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New Findings What is the central question of this study? Skeletal muscle extracellular vesicles likely act as pro‐angiogenic signalling factors: does overexpression of peroxisome proliferator‐activated receptor γ coactivator 1‐α (PGC‐1α) alter skeletal muscle myotube extracellular vesicle release, contents and angiogenic potential? What is the main finding and its importance? Overexpression of PGC‐1α results in secretion of extracellular vesicles that elevate measures of angiogenesis and protect against acute oxidative stress in vitro. Skeletal muscle with high levels of PGC‐1α expression, commonly associated with exercise induced angiogenesis and high basal capillarization, may secrete extracellular vesicles that support capillary growth and maintenance. Skeletal muscle capillarization is proportional to muscle fibre mitochondrial content and oxidative capacity. Skeletal muscle cells secrete many factors that regulate neighbouring capillary endothelial cells (ECs), including extracellular vesicles (SkM‐EVs). Peroxisome proliferator‐activated receptor γ coactivator 1‐α (PGC‐1α) regulates mitochondrial biogenesis and the oxidative phenotype in skeletal muscle. Skeletal muscle PGC‐1α also regulates secretion of multiple angiogenic factors, but it is unknown whether PGC‐1α regulates SkM‐EV release, contents and angiogenic signalling potential. PGC‐1α was overexpressed via adenovirus in primary human myotubes. EVs were collected from PGC‐1α‐overexpressing myotubes (PGC‐EVs) as well as from green fluorescent protein‐overexpressing myotubes (GFP‐EVs), and from untreated myotubes. EV release and select mRNA contents were measured from EVs. Additionally, ECs were treated with EVs to measure angiogenic potential of EVs in normal conditions and following an oxidative stress challenge. PGC‐1α overexpression did not impact EV release but did elevate EV content of mRNAs for several antioxidant proteins (nuclear factor erythroid 2‐related factor 2, superoxide dismutase 2, glutathione peroxidase). PGC‐EV treatment of cultured human umbilical vein endothelial cells (HUVECs) increased their proliferation (+36.6%), tube formation (length: +28.1%; number: +25.7%) and cellular viability (+52.9%), and reduced reactive oxygen species levels (−41%) compared to GFP‐EVs. Additionally, PGC‐EV treatment protected against tube formation impairments and induction of cellular senescence following acute oxidative stress. Overexpression of PGC‐1α in human myotubes increases the angiogenic potential of SkM‐EVs. These angiogenic benefits coincided with increased anti‐oxidative capacity of recipient HUVECs. High PGC‐1α expression in skeletal muscle may prompt the release of SkM‐EVs that support vascular redox homeostasis and angiogenesis.

Emerging infectious diseases (EIDs) caused by viruses are increasing in frequency, causing a high disease burden and mortality world-wide. The COVID-19 pandemic caused by the novel SARS-like coronavirus (SARS-CoV-2) underscores the need to innovate and accelerate the development of effective vaccination strategies against EIDs. Human leukocyte antigen (HLA) molecules play a central role in the immune system by determining the peptide repertoire displayed to the T-cell compartment. Genetic polymorphisms of the HLA system thus confer a strong variability in vaccine-induced immune responses and may complicate the selection of vaccine candidates, because the distribution and frequencies of HLA alleles are highly variable among different ethnic groups. Herein, we build on the emerging paradigm of rational epitope-based vaccine design, by describing an immunoinformatics tool (Predivac-3.0) for proteome-wide T-cell epitope discovery that accounts for ethnic-level variations in immune responsiveness. Predivac-3.0 implements both CD8+ and CD4+ T-cell epitope predictions based on HLA allele frequencies retrieved from the Allele Frequency Net Database. The tool was thoroughly assessed, proving comparable performances (AUC ~0.9) against four state-of-the-art pan-specific immunoinformatics methods capable of population-level analysis (NetMHCPan-4.0, Pickpocket, PSSMHCPan and SMM), as well as a strong accuracy on proteome-wide T-cell epitope predictions for HIV-specific immune responses in the Japanese population. The utility of the method was investigated for the COVID-19 pandemic, by performing in silico T-cell epitope mapping of the SARS-CoV-2 spike glycoprotein according to the ethnic context of the countries where the ChAdOx1 vaccine is currently initiating phase III clinical trials. Potentially immunodominant CD8+ and CD4+ T-cell epitopes and population coverages were predicted for each population (the Epitope Discovery mode), along with optimized sets of broadly recognized (promiscuous) T-cell epitopes maximizing coverage in the target populations (the Epitope Optimization mode). Population-specific epitope-rich regions (T-cell epitope clusters) were further predicted in protein antigens based on combined criteria of epitope density and population coverage. Overall, we conclude that Predivac-3.0 holds potential to contribute in the understanding of ethnic-level variations of vaccine-induced immune responsiveness and to guide the development of epitope-based next-generation vaccines against emerging pathogens, whose geographic distributions and populations in need of vaccinations are often well-defined for regional epidemics.

