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• Short-term fasting before living kidney donation has an immune-modulatory effect

  Background: Short-Term Fasting (STF) is an intervention reducing the intake of calories, without causing undernutrition or micronutrient-related malnutrition. It aims to systemically improve resilience against acute stress. Several (pre-)clinical studies have suggested protective effects of STF, marking the systemic effects STF can induce in respect to surgery and ischemia-reperfusion injury. In addition, STF also affects the number of circulating immune cells. We aim to determine the effect of STF on the abundance and phenotype of different immune cell populations. Methods: Thirty participants were randomly selected from the FAST clinical trial, including living kidneys donors, randomised to a STF-diet or control arm. In an observational cohort sub-study we prospectively included 30 patients who donated blood samples repeatedly during study runtime. Using flow cytometry analyses, immune cell phenotyping was performed on peripheral blood mononuclear cells. Three panels were designed to investigate the presence and activation status of peripheral T cells, B cells, dendritic cells (DCs) and myeloid cells. Results: Eight participants were excluded due to sample constraints. Baseline characteristics showed no significant differences, except for fasting duration. Weight changes were minimal and non-significant across different time intervals, with slight trends towards long-term weight loss pre-surgery. Glucose, insulin, and β-hydroxybutyrate levels differed significantly between groups, reflecting adherence to the fasting diet. Flow cytometry analysis revealed no baseline differences between groups, with high variability within each group. We observed significant changes in immune cell populations due to fasting, particularly in B cells, T cells, and DCs. Discussion: In this study, we found that STF changes the levels and phenotype of immune cells, reducing abundance and activation of T cells and regulatory T cells, increased presence of (naïve) B cells, and elevation of type 1 conventional DCs. Further research should focus on the clinical implications of the changes in immune cell populations and significance of these observed immunological changes. Background: Short-Term Fasting (STF) is an intervention reducing the intake of calories, without causing undernutrition or micronutrient-related malnutrition. It aims to systemically improve resilience against acute stress. Several (pre-)clinical studies have suggested protective effects of STF, marking the systemic effects STF can induce in respect to surgery and ischemia-reperfusion injury. In addition, STF also affects the number of circulating immune cells. We aim to determine the effect of STF on the abundance and phenotype of different immune cell populations. Methods: Thirty participants were randomly selected from the FAST clinical trial, including living kidneys donors, randomised to a STF-diet or control arm. In an observational cohort sub-study we prospectively included 30 patients who donated blood samples repeatedly during study runtime. Using flow cytometry analyses, immune cell phenotyping was performed on peripheral blood mononuclear cells. Three panels were designed to investigate the presence and activation status of peripheral T cells, B cells, dendritic cells (DCs) and myeloid cells. Results: Eight participants were excluded due to sample constraints. Baseline characteristics showed no significant differences, except for fasting duration. Weight changes were minimal and non-significant across different time intervals, with slight trends towards long-term weight loss pre-surgery. Glucose, insulin, and β-hydroxybutyrate levels differed significantly between groups, reflecting adherence to the fasting diet. Flow cytometry analysis revealed no baseline differences between groups, with high variability within each group. We observed significant changes in immune cell populations due to fasting, particularly in B cells, T cells, and DCs. Discussion: In this study, we found that STF changes the levels and phenotype of immune cells, reducing abundance and activation of T cells and regulatory T cells, increased presence of (naïve) B cells, and elevation of type 1 conventional DCs. Further research should focus on the clinical implications of the changes in immune cell populations and significance of these observed immunological changes.

  Study EGAS00001008034

• Genome_wide_association_study_of_vaccine_responses_in_infants_living_in_the_Developing_World__VaccGene___Phase_II_African_Cohorts

