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ANTIBODIES & COMPLEMENT IN MYOPATHIES AND NMJ DISORDERS

General topics Antibodies   Acetylcholine receptor   ANCA   Cytoplasmic   DNA   Myositis-overlap   Myositis-specific   Nuclear   Nucleolar   Ribonucleoproteins   Striational   tRNA synthetases   Types Complement Connective tissue disorders Lambert-Eaton myasthenia M-protein   Amyloid: Light chain   Anti-Decorin myopathy: IgM   Scleromyxedema: IgG Myasthenia gravis Neuropathy Scleroderma

Specific antibodies 56 kD RNP component CADM-140 Centromere Double-stranded DNA Fibrillarin Fibroblast Giantin Histones Jo-1 Ku Mi-2 Nucleosome p155 Phospholipid PM-Scl Rheumatoid factors RNP   Nuclear     U1-nRNP     U2-nRNP     U3-RNP   Ribosomal Signal Recognition Particle Sm SS-56 SSA (Ro) SSB (La)

Normal IgG

• Anti-Acetylcholine Receptor (IgG)
• MuSK (IgG)
• Anti-Striational (IgG): With Thymoma

• Anti-P-type Calcium Channel (IgG)

• Clinical features
  • Onset: 7th deacde
  • Weakness
    • Proximal
    • Symmetric
  • Tendon reflexes: Present
  • Course: Slowly progressive to moderate disability
• Laboratory
  • Serum CK: High, ~ 1,000
  • IgMκ M-protein ± Waldenström's macroglobulinemia
  • Serum IgM vs Decorin (BJ antigen): Testing
    • Decorin antigen
      • Proteoglycan
      • Located in muscle basal lamina
    • Antigenic epitope: Chondroitin sulfate carbohydrate moiety
  • EMG: Myopathic ± Spontaneous activity
  • Muscle pathology
    • Variable muscle fiber size
    • Endomysial fibrosis
    • Large, irregularly shaped myonuclei
    • Endomysial deposition of IgM
    • Serum IgM binding to muscle endomysium

ANTIBODIES IN CONNECTIVE TISSUE DISORDERS¹

MYOSITIS-ASSOCIATED ANTIBODIES
Autoantibody	Antigen	Antigen function	Clinical associations	HEp-2 IIF pattern
Aminoacyl t-RNA synthetase antibodies
Jo-1	Histidyl-tRNA synthetase	Protein synthesis   Intracytoplasmic Amino acid binding   to cognate tRNA	PM 30%; DM-like 13% Lung fibrosis Mechanics hands Raynauds Arthritis Fever Specificity > 95% HLA DRw52	Cytoplasm: Speckled
PL-7	Threonyl-tRNA synthetase		PM/DM 3%-5% Lung fibrosis Similar to Jo-1	Cytoplasm: Speckled
PL-12	Alanyl-tRNA synthetase		Lung fibrosis PM/DM 3% Similar to KS	Cytoplasm Speckled (dense)
EJ	Glycyl-tRNA synthetase		PM <3% Antibody +: DM 80% Lung fibrosis	Cytoplasm
OJ	Isoleucyl-tRNA synthetase		PM/DM <3% Lung fibrosis	Cytoplasm
KS	Asparaginyl-tRNA synthetase12		Interstitial lung disease Myositis: Some patients Japanese	Cytoplasm
Zo	Phenylalanyl-tRNA synthetase		Antisynthetase syndrome	Cytoplasm
YRS	Tyrosyl-tRNA synthetase		Antisynthetase syndrome	Cytoplasm
Other antibodies
SRP	Signal recognition particle   54 kD protein   In 7SL-RNAcomplex	Protein translocation   Intracytoplasmic   (Endoplasmic reticulum)	PM 5%; Specificity 93% Acute onset; Severe HLA DRw52	Cytoplasm: Speckled Nucleolus
Mi-2	Helicase protein part of   NuRD complex	Nuclear transcription	DM 15% -35% PM 5%-9% HLA DRw53 Nailfold lesions	Nucleoplasm Fine speckled
PMS1 6	PMS1	DNA mismatch   repair enzyme	Immune myopathy (7%) DM/PM/Fasciitis
p155/140 14	Transcriptional intermediary   factor 1γ	Nuclear transcription Cellular differentiation	Dermatomyositis (20%) Skin ulceration & edema Cancer-associated DM (75%)	Nucleus
CADM-14015	MDA5 (IFIH1)	Immunity vs viral infection Detection of viral dsDNA	Dermatomyositis, Amyopathic Lung disease, rapidly progressive	Cytoplasmic
MJ (p140) MU	NXP-2; 140 kD protein	Nuclear transcription	Dermatomyositis, Juvenile   Calcinosis	Cytoplasm
SAE	Small-ubiquitin-like   modifier enzyme (SUMO)	Post-translational modification   Nuclear transcription factors	Dermatomyositis, adult   Amyopathic early   Dysphagia
Decorin	Chondroitin sulfate		Immune myopathy	Basal lamina
Ku	Thyroid autoantigen, 70-kD   G22P1	DNA protein kinase   regulatory subunit	Systemic sclerosis PM/Scleroderma Japanese	Nucleolus & Nucleus Homogeneous
KJ		? Translation factor	PM < 1%; Raynaud's Lung Fibrosis	Cytoplasm Speckled (dense)

