Uncovering the significance of expanded CD8+ large granular lymphocytes in inclusion body myositis: Insights into T cell phenotype and functional alterations, and disease severity

IntroductionInclusion body myositis (IBM) is a progressive inflammatory myopathy characterised by skeletal muscle infiltration and myofibre invasion by CD8+ T lymphocytes. In some cases, IBM has been reported to be associated with a systemic lymphoproliferative disorder of CD8+ T cells exhibiting a highly differentiated effector phenotype known as T cell Large Granular Lymphocytic Leukemia (T-LGLL). MethodsWe investigated the incidence of a CD8+ T-LGL lymphoproliferative disorder in 85 IBM patients and an aged-matched group of 56 Healthy Controls (HC). Further, we analysed the phenotypical characteristics of the expanded T-LGLs and investigated whether their occurrence was associated with any particular HLA alleles or clinical characteristics. ResultsBlood cell analysis by flow cytometry revealed expansion of T-LGLs in 34 of the 85 (40%) IBM patients. The T cell immunophenotype of T-LGLHIGH patients was characterised by increased expression of surface molecules including CD57 and KLRG1, and to a lesser extent of CD94 and CD56 predominantly in CD8+ T cells, although we also observed modest changes in CD4+ T cells and γδ T cells. Analysis of Ki67 in CD57+ KLRG1+ T cells revealed that only a small proportion of these cells was proliferating. Comparative analysis of CD8+ and CD4+ T cells isolated from matched blood and muscle samples donated by three patients indicated a consistent pattern of more pronounced alterations in muscles, although not significant due to small sample size. In the T-LGLHIGH patient group, we found increased frequencies of perforin-producing CD8+ and CD4+ T cells that were moderately correlated to combined CD57 and KLRG1 expression. Investigation of the HLA haplotypes of 75 IBM patients identified that carriage of the HLA-C*14:02:01 allele was significantly higher in T-LGLHIGH compared to T-LGLLOW individuals. Expansion of T-LGL was not significantly associated with seropositivity patient status for anti-cytosolic 5'-nucleotidase 1A autoantibodies. Clinically, the age at disease onset and disease duration were similar in the T-LGLHIGH and T-LGLLOW patient groups. However, metadata analysis of functional alterations indicated that patients with expanded T-LGL more frequently relied on mobility aids than T-LGLLOW patients indicating greater disease severity. ConclusionAltogether, these results suggest that T-LGL expansion occurring in IBM patients is correlated with exacerbated immune dysregulation and increased disease burden

Adolescent transport and unintentional injuries: a systematic analysis using the Global Burden of Disease Study 2019

Background: Globally, transport and unintentional injuries persist as leading preventable causes of mortality and morbidity for adolescents. We sought to report comprehensive trends in injury-related mortality and morbidity for adolescents aged 10–24 years during the past three decades. Methods: Using the Global Burden of Disease, Injuries, and Risk Factors 2019 Study, we analysed mortality and disability-adjusted life-years (DALYs) attributed to transport and unintentional injuries for adolescents in 204 countries. Burden is reported in absolute numbers and age-standardised rates per 100 000 population by sex, age group (10–14, 15–19, and 20–24 years), and sociodemographic index (SDI) with 95% uncertainty intervals (UIs). We report percentage changes in deaths and DALYs between 1990 and 2019. Findings: In 2019, 369 061 deaths (of which 214 337 [58%] were transport related) and 31·1 million DALYs (of which 16·2 million [52%] were transport related) among adolescents aged 10–24 years were caused by transport and unintentional injuries combined. If compared with other causes, transport and unintentional injuries combined accounted for 25% of deaths and 14% of DALYs in 2019, and showed little improvement from 1990 when such injuries accounted for 26% of adolescent deaths and 17% of adolescent DALYs. Throughout adolescence, transport and unintentional injury fatality rates increased by age group. The unintentional injury burden was higher among males than females for all injury types, except for injuries related to fire, heat, and hot substances, or to adverse effects of medical treatment. From 1990 to 2019, global mortality rates declined by 34·4% (from 17·5 to 11·5 per 100 000) for transport injuries, and by 47·7% (from 15·9 to 8·3 per 100 000) for unintentional injuries. However, in low-SDI nations the absolute number of deaths increased (by 80·5% to 42 774 for transport injuries and by 39·4% to 31 961 for unintentional injuries). In the high-SDI quintile in 2010–19, the rate per 100 000 of transport injury DALYs was reduced by 16·7%, from 838 in 2010 to 699 in 2019. This was a substantially slower pace of reduction compared with the 48·5% reduction between 1990 and 2010, from 1626 per 100 000 in 1990 to 838 per 100 000 in 2010. Between 2010 and 2019, the rate of unintentional injury DALYs per 100 000 also remained largely unchanged in high-SDI countries (555 in 2010 vs 554 in 2019; 0·2% reduction). The number and rate of adolescent deaths and DALYs owing to environmental heat and cold exposure increased for the high-SDI quintile during 2010–19. Interpretation: As other causes of mortality are addressed, inadequate progress in reducing transport and unintentional injury mortality as a proportion of adolescent deaths becomes apparent. The relative shift in the burden of injury from high-SDI countries to low and low–middle-SDI countries necessitates focused action, including global donor, government, and industry investment in injury prevention. The persisting burden of DALYs related to transport and unintentional injuries indicates a need to prioritise innovative measures for the primary prevention of adolescent injury. Funding: Bill & Melinda Gates Foundation

