Diagnostic utility of cytoplasmic 5’-nucleotidase autoantibodies to identify inclusion body myositis patients

Bundell, Christine; Shakya, Rakesh; Brusch, Anna; McLean‐Tooke, Andrew; Hollingsworth, Peter; Needham, Merrilee

doi:10.1016/j.pathol.2016.12.327

Cited by 4 publications

(6 citation statements)

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“…Anti-cN1A antibodies were measured using a semi-quantitative ELISA adapted from Bundell et al. ( 42 ). 96-well plates (Maxisorp, Nunc, Roskilde, Denmark) were coated with 10 µg/ml of cN1A protein (GenScript, NJ, SA) diluted in 50 mM carbonate-bicarbonate buffer (pH 9.6) for 2 hours at ambient temperature.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2023

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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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2023

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“…Anti‐cN1A antibodies were assessed using a semi‐quantitative ELISA protocol as outlined in McLeish et al 35 . and Bundell et al 39 . Briefly, 10 μg mL −1 of cN1A protein (GenScript, NJ, SA) were coated onto 96‐well flat‐bottom plates in 50 mM carbonate–bicarbonate buffer pH 9.6 for 2 h at RT.…”

Section: Methodsmentioning

confidence: 99%

“…Test sample measures were recorded as fold change relative to averaged negative control values. Positivity cut‐off aligned with the 99th percentile of healthy serum pool samples 39 …”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, the cells underwent both surface and intracellular staining procedures as described above for whole blood samples. Anti-cN1A ELISA Anti-cN1A antibodies were assessed using a semiquantitative ELISA protocol as outlined in McLeish et al 35 and Bundell et al 39 Briefly, 10 lg mL À1 of cN1A protein (GenScript, NJ, SA) were coated onto 96-well flat-bottom plates in 50 mM carbonate-bicarbonate buffer pH 9.6 for 2 h at RT. Plates were blocked overnight with 5% skim milk powder in PBS Tween at 4°C.…”

Section: Immunophenotyping Of Peripheral Blood Immune Cellsmentioning

confidence: 99%

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Identification of distinct immune signatures in inclusion body myositis by peripheral blood immunophenotyping using machine learning models

McLeish,

Sooda,

Slater

et al. 2024

Clin & Trans Imm

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ObjectiveInclusion body myositis (IBM) is a progressive late‐onset muscle disease characterised by preferential weakness of quadriceps femoris and finger flexors, with elusive causes involving immune, degenerative, genetic and age‐related factors. Overlapping with normal muscle ageing makes diagnosis and prognosis problematic.MethodsWe characterised peripheral blood leucocytes in 81 IBM patients and 45 healthy controls using flow cytometry. Using a random forest classifier, we identified immune changes in IBM compared to HC. K‐means clustering and the random forest one‐versus‐rest model classified patients into three immunophenotypic clusters. Functional outcome measures including mTUG, 2MWT, IBM‐FRS, EAT‐10, knee extension and grip strength were assessed across clusters.ResultsThe random forest model achieved a 94% AUC ROC with 82.76% specificity and 100% sensitivity. Significant differences were found in IBM patients, including increased CD8+ T‐bet+ cells, CD4+ T cells skewed towards a Th1 phenotype and altered γδ T cell repertoire with a reduced proportion of Vγ9+Vδ2+ cells. IBM patients formed three clusters: (i) activated and inflammatory CD8+ and CD4+ T‐cell profile and the highest proportion of anti‐cN1A‐positive patients in cluster 1; (ii) limited inflammation in cluster 2; (iii) highly differentiated, pro‐inflammatory T‐cell profile in cluster 3. Additionally, no significant differences in patients' age and gender were detected between immunophenotype clusters; however, worsening trends were detected with several functional outcomes.ConclusionThese findings unveil distinct immune profiles in IBM, shedding light on underlying pathological mechanisms for potential immunoregulatory therapeutic development.

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“…Anti-NT5C1A antibodies were analysed in the serum using an in-house semi-quantitative ELISA methodology adapted from Bundell et al 54 Flat bottom microtitre 96-well plates (Maxisorp, Nunc, Roskilde, Denmark) were coated with 10 lg mL À1 of NT5C1A protein (GenScript, NJ, SA) diluted in 50 mMM carbonate-bicarbonate buffer pH 9.6 for 2 h at ambient temperature. Wells were washed with PBS/0.1% Tween (PBST) and saturated with blocking buffer (PBST / 5% skim milk powder) overnight at 4°C.…”

Section: Anti-nt5c1a Antibodies Elisamentioning

confidence: 99%

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

et al. 2022

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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, crossover 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.

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Diagnostic utility of cytoplasmic 5’-nucleotidase autoantibodies to identify inclusion body myositis patients

Cited by 4 publications

References 0 publications

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

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

Identification of distinct immune signatures in inclusion body myositis by peripheral blood immunophenotyping using machine learning models

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

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