MYOSITIS-OVERLAP ANTIBODIES Associated with PM and other connective tissue disorders Sensitivity for connective tissue disorders up to 95% Antibody class: IgG
Autoantibody	Antigen	Clinical associations	HEp-2 IIF pattern
PM-Scl	Exosome: RNA processing complex PM/Scl-100 PM/Scl-75	PM 8%-12% Scleroderma 25% Caucasian	Nucleolus & Nucleus Homogeneous
56 kD	RNP component	PM/DM 87% Other CTD 10% High with active disease	?
SSA/Ro	2 proteins   & 4 small RNAs	PM/DM 5%-10% Sjögren's 90%	Nucleoplasm Fine speckled
U1-nRNP	3 proteins:   22, 33 & 70 kD	PM/DM 4%-17% SLE & Scleroderma 30% MCTD 95%	Nucleoplasm Speckled
U2-nRNP	Small nuclear RNP	PM/DM 4%-17% SLE & Scleroderma 30%	Nucleoplasm Speckled
Fer	Elongation factor 1a	PM/DM (rare)	?
MAS	tRNA bindingSer   protein	PM/DM (rare) HLA DRw53 Rhabdomyolysis Myalgias	Diffuse cytoplasm

• anti-tRNA synthetases (IgG)
  • Antigen: Jo-1 = histidyl tRNA synthetase : 50-52 kD
    • Clinical syndrome
      • Myositis: Perimysial connective tissue damage
      • Interstitial Lung Disease
      • Raynaud's
      • Arthritis
      • Onset: Spring
    • ANA: Cytoplasmic (speckled)
    • Immunology
      • Associated with HLA-DR3/-DRw52
        
      • Antibody is IgG1 isotype
    • Pathology: Immune myopathy with perimysial pathology
    • Therapy
      • Myopathy: Often improves with corticosteroids
      • Lung disease: Less responsive
        
      • Prominent flares when tapering drugs
  • Other target antigens: Associated with similar clinical syndrome & cytoplasmic ANA
    • PL-12 = alanyl :
    • PL-7 = threonyl
    • EJ = glycyl
    • OJ = isoleucyl
    • KS = asparaginyl
    • Lysyl
• anti-KJ
  • ANA: Cytoplasm (dense speckled)
  • Antigen: Unidentified factor in protein translation
  • Clinical: Similar to anti-synthetase antibodies
• anti-Signal Recognition Particle
  • ANA: Cytoplasm (speckled) and nucleolus
  • Antigen: 54 kD protein
    • In signal recognition particle with 6 proteins and 7SL-RNA
    • Transfers secretory proteins from ribosome to TRAM (translocating chain-associating membrane protein)
    • Interacts with RNPS1
  • HLA DRw52
  • Clinical
    • Onset: Acute; Fall
    • Severe myositis
    • No overlap with other connective tissue diseases
    • Steroid responsive early; Resistant to treatment later
• anti-Ribosomal RNP
  • Antigens: Phosphoproteins in elongation step of protein synthesis
  • ANA: Cytoplasmic (dense); Nucleolar (homogeneous)
  • Clinical
    • Clinical associations: SLE
      • Prevalence: 10% to 20%
        