Inhibition of natural killer (NK) cells is mediated by MHC class I receptors including the killer cell Ig-like receptor (KIR). We demonstrate that HLA-C binding peptides can function as altered peptide ligands for KIR and antagonize the inhibition mediated by KIR2DL2/KIR2DL3. Antagonistic peptides promote clustering of KIR at the interface of effector and target cells, but do not result in inhibition of NK cells. Our data show that, as for T cells, small changes in the peptide content of MHC class I can regulate NK cell activity.

High resolution typing of the HLA-DPB1 locus for patient who requested for hematopoietic stem cell transplantation (HSCT) workup has recently become mandatory by the National Marrow Donor Program (NMDP) in order to facilitate matching between donors and recipients for better outcomes. The likelihood of identifying HLA matched donors in Hong Kong, on top of the existing HLA-A, -B, -C, and -DRB1 loci, is revisited in this study. HLA-A, -B, -C, -DRB1 and -DPB1 genotypes of 5,266 volunteer unrelated Chinese donors from the Hong Kong Bone Marrow Donor Registry (HKBMDR), were included in this study. Matching models were employed to determine the matching probabilities for 10/10(DPB1) and 9/10(DPB1) HLA match. The matching probabilities are 20% at 10/10(DPB1) HLA match and 55% at 9/10(DPB1) match, based on the existing 130,000 donors in the HKBMDR. The likelihoods of match become 27% and 65% respectively, by increasing the registry to 250,000. However, if DPB T-cell-epitope (TCE) model is considered in the matching, the probability will increase to 46% at 10/10 DPB1 permissive mismatching. Our findings provide vital information about the future planning on the targeted recruitment size, HLA typing and search strategies of the donor registry and arose the transplant physicians’ acceptability to 9/10(DBP1) or 10/10(DBP1) HLA match. Nevertheless, the marrow donor registry has planned for increasing the registry size and bringing down the age of recruited donors which will ultimately enhance patient outcome.

There is a great deal of interest in a fine-scale population structure in the UK, both as a signature of historical immigration events and because of the effect population structure may have on disease association studies. Although population structure appears to have a minor impact on the current generation of genome-wide association studies, it is likely to have a significant part in the next generation of studies designed to search for rare variants. A powerful way of detecting such structure is to control and document carefully the provenance of the samples involved. In this study, we describe the collection of a cohort of rural UK samples (The People of the British Isles), aimed at providing a well-characterised UK-control population that can be used as a resource by the research community, as well as providing a fine-scale genetic information on the British population. So far, some 4000 samples have been collected, the majority of which fit the criteria of coming from a rural area and having all four grandparents from approximately the same area. Analysis of the first 3865 samples that have been geocoded indicates that 75% have a mean distance between grandparental places of birth of 37.3 km, and that about 70% of grandparental places of birth can be classed as rural. Preliminary genotyping of 1057 samples demonstrates the value of these samples for investigating a fine-scale population structure within the UK, and shows how this can be enhanced by the use of surnames.

Epstein-Barr virus (EBV) is the major environmental factor associated with Hodgkin's lymphoma (HL), a common lymphoma in young adults. Natural killer (NK) cells are key actors of the innate immune response against viruses. The regulation of NK cell function involves activating and inhibitory Killer cell Immunoglobulin-like receptors (KIRs), which are expressed in variable numbers on NK cells. Various viral and virus-related malignant disorders have been associated with the presence/absence of certain KIR genes in case/control studies. We investigated the role of the KIR cluster in HL in a family-based association study. We included 90 families with 90 HL index cases (age 16-35 years) and 255 first-degree relatives (parents and siblings). We developed a procedure for reconstructing full genotypic information (number of gene copies) at each KIR locus from the standard KIR gene content. Out of the 90 collected families, 84 were informative and suitable for further analysis. An association study was then carried out with specific family-based analysis methods on these 84 families. Five KIR genes in strong linkage disequilibrium were found significantly associated with HL. Refined haplotype analysis showed that the association was supported by a dominant protective effect of KIR3DS1 and/or KIR2DS1, both of which are activating receptors. The odds ratios for developing HL in subjects with at least one copy of KIR3DS1 or KIR2DS1 with respect to subjects with neither of these genes were 0.44[95% confidence interval 0.23-0.85] and 0.42[0.21-0.85], respectively. No significant association was found in a tentative replication case/control study of 68 HL cases (age 18-71 years). In the familial study, the protective effect of KIR3DS1/KIR2DS1 tended to be stronger in HL patients with detectable EBV in blood or tumour cells. This work defines a template for family-based association studies based on full genotypic information for the KIR cluster, and provides the first evidence that activating KIRs can have a protective role in HL.

