronic, low-grade inflammation. Epigenetic, transcriptional, and functional changes in monocytes occur with aging, though most studies to date have focused on differences between young adults and the elderly in populations with European ancestry; few data exist regarding changes that occur in circulating monocytes during the first few decades of life or in African populations. We analyzed DNA methylation profiles, cytokine production, and inflammatory gene expression profiles in monocytes from young adults and children from western Kenya. Results: We identified several hypo- and hyper-methylated CpG sites in monocytes from Kenyan young adults vs. children that replicated findings in the current literature of differential DNA methylation in monocytes from elderly persons vs. young adults across diverse populations. Differentially methylated CpG sites were also noted in gene regions important to inflammation and innate immune responses. Monocytes from Kenyan young adults vs. children displayed increased production of IL-8, IL-10, and IL-12p70 in response to TLR4 and TLR2/1 stimulation as well as distinct inflammatory gene expression profiles. Conclusions: These findings complement previous reports of age-related methylation changes in isolated monocytes and provide novel insights into the role of age-associated changes in innate immune functions.
Project description:Characterisation of the forehead and armpit skin microbiome of healthy individuals using shotgun metagenomics and 16S rRNA gene sequencing
Project description:Patients deficient in the guanine nucleotide exchange factor DOCK8 have decreased numbers and impaired in vitro function of T regulatory (Treg) cells and make autoantibodies, but seldom develop autoimmunity. We show that similarly, Dock8-/- mice have decreased numbers and impaired in vitrofunction of Treg cells, but do not develop autoimmunity. In contrast, mice with selective DOCK8 deficiency in Treg cells develop lymphoproliferation, autoantibodies, and gastrointestinal inflammation, despite normal percentage and in vitro function of Treg cells, suggesting that deficient T effector cell function might protect DOCK8 deficient patients from autoimmunity. We demonstrate that DOCK8 associates with STAT5 and is important for IL-2 driven STAT5 phosphorylation in Treg cells. DOCK8 localizes within the lamellar actin ring of the Treg cell immune synapse (IS). Dock8-/- Treg cells have abnormal TCR-driven actin dynamics, decreased adhesiveness, altered gene expression profile, an unstable IS with decreased recruitment of signaling molecules, and impaired transendocytosis of the co-stimulatory molecule CD86. These data suggest that DOCK8 enforces immunological tolerance by promoting IL-2 signaling, TCR-driven actin dynamics, and the IS in Treg cells.