      • Cerebritis with psychosis: 56% to 90%
• 56 kD RNP component
  • ANA: ?
  • Clinical associations
    • Polymyositis/Dermatomyositis: 87%
    • Other connective tissue diseases: 10%
    • Titers related to disease activity
• Giantin¹⁰
  • Location: Golgi
  • Molecular weight: 372 kD
  • Disease associations
    • Myopathy with reduced α-dystroglycan
    • Connective tissue disorders: Sjögren; Scleroderma; Rheumatoid arthritis
    • HIV positive: Low titer
• p140¹⁵
  • p140 antigen
    • Identity: NXP-2 protein
    • Location: Cytoplasm
  • Clinical associations: Amyopathic dermatomyositis with interstitial lung disease
    • Clinical
      • Rash: Typical dermatomyositis pattern; Heliotrope erythema
      • Gottron’s sign
      • Strength: Normal
      • No muscle pain
      • Juvenile DM: Calcinosis
      • Adults: Interstitial lung disease; Often acute & progressive
    • Laboratory
      • Serum CK: Normal
      • HLA association: DRB1*08

• anti-Centromere antibodies¹³
  • ANA: Diffuse speckled nuclear
  • HLA type association: Non-leucine residue at position 26 of HLA-DQB1 chain
  • Protein target: Antigens localized at the kinetochore plates
    • Primary Scleroderma: Centromere protein-B (CENP-B)
    • Other proteins: CENP-A ; CENP-C
  • Clinical
    • Systemic scleroderma, Primary
      • Sensitivity: 5% to 33%
      • Specificity: High (99%)
      • Higher frequency of multisystem involvement
        • Raynaud’s phenomenon
        • Xerophthalmia
        • Peripheral neuropathy (25% vs 2%)
        • Primary biliary cirrhosis
      • Less frequent: Anti-SSA or SSB antibodies
    • Scleroderma variants
      • CREST (60% to 80%)
      • Raynaud's: Primary
    • Other associations
      • Raynaud’s disease with primary biliary cirrhosis (30%)
      • Systemic lupus erythematosus (4%)
      • Mixed connective tissue disease(8%)
      • Rheumatoid arthritis (1% to 7%)
• anti-dsDNA (Double-stranded DNA) antibodies
  • ANA: Homogeneous nuclear pattern
  • Clinical
    • Active SLE (40% to 70%)
    • Nephropathy: High avidity antibody
    • CNS: Low avidity antibody
  • In vivo target: Nucleosome
• anti-Mi-2 antibody
  • ANA: Speckled nuclear pattern
  • Target Antigens: Mi-2α (CHD3) & Mi-2β (CHD4)
    • SNF2-superfamily helicase: Contains zinc fingers & helicase motifs
    • DNA-binding: Transcriptional regulation at chromosomal level
    • Component of nucleosome remodeling & deacetylase machinery
    • 220 to 260 kD
  • HLA type associations
    • DRw53; DR7 in most
    • Tryptophan at position 9 of DRβ chain
  • Clinical associations
    • Dermatomyositis without other associated connective tissue disorders
    • Onset: Acute; Adult
    • Nailfold involvement
    • ANA + (100%)
• anti-Ro/SSA
  • hY RNP peptide (60 kD) antigen
    • Associated with hY RNA (short & uridine rich) in ribonucleoproteins
    • Location
      • Mostly in cytoplasmic compartment (70%)
      • Few in nuclear compartment (30%)
      • Cytoplasmic membrane or in small blebs during apoptosis
    • ? Involved in ribosomal RNA discard pathway
      • Binds misfolded 5S rRNA precursors
      • May function in quality-control pathway for ribosome biogenesis
    • ? Protect against autoantibody development
      • Mechanism: Sequester defective ribonucleoproteins from immune surveillance
    • Knockout mice: Develop autoimmune syndrome
      • Antibodies: Anti-ribosome; Antichromatin
      • Glomerulonephritis
      • Some strains: Sensitivity to irradiation with UV light
  • Other components of Ro/SSA
    • 52 kD antigen
      • Some antibodies also bind to this polypeptide
      • Interrupts atrioventricular conduction & L-calcium channel influx of fetal cardiocytes
      • Associated with Anti-Jo-1 antibody
    • 54 kD peptide
  • ANA: Fine speckled
  • Clinical
    • Sjögren's syndrome, Primary (50% to 60%): Increased frequency of
      • Earlier disease onset
      • Longer disease duration
      • Salivary gland: Enlargement; Severity of lymphocytic infiltration of minor salivary glands
      • Extraglandular manifestations: Lymphadenopathy; Purpura; Vasculitis
    • Lupus (35% to 50%): Specific syndromes
      • Cutaneous (70%)
      • Neonatal (born of SSA mothers) associations
        • Congenital complete heart block
        • QT interval prolongation
      • Old onset (> 90%)
      • C2 & C4 deficiencies
      • ANA-negative
      • Asian (50% to 60%)
    • Immune myopathies: Common in Koreans (24%)
• anti-La/SSB
  • 48 kD antigen: Termination factor for RNA polymerase III
  • ANA: Fine speckled
  • HLA association: Leucine at position 26 of HLA-DQB1 chain
  • Clinical
    • Lupus; Especially neonatal
    • Sjögren's syndrome
• anti-SS-56
  • Structurally related to 52-kDa Ro/SSA antigen
  • Localization: Perinuclear cytoplasmic
  • Associated with: Visceral complications in SLE
  