  Human genetic heterogeneity accounts for approximately 70% of the observed variability inresponse to common childhood vaccinations. Identifying the elements of this heterogeneitywill enable the development of vaccines which have improved clinical and cost effectivenessthrough rational adjuventation approaches, or by targeting particular at-risk or responsivesubgroups. The studies published to date have been limited in terms of the number of vaccinesubtypes analysed, diversity of the populations included, sample sizes and breadth of otherenvironmental and lifestyle variables which may explain some of the remainder of variation inthe response to each vaccine. Nevertheless, these studies have proposed that the geneswhich harbour the variants associated with vaccine responsiveness reside within regions ofthe genome associated with immune function such as the HLA locus and FOXP1and ITGAL.We are proposing one of the largest genome-wide association studies of vaccine responseever undertaken, which will provide unparalleled power to identify the genetic factorsassociated with the response to all of the most commonly used childhood vaccinesworldwide. For the first phase of the project (Prelim I0308) we genotyped ca. 1,400 childrenfrom the Entebbe Mother and Baby (EMaB) study, an ongoing birth cohort in southernUganda, using the Illumina HumanOmni2.5-8 chip. Analyses for response to vaccinationagainst diphtheria, tetanus, pertussis, Haemophilus influenzae, measles, hepatitis B and BCGare now underway. Working alongside other ongoing studies, we plan for the second phaseof the project to expand sample sizes to up to 7,000 individuals in total. This large cohort willinclude 1,500 South African infants, 500 Burkina Faso infants, 1,800 children from Ecuadorand up to 1,800 from a variety of countries (MAL-ED Consortium). The principal measuredphenotypes will be the serological responses at 6 or 12 months of age to the vaccinesadministered to all enrolled children as part of expanded programme of immunisation in allcountries. As for the primary Ugandan study the responses will be analysed as quantitativetraits using mixed model (GEMMA) analyses following imputation using a combined 1000Genome and African Genome Variation Project reference panel. The data will be analysed asone large discovery mega-analysis to maximise the power of finding the relevant associatedvariants. Interim GWAS analyses will be performed on each population to test for populationspecific signals. Other phenotypes that will be assessed in the new studies includequantitative response to BCG, rotavirus and pneumococcal vaccines (South Africa, Ecuadorand Uganda), clinical and immunological response to malaria vaccines (Burkina),immunological correlates of atopy (Ecuador and Uganda) and malaria infection (Burkina,Ecuador and Uganda) in addition to non-communicable disease traits such as blood pressurepage 1[A8] If applicable, provide text (abstract) which will accompany data for this study in the ENA/EGA.[A9] Does this project use samples?[A10] Please choose which types of sample you will be using[A12] Please state the anticipated date (month/year) samples will be available in-house (if known). If samples arealready in-house, type 'in-house' here. Please note that for sequencing, genotyping and microarrays this is essentialfor scheduling the work.[A13] Please state the anticipated project start date (month/year).[A14] Please state the project duration (months).[A15] Please read WTSI's Data Sharing Policy and Guidelines, then explain your data sharing plans for the project[A16] Are the data sharing plans of this project compliant with the Institute's Guidelines?[A17] Are there any conflicts of interest related to this proposal?(Uganda).We are requesting funds to genotype on the Illumina HumanOmni2.5-8 platform 2,000 Africaninfants recruited through ongoing maternal and child vaccine clinical trials (againstpneumococcus in Soweto, South Africa, and malaria in Banfora, Burkina Faso). The DNAfrom the Burkina cohort is at the University of Oxford and ready to be shipped, while the DNAfrom the South African cohort will be available by mid-2014. Appropriate consent for geneticanalyses has been acquired for the proposed study cohorts from both local ethics committeesand UK-based Committees.

  Study EGAS00001000918

• COVID-19: Post-Hospital Thromboprophylaxis A Multicenter, Adaptive, Prospective, Randomized Trial Evaluating the Efficacy and Safety of Antithrombotic Strategies in Patients with COVID-19 Following Hospital Discharge (ACTIV-4C)

  This study is an adaptive, prospective, randomized trial designed to compare the effectiveness and safety of antithrombotic therapy with no antithrombotic therapy after hospitalization for 48 hours or longer for COVID-19. For Stage 1 of this study, participants will be randomized to either prophylactic anticoagulation or no anticoagulant therapy for 30 days, and then followed for an additional 60 days after the completion of treatment (total duration of follow-up, approximately 90 days). Biobanking of samples for future biomarker and mechanistic studies will be available for centers able to participate and collect samples from eligible participants. Samples will be collected at the time of enrollment and after the completion of 30 days of therapy.

  Study phs003063

• Spatiotemporal single-cell analysis reveals a time-dependent immunological modulation by multi-fractionated radiotherapy in esophageal cancer

  Description Here, by integrative spatial and single-cell analyses using spatial transcriptome (VISIUM) and single-cell RNA sequencing (scRNA-seq), we examined the spatiotemporal dynamics of immune-response in the tumor microenvironment of esophageal squamous cell carcinoma (ESCC) before, during and after RT. The comprehensive single-cell analyses demonstrated the infiltration of immune cells into tumor with the dynamic change of immune-stimulatory and -suppressive gene expression after RT. ScRNA-seq analysis uncovered the time- and cell-type-dependent alteration of gene expression profile from during to after RT. This datasets in the present study will provide the information of RT-induced gene signature to seek an appropriate target of personalized immunotherapy combined with radiotherapy and of determining the timing of the combination therapy.

  Study JGAS000712

• WGoxBS and SureSelect data of the POPS placenta samples

  We carried out whole-genome oxidative bisulfite sequencing (WGoxBS) in the placentas of two healthy female and two healthy male pregnancies generating an average genome depth of coverage of 25x. The sex-specific differential methylation pattern observed in this region was validated in additional 8 healthy placentas (including 2 from the WGoxBS) using SureSelect in-solution target capture. For WGoxBS, placental genomic DNA (4 µg) from 4 healthy pregnancies was processed to achieve 10 kb fragments with the g-Tube (Covaris), according to the manufacturer's instructions. To increase the number of uniquely sequenced reads, two independent libraries were generated for each individual. Multiplexed sequencing was carried out on the Illumina MiSeq, HiSeq 2000, and HiSeq 2500 instruments with 2x100, 2x50 and 2x125 cycles using MiSeq Reagent Kit v3, HiSeq SBS Kit v3 and HiSeq SBS Kit v4, respectively. For SureSelect in-solution capture, placental genomic DNA (3.5 µg) from 8 healthy pregnancies (including 2 from the WGoxBS) was fragmented by the Covaris S220 system according to the SureSelect Methyl-Seq target enrichment protocol (Agilent). All 8 libraries were pooled and sequenced on the Illumina HiSeq 2500 instrument with 2 × 125 cycles using HiSeq SBS Kit v4 and a single lane of the Illumina HiSeq 4000 instrument with 2 × 150 cycles using HiSeq 3000/4000 SBS Kit following Illumina's guidelines (Illumina Application Note: Epigenetics February 2016).