Whole genome comparisons identified introgression from archaic to modern humans. Our analysis of highly polymorphic human leukocyte antigen (HLA) class I, vital immune system components subject to strong balancing selection, shows how modern humans acquired the HLA-B*73 allele in west Asia through admixture with archaic humans called Denisovans, a likely sister group to the Neandertals. Virtual genotyping of Denisovan and Neandertal genomes identified archaic HLA haplotypes carrying functionally distinctive alleles that have introgressed into modern Eurasian and Oceanian populations. These alleles, of which several encode unique or strong ligands for natural killer cell receptors, now represent more than half the HLA alleles of modern Eurasians and also appear to have been later introduced into Africans. Thus, adaptive introgression of archaic alleles has significantly shaped modern human immune systems.

There are few reported studies on Saudi population for human leukocyte antigens (HLA) genes. We investigated allele lineages (two-digit) and haplotype frequencies of HLA-A, -B, -C, -DRB1, and -DQB1 loci in 499 healthy unrelated individuals, selected from potential bone marrow transplant (BMT) families' donors at King Fahad Medical City (KFMC), Saudi Arabia (SA). Genotyping was performed by Sequence Specific Oligonucleotide Probe (SSOP) utilizing a Luminex-based method. Allele lineages and haplotype frequencies were evaluated along with principal component analysis (PCA) to compare findings with previously reported data on Arab related populations. A total of 18 allele lineages for HLA-A, 28 for -B, 14 for -C, 13 for -DRB1, and 5 for -DQB1 were detected. High values for linkage disequilibrium indicators were found for B:C (D' = 0.86599) and DRB1:DQB1 (D = 0.89468) loci. Additionally, PCA results confirmed previous findings on this population, but also indicated some genetic distances from other Arab related populations. The present study helps in further investigations of this population in anthropological analysis and HLA-associated disease studies. KEYWORDS: HLA, allele, frequencies, Saudis

Skeletal muscle functions as an endocrine organ. Exosomes, small vesicles containing mRNAs, miRNAs, and proteins, are secreted from muscle cells and facilitate cell-to-cell communication. Our recent work found greater exosome release from oxidative compared to glycolytic muscle. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key driver of mitochondrial biogenesis, a characteristic of oxidative muscle. It was hypothesized that PGC1α regulates exosome biogenesis and secretion in skeletal muscle. The purpose of this study is to determine if PGC-1α regulates skeletal muscle exosome biogenesis and secretion. On day 4 of differentiation, human primary myotubes from vastus lateralis biopsies from lean donors (BMI < 25.0 kg/m2) were exposed to adenovirus encoding human PGC-1α or GFP control. On day 6 of differentiation, culture media was replaced with exosome-free media. On day 8, cells were collected for mRNA and protein analysis, and culture media was collected for exosome isolation. Overexpression of PGC-1α increases regulators of exosome biogenesis in the endosomal sorting complexes required for transport (ESCRT) pathway: Alix (CON: 1.0 ± 0.2 vs. PGC-1α: 7.6 ± 3.8), TSG-101 (CON: 1.0 ± 0.1 vs. PGC-1α: 7.3 ± 2.1), CD63 (CON: 1.0 ± 0.17 vs. PGC-1α: 3.7 ± 0.4), Clathrin (CON: 1.0 ± 0.2 vs. PGC-1α: 11.6 ± 2.5), and the secretion pathway: Rab27b (CON: 1.0 ± 0.3 vs. PGC-1α: 3.2 ± 0.3), STAM (CON: 1.0 ± 0.3 vs. PGC-1α: 7.3 ± 0.6), and VTA1 (CON: 1.0 ± 0.1 vs. PGC-1α: 7.3 ± 2.4). Exosome count and total extracellular vesicle count were not significantly different from control. Overexpression of PGC-1α increases gene expression of regulators of exosome biogenesis and secretion in human primary myotubes. In the future, in vitro studies assessing exosomal content from PGC-1 OE cells as well as in vivoeffects of PGC-1 OE on exosome production and release should be investigated to further understand the role PGC-1 plays in exosome secretion.