  
• anti-Histones
  • ANA: Homogeneous nuclear pattern
  • Clinical
    • Drug induced SLE (95%)
    • Systemic sclerosis with lung fibrosis
• anti-Nucleosomes³
  • Definition: Fundamental unit of chromatin
  • Composition
    • Octamer of 2 copies each of Histones H2A, H2B, H3 & H4
    • 146 base pair helical DNA wraps structure
  • Generated during apoptosis
  • Nucleosomes are immunogen for & in vivo target of lupus anti-dsDNA & anti-histone antibodies
  • Disease sensitivity & specificity of anti-nucleosome antibodies
    • IgG: SLE (72%); Scleroderma (46%); Mixed connective tissue disease (45%)
    • IgM: SLE (66%); Scleroderma (22%); Mixed connective tissue disease (45%); Antiphospholipid syndrome (25%)
    • Overall presence in SLE
      • Frequency: 80% to 90%
      • Disease activity: Present in active & inactive disease; Titers correlate with activity
      • IgG3 anti-nucleosome antibodies may play role in pathogenesis of lupus
• anti-Sm antibodies
  • Targets: Core proteins of U-snRNPs
    • Proteins: Sm-B/B'; D
    • May cross-react with U-nRNPs
  • ANA: Coarse speckled nuclear pattern
  • Clinical association: SLE
    • Prevalence: 15% to 30%
      
    • Specificity: 99%
      
    • Correlation with: Disease activity; CNS involvement
• anti-U1-nRNP : 70 kDa polypeptide
  • ANA: Coarse speckled nuclear pattern
  • Mixed connective tissue disease: 95% to 100%
    • Swollen hands & fingers
    • Synovitis
    • Muscle: Myositis or Myalgia
    • Raynaud's
    • ± Acrosclerosis
  • Associated disorders: Antibody is not specific
    • SLE: 30%
    • Scleroderma (Systemic Sclerosis): 15%
    • Rheumatoid arthritis
• anti-U2-nRNP
  • ANA: Speckled ribonuclear pattern
  • Clinical association: Scleroderma
• anti-U3-RNP¹⁶
  • Clinical syndrome association: Scleroderma (Systemic sclerosis (SS))
  • More common with
    • African American race
    • Male sex
  • SS higher frequency of
    • Skeletal muscle involvement
    • Pulmonary arterial hypertension
• anti-Topoisomerase (Scl-70)
  • Nicking closing activity on DNA
  • Disease associations
    • Systemic sclerosis
    • SLE (26%): Correlates with Disease activity, Pulmonary hypertension, & Nephritis
• p155 antibody¹⁴
  • Antigen: Transcriptional intermediary factor 1γ
  • Location: Nuclear
  • Clinical syndrome: Dermatomyositis with cancer
    • V-sign rash and shawl sign
    • No interstitial lung disease
  • Syndrome associations
    • Juvenile dermatomyositis (29%)
    • Juvenile CTD-associated myositis (33%)
    • Adult dermatomyositis (21%)
    • Adult CTD-associated myositis (15%)
    • Cancer-associated dermatomyositis (75%)
  • HLA risk factor: DQA1*0301 in Caucasian patients