Global, regional, and national mortality among young people aged 10-24 years, 1950-2019: a systematic analysis for the Global Burden of Disease Study 2019

Background Documentation of patterns and long-term trends in mortality in young people, which reflect huge changes in demographic and social determinants of adolescent health, enables identification of global investment priorities for this age group. We aimed to analyse data on the number of deaths, years of life lost, and mortality rates by sex and age group in people aged 10-24 years in 204 countries and territories from 1950 to 2019 by use of estimates from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019. Methods We report trends in estimated total numbers of deaths and mortality rate per 100 000 population in young people aged 10-24 years by age group (10-14 years, 15-19 years, and 20-24 years) and sex in 204 countries and territories between 1950 and 2019 for all causes, and between 1980 and 2019 by cause of death. We analyse variation in outcomes by region, age group, and sex, and compare annual rate of change in mortality in young people aged 10-24 years with that in children aged 0-9 years from 1990 to 2019. We then analyse the association between mortality in people aged 10-24 years and socioeconomic development using the GBD Socio-demographic Index (SDI), a composite measure based on average national educational attainment in people older than 15 years, total fertility rate in people younger than 25 years, and income per capita. We assess the association between SDI and all-cause mortality in 2019, and analyse the ratio of observed to expected mortality by SDI using the most recent available data release (2017). Findings In 2019 there were 1.49 million deaths (95% uncertainty interval 1.39-1.59) worldwide in people aged 10-24 years, of which 61% occurred in males. 32.7% of all adolescent deaths were due to transport injuries, unintentional injuries, or interpersonal violence and conflict; 32.1% were due to communicable, nutritional, or maternal causes; 27.0% were due to non-communicable diseases; and 8.2% were due to self-harm. Since 1950, deaths in this age group decreased by 30.0% in females and 15.3% in males, and sex-based differences in mortality rate have widened in most regions of the world. Geographical variation has also increased, particularly in people aged 10-14 years. Since 1980, communicable and maternal causes of death have decreased sharply as a proportion of total deaths in most GBD super-regions, but remain some of the most common causes in sub-Saharan Africa and south Asia, where more than half of all adolescent deaths occur. Annual percentage decrease in all-cause mortality rate since 1990 in adolescents aged 15-19 years was 1.3% in males and 1.6% in females, almost half that of males aged 1-4 years (2.4%), and around a third less than in females aged 1-4 years (2.5%). The proportion of global deaths in people aged 0-24 years that occurred in people aged 10-24 years more than doubled between 1950 and 2019, from 9.5% to 21.6%. Interpretation Variation in adolescent mortality between countries and by sex is widening, driven by poor progress in reducing deaths in males and older adolescents. Improving global adolescent mortality will require action to address the specific vulnerabilities of this age group, which are being overlooked. Furthermore, indirect effects of the COVID-19 pandemic are likely to jeopardise efforts to improve health outcomes including mortality in young people aged 10-24 years. There is an urgent need to respond to the changing global burden of adolescent mortality, address inequities where they occur, and improve the availability and quality of primary mortality data in this age group. Copyright (C) 2021 The Author(s). Published by Elsevier Ltd

In vitro selection of DNA aptamers that neutralise autoantibodies to cytosolic 5’-nucleotidase-1A