  Dataset EGAD00001003136

• Validation of AML Mutational Screening

  We recently worked-up a pulldown protocol for studying 21 genes recurrently mutated in AML (Study1770). Our manuscript is currently under revision and to address the reviewers' comments we need to validate some mutations by re-sequencing. In this add-on study we will be using PCR followed by MiSeq for this purpose.

  Dataset EGAD00001000445

• Whole Exome sequencing of colorectal cancer patients (SG-BULK)

  DNA-seq libraries were captured to exome regions using xGen Exome Research Panel v1.0 (IDT), and libraries were prepared using the KAPA Hyper prep kit. DNA libraries were sequenced to a target depth of ×200 for tumor sample, ×100 for normal samples on the Illumina HiSeq platform.

  Dataset EGAD00001008543

• scRNA-seq of total bone marrow and T cells from multiple myeloma long-term survivors

  scRNA This dataset contains 50 scRNA-seq samples from bone marrow aspirates of 11 multiple myeloma patients experiencing long-term survival and 3 healthy donors. For each donor, total bone marrow and CD3+ T cells were sequenced. For multiple myeloma patients, paired samples were collected at initial diagnosis and between 7-17 years after first-line therapy. Bone marrow mononuclear cells were isolated by Ficoll density gradient centrifugation. For sorting of total bone marrow cells singlet, live cells were gated and sorted, for sorting of T cells CD45+, CD3+ cells were gated and sorted on either FACSAria Fusion or FACSAria II. Single-cell RNA sequencing were generated using 10x Genomics single-cell RNAseq technology (Chromium Single Cell 3’ Solution v2) according to the manufacturer’s protocol and sequenced on an Illumina HiSeq4000 (paired end, 26 and 74 bp). Bulk RNA Singlet, live CD3+CD4- CXCR3+CD8+ and CD3+CD4- CXCR3-CD8+ cells were sorted from 7 bone marrow and 3 peripheral blood samples of 7 multiple myeloma patients using a FACSAria Fusion machine. Bulk-RNA sequencing libraries were generated using the SMART Seq Stranded Total RNA-Seq kit (Takara) and sequenced using the Illumina NovaSeq 6000 platform (2 x 100 bp).

  Dataset EGAD00001010025

• Influence of the Microbiome on Epigenetic Mechanisms in IBD

  Live CD4 T cells were sorted from inflamed and non-inflamed tissue samples of IBD patients or from healthy and IBD blood samples. ATAC-Seq libraries were generated from live CD4 T cells sorted from i) inflamed and non-inflamed tissue samples, ii) healthy and IBD blood samples, or from iii) CD4 T cell subsets polarised from healthy blood samples. After isolating crude nuclei, live CD4+ T cells were treated with Tagment DNA buffer and Tagment DNA Enzyme (Nextera DNA Library Prep Kit, Illumina), and then the DNA was purified by MinElute PCR Purification Kit (Qiagen). Transposed DNA fragments were amplified using specific adapters followed by purification with MinElute PCR Purification Kit (Qiagen). Fragments from 240-360pb were selected in the PippinHT system (Sage Science). The quality of the library and its DNA concentration were assessed by Bioanalyzer instruments (Agilent Technologies) and ultimately submitted for sequencing using Illumina HiSeq 2500 sequencer, V4 chemistry. On the other hand, single cell RNA-Seq libraries were generated exclusively from inflamed and non-inflamed tissue samples of Crohn’s disease patients. Briefly, live CD4 T cells were captured and encapsulated before cDNA amplification using the 10X Genomics Chromium Platform. Samples were prepared as outlined by 10x genomics Single Cell 3’ Reagent Kits v2 user guide. Samples were sequenced on a HiSeq 2500 with the following run parameters: Read 1 – 26 cycles, read 2 – 98 cycles, index 1 – 8 cycles.

  Dataset EGAD00001011066

• An integrated single-cell reference atlas of the human endometrium

  This dataset includes both the anchor scRNA-seq and snRNA-seq datasets used to build the Human Endometrial Cell Atlas (HECA). HECA provides a comprehensive definition of endometrial cell types and states throughout the menstrual cycle in donor with and without endometriosis. It identifies consensus cell types across datasets generated by teams worldwide, as well as previously unreported cell types, all of which are validated and mapped in situ using spatial transcriptomics. Processed data is available from ArrayExpress with accession number E-MTAB-14039. See Marečková, M., Garcia-Alonso, L., Moullet, M. et al. An integrated single-cell reference atlas of the human endometrium. Nat Genet 56, 1925–1937 (2024) https://doi.org/10.1038/s41588-024-01873-w for more information.

  Dataset EGAD00001015446