• anti-PM-Scl (IgG) antibody⁹
  • Target antigens:
    • PM-Scl proteins
      • Location: Exosome; Nucleolar & nuclear protein complex (11 proteins)
      • Exoribonucleases: Involved in the degradation & processing of many different RNA species
    • 100 kD protein
      • Major antigenic protein for many sera
      • Associated with PM-Scl clinical syndrome
    • 40 kD protein (PM/Scl-75)
      • Common target antigen for PM/Scleroderma overlap syndrome (28%)
    • Other PM-Scl protein antigenic targets
      • hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p
  • ANA features of antibody
    • Pattern: Homogeneous
    • Location: Nucleolar & Nuclear
  • Epidemiology
    • Common in Polish immune myopathy cohorts
    • Absent in Japan
    • Associated with HLA-DR3: 75% to 100%
    • Frequency
      • PM/Scleroderma overlap syndrome: 24%
      • Scleroderma: 2%
      • PM: 6%
      • PM-Scl antibodies: 43% to 88% have PM/Scleroderma overlap syndrome
    • Relation to dsDNA antibodies
      • PM-Scl mainly associated with Myositis & Scleroderma when anti-dsDNA antibodies are absent
  • Clinical associations: Polymyositis + Scleroderma
    • Weakness
    • Skin
      • Dermatomyositis-like
      • Machinist's hands
    • Raynaud's phenomenon
    • Calcinosis
    • Telangectasia
    • Other: Arthritis; Pulmonary disease
  • Course
    • Treatment: Syndromes often improve with immunosuppression
    • Prognosis: Often good
• anti-Fibrillarin (U3-RNP)
  • ANA: Clumpy nucleolar
  • Antigen: 34 kD protein in U3-RNP particle
  • Clinical association
    • CREST
    • Systemic sclerosis: Skeletal muscle; Small bowel; Pulmonary hypertension
• anti-Ku (IgG)
  • ANA: Speckled & nucleolar; 100%
  • Antigens: 60-70 & 80-86 kD proteins
  • Antibody occurrence
    • More common in females
    • Age: Mean 60 years; Range 19 to 83
  • Clinical associations: Varied
    • Polymyositis + Scleroderma in Japan
    • Raynaud's phenomenon: 79%
    • Myositis: 50%
    • Arthralgias: 86%
    • Esophageal dysmotility: 35%
    • Pulmonary fibrosis: 40%

• Double stranded (dsDNA)
• Native: SLE > > other connective tissue disease
• Single stranded
  • 70% in active SLE
  • Many connective tissue disorders
  • ANA: Negative
• In vivo target: Nucleosome

• U1RNP
• U2RNP
• Sm: SLE
• SSA (Ro) & SSB (La)
• anti-56kD

• Target antigens
  • β₂-glycoprotein I (β2GPI; Apolipoprotein H)
    • Antibodies account for anti-cardiolipin reactivity
    • Associated with lupus anticoagulant activity: Especially IgG
  • Prothrombin (Coagulation factor II)
  • Other antigens
    • Phosphatidyl-ethanolamine/kininogens: Early recurrent pregnancy loss
    • Lamin B1
• Clinical features
  • Vascular thrombosis: Arterial, Venous, or Small vessel; No inflammation
  • Pregnancy morbidity
• Laboratory
  • Anti-cardiolipin antibody
  • Lupus anti-coagulant
• Treatment: Anticoagulation
  • Warfarin
  • Heparin/Low-dose aspirin

• Induce fibroblast activation in vitro
• Present in 46% of patients with Systemic sclerosis (SSc)
• Stimulate monocyte adhesion to fibroblasts by
  • Up-regulation
    • Intercellular adhesion molecule 1 (ICAM-1)
    • Fibroblast secretion of interleukin-1 (IL-1) and monocyte chemotactic protein 1
  • Steady-state increase of messenger RNA for cytokines and chemokines