Background. Inclusion Body Myositis (IBM) is the most commonly diagnosed inflammatory muscle disease associated with aging. It’s characterised by degeneration of skeletal muscles that inevitably leads to the loss of mobility. The mechanisms that drive IBM pathogenicity remain poorly understood. However, studies reveal that on average 50% of IBM sera contains circulating autoantibodies targeting cytosolic 5’-nucleotidase 1A (cN1A) – a metabolic enzyme that is highly expressed in skeletal muscles. Accumulating evidence suggests that anti-cN1A antibodies may play a role in IBM pathogenesis making them an attractive target for aptamer development. Problem. IBM remains refractive to current immunosuppressive treatments. Preventing anti-cN1A antibodies binding to their target protein may reduce the severity of IBM symptoms and halt the disease progression. Aim. Within this project, we aim to select DNA aptamers that conjugate with anti-cN1A binding sites and prevent their interaction with cN1A protein. Results. Sera of forty-eight IBM patients were screened with enzyme-linked immunosorbent assay (ELISA) against purified cN1A protein. Eighteen patients (38%) were found seropositive. 6.5 mg of polyclonal anti-cN1A were purified out of 90 ml of a selected IBM patient’s blood. Their biological activity against cN1A was confirmed by ELISA. Purified anti-cN1A were used as a target for aptamer selection. Initial library of single-stranded DNA fragments (aptamers) was synthesised with a 40-nucleotide randomised region flanked by constant primer-binding sites. Neutralising aptamers were selected through the process of systematic evolution of ligands by exponential enrichment (SELEX). Two complete cycles of selection against anti-cN1A were performed. Selected aptamers were assayed for their neutralising capacity by ELISA. Anti-cN1A were pre-incubated with the aptamers at various concentrations. Following a one-hour incubation, the ELISA-obtained absorbance remained unchanged or increased in a dose-dependent manner. While upon an extended overnight incubation, a 25% decrease in absorbance values corresponding to a 35% decrease in anti-cN1A binding was observed. Conclusions. The methodology developed during this project resulted in the generation of aptamers that conjugate with anti-cN1A binding sites. Following two SELEX cycles, the selected aptamers demonstrate 35% neutralisation capacity upon prolonged incubation with the immunoglobulins. Our results strongly suggest that the aptamers target monomeric IgG and IgA while having low or no affinity for IgM anti-cN1A

Isolation of live leukocytes from human inflammatory muscles

In inflammatory myopathies, the self-reactive immune cells involved in muscle aggression have been studied mostly using histological assessment of muscle biopsy sections; this methodology provides the advantage of visualizing and identifying cells within the tissue, but it does not allow further investigation. To gain access to live and isolated cells, many studies utilized blood samples; however, in the absence of biological tools to discriminate the leukocytes associated with the autoimmune process from those that emerged from responses against pathogens, the information observed on circulating immune cells often lacks in specificity, and thus result interpretation may prove difficult. In order to selectively retrieve self-reactive immune cells, we developed a protocol to isolate live leukocytes from human muscle biopsies, which allows for further analysis using a large range of methodologies. The protocol uses enzymatic digestion to release live leukocytes from freshly collected skeletal muscle samples, followed by filtration and separation of the leukocytes from the myocytes by density gradient centrifugation. The isolated cells can be submitted immediately to various analysis strategies to characterize ex vivo the specific cellular and molecular mechanisms responsible for self-directed immune muscle aggression or may be placed in culture for expansion

Uncovering the significance of expanded CD8+ large granular lymphocytes in inclusion body myositis: Insights into T cell phenotype and functional alterations, and disease severity

Introduction: Inclusion body myositis (IBM) is a progressive inflammatory myopathy characterised by skeletal muscle infiltration and myofibre invasion by CD8+ T lymphocytes. In some cases, IBM has been reported to be associated with a systemic lymphoproliferative disorder of CD8+ T cells exhibiting a highly differentiated effector phenotype known as T cell Large Granular Lymphocytic Leukemia (T-LGLL). Methods: We investigated the incidence of a CD8+ T-LGL lymphoproliferative disorder in 85 IBM patients and an aged-matched group of 56 Healthy Controls (HC). Further, we analysed the phenotypical characteristics of the expanded T-LGLs and investigated whether their occurrence was associated with any particular HLA alleles or clinical characteristics. Results: Blood cell analysis by flow cytometry revealed expansion of T-LGLs in 34 of the 85 (40%) IBM patients. The T cell immunophenotype of T-LGLHIGH patients was characterised by increased expression of surface molecules including CD57 and KLRG1, and to a lesser extent of CD94 and CD56 predominantly in CD8+ T cells, although we also observed modest changes in CD4+ T cells and γδ T cells. Analysis of Ki67 in CD57+ KLRG1+ T cells revealed that only a small proportion of these cells was proliferating. Comparative analysis of CD8+ and CD4+ T cells isolated from matched blood and muscle samples donated by three patients indicated a consistent pattern of more pronounced alterations in muscles, although not significant due to small sample size. In the T-LGLHIGH patient group, we found increased frequencies of perforin-producing CD8+ and CD4+ T cells that were moderately correlated to combined CD57 and KLRG1 expression. Investigation of the HLA haplotypes of 75 IBM patients identified that carriage of the HLA-C*14:02:01 allele was significantly higher in T-LGLHIGH compared to T-LGLLOW individuals. Expansion of T-LGL was not significantly associated with seropositivity patient status for anti-cytosolic 5\u27-nucleotidase 1A autoantibodies. Clinically, the age at disease onset and disease duration were similar in the T-LGLHIGH and T-LGLLOW patient groups. However, metadata analysis of functional alterations indicated that patients with expanded T-LGL more frequently relied on mobility aids than T-LGLLOW patients indicating greater disease severity. Conclusion: Altogether, these results suggest that T-LGL expansion occurring in IBM patients is correlated with exacerbated immune dysregulation and increased disease burden