• General
  • RF definition: Antibodies binding to Fc portion of IgG
  • Other disorders with RF
    • Systemic lupus erythematosus: 15%–35%
    • Sjögren’s syndrome: 20%–30%
    • Cryoglobulinemia: 40%–100%
    • Systemic sclerosis: 20%–30%
    • Polymyositis/dermatomyositis: 5%–10%
    • Healthy individuals: 2%–10%
  • Correlations with higher titers
    • Systemic symptoms
    • Vasculitis
    • More severe disease
• IgM: Present in 90% of patients with rheumatoid arthritis
• IgG
  • Present in 65% of patients with rheumatoid arthritis
  • Associated with localized or monoarticular disease
• IgA
  • Present in 65% of patients with rheumatoid arthritis
  • Associated with bone erosions

• Antibodies: Centromere & Fibrillarin
• Neuropathy: Vasculitic (1%)

COMPLEMENT IN HUMAN MUSCLE DISEASE²

• Proteins
  • Several proteins in the complement cascade are proteolytically activated serine proteases
• Classical pathway
  • Triggered by antigen-antibody complexes
  • C1 binds complexes & is activated
  • C4b2a formation is catalyzed by activated C1
  • C3b: Produced from C3 ; binds C4b2a
  • C5b: Produced from C5 ; Catalyzed by C4b2a3b
  • Membrane attack complex (MAC):
    • Formed by the binding of C6 , C7 , C8 & C9 to C5b
• Alternative pathway
  • Spontaneously available C3b binds Factor B
  • C3bBb: Formed by cleavage catalyzed by Factor D
  • C3b: Produced from C3; Catalyzed by & binds C3bBb
  • C5b: Produced from C5; Catalyzed by C3Bb3b; With properdin
  • MAC: Formed from the binding of C6, C7, C8 & C9 to C5b

• Membrane attack complex (MAC): Complex of C5b-9
  • Induces cytolysis
  • Main mechanism playing a role in muscle diseases
• Biologic effects of proteolytic fragments of complement
  • Phagocytosis: Opsonization & promotion
  • Inflammation mediated by anaphylotoxins (fragments of C3, C4 & C5): Activation
  • Immune complexes: Solubilization & phagocytic clearance
  • Leukocyte recruitment: C3a, C4a, C5a
  • Phagocytosis enhancement:
    • C3b, C4b, iC3b, vitronectin interact with complement receptors

• Necrosis of muscle fibers in myopathies
  • MAC binding: Intracellular organelles
  • Complement activation:
    • ? alternative pathway
    • Possibly facilitated by Ca⁺⁺-active neutral proteases
  • Effects
    • Removal of necrotic debris
    • Allows myofiber regeneration
• Damage to post synaptic membranes in myasthenia gravis
  • MAC binding: Post-synaptic membrane
  • Associated with IgG binding
  • Effects
    • Simplification of post-synaptic membrane folding
    • Reduction in # of AChRs
    • Widening of synaptic cleft
• Dermatomyositis: Especially childhood
  • MAC binding: Intramuscular capillaries, venules ± arterioles
  • IV Ig treatment: Reduces MAC binding
  • Effect: Perifascicular myopathy
• Necrotizing myopathy with pipestem capillaries
  • MAC binding: Capillaries
  • Effect: Necrotic myofibers
• Paraneoplastic necrotic myopathy
  • MAC binding: Necrotic myofibers
• X-linked vacuolated myopathy
  • MAC binding
    • Sarcolemma & internal vacuoles; Granular appearance
    • Vessels
  • Effect: Shedding of sarcolemma into internal vesicles
• Complement factor C2 deficiency
  • ? Associated with increased frequency of
    • Penicillamine-induced polymyositis
    • Spontaneous polymyositis: HLA-A25, B18, C2 null DR2 supratype

Myopathies: Antibody Testing Laboratories

• Myositis-specific antibodies and lupus screen are evaluated at:
  
  Oklahoma Medical Research Foundation
  Clinical Immunology Laboratory
  825 N.E. 13th Street, C-323
  Oklahoma City OK 73104
  Phone: (405) 271-7395; (800) 522-0211
  FAX: (405) 271-4110
  
  
• Anti-Decorin (BJ antigen) antibodies are evaluated at:
  
  Neuromuscular Clinical Laboratory
  Washington University, St. Louis