High-resolution HLA genotyping in inclusion body myositis refines 8.1 ancestral haplotype association to DRB1*03:01:01 and highlights pathogenic role of arginine-74 of DR?1 chain

Objectives: Inclusion body myositis (IBM) is a progressive inflammatory-degenerative muscle disease of older individuals, with some patients producing anti-cytosolic 5\u27-nucleotidase 1A (NT5C1A, aka cN1A) antibodies. Human Leukocyte Antigens (HLA) is the highest genetic risk factor for developing IBM. In this study, we aimed to further define the contribution of HLA alleles to IBM and the production of anti-cN1A antibodies. Methods: We HLA haplotyped a Western Australian cohort of 113 Caucasian IBM patients and 112 ethnically matched controls using Illumina next-generation sequencing. Allele frequency analysis and amino acid alignments were performed using the Genentech/MiDAS bioinformatics package. Allele frequencies were compared using Fisher\u27s exact test. Age at onset analysis was performed using the ggstatsplot package. All analysis was carried out in RStudio version 1.4.1717. Results: Our findings validated the independent association of HLA-DRB1*03:01:01 with IBM and attributed the risk to an arginine residue in position 74 within the DRβ1 protein. Conversely, DRB4*01:01:01 and DQA1*01:02:01 were found to have protective effects; the carriers of DRB1*03:01:01 that did not possess these alleles had a fourteenfold increased risk of developing IBM over the general Caucasian population. Furthermore, patients with the abovementioned genotype developed symptoms on average five years earlier than patients without. We did not find any HLA associations with anti-cN1A antibody production. Conclusions: High-resolution HLA sequencing more precisely characterised the alleles associated with IBM and defined a haplotype linked to earlier disease onset. Identification of the critical amino acid residue by advanced biostatistical analysis of immunogenetics data offers mechanistic insights and future directions into uncovering IBM aetiopathogenesis

Immunoregulatory effects of testosterone supplementation combined with exercise training in men with Inclusion Body Myositis: a double-blind, placebo-controlled, cross-over trial

Objectives Sporadic Inclusion Body Myositis (IBM) is an inflammatory muscle disease affecting individuals over the age of 45, leading to progressive muscle wasting, disability and loss of independence. Histologically, IBM is characterised by immune changes including myofibres expressing major histocompatibility complex molecules and invaded by CD8+ T cells and macrophages, and by degenerative changes including protein aggregates organised in inclusion bodies, rimmed vacuoles and mitochondrial abnormalities. There is currently no cure, and regular exercise is currently the only recognised treatment effective at limiting muscle weakening, atrophy and loss of function. Testosterone exerts anti-inflammatory effects, inhibiting effector T-cell differentiation and pro-inflammatory cytokine production. Methods We conducted a double-blind, placebo-controlled, cross-over trial in men with IBM, to assess whether a personalised progressive exercise training combined with application of testosterone, reduced the inflammatory immune response associated with this disease over and above exercise alone. To assess intervention efficacy, we immunophenotyped blood immune cells by flow cytometry, and measured serum cytokines and chemokines by Luminex immunoassay. Results Testosterone supplementation resulted in modest yet significant count reduction in the classical monocyte subset as well as eosinophils. Testosterone-independent immunoregulatory effects attributed to exercise included altered proportions of some monocyte, T- and B-cell subsets, and reduced IL-12, IL-17, TNF-α, MIP-1β and sICAM-1 in spite of interindividual variability. Conclusion Overall, our findings indicate anti-inflammatory effects of exercise training in IBM patients, whilst concomitant testosterone supplementation provides some additional changes. Further studies combining testosterone and exercise would be worthwhile in larger cohorts and longer testosterone administration periods