MI-2 & MDA-5.pptx sms mc Jaipur Rajasthan

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1. MYOSITIS ANTIBODIES

2. McHugh, Neil J.; Tansley , Sarah L. (2018). Autoantibodies in myositis. Nature Reviews Rheumatology, 14(5), 290–302. doi:10.1038/nrrheum.2018.56 

3. Mi2

4. Ghirardello , Anna; Zampieri , Sandra; Iaccarino , Luca; Tarricone , Elena; Bendo *, Raffaele; Gambari, Pier Franca; Doria , Andrea (2005).  Anti-Mi-2 antibodies. Autoimmunity, 38(1), 79–83. doi:10.1080/08916930400022681 

5. Mi-2/ NuRD  (Nucleosome Remodeling Deacetylase)  complex , group of associated proteins with  ATP dependent  chromatin remodeling  and  histone deacetylase  activities It contains seven major polypeptides Mi2 is a SWI2/SNF2 type helicase/ atpase domain-containing protein Responsible for the chromatin remodeling activity of the NuRD complex Dna -dependent, nucleosome-stimulated atpase Efficiently disrupts histone– dna contact in an atp -dependent manner

6. Packaging of DNA into chromatin blocks access to DNA by proteins. Dynamic changes in chromatin structure play important roles in transcription, replication, recombination and repair Certain protein complexes are capable of altering chromatin structure to allow protein factors access to nucleosomal DNA A common feature of the nucleosome remodeling complexes is the presence of a subunit belonging to the SWI2/SNF2 family of atpases The presence of an atpase domain in the mi2 protein suggests that mi2 is likely responsible for the atpase activity of the nurd complex

7. Anti-Mi-2 autoantibodies immunoprecipitate two proteins, Mi-2 a (220 kDa) Mi-2 b ( 218 kDa)

8. I n DM patients adults (11 - 59%) children (4 - 10%) The Mi-2 protein is over-regulated during muscle regeneration in DM patients thought to be related to ultraviolet exposure sex HLA DRB1*0302 and DRB1*0701 A cohort study conducted in the USA showed a positive association between anti-Mi-2 antibodies and UV exposure in women

9. DM with Mi2  31% DM in Mi2  90% Anti-Mi-2  4 – 10% of JDM patients

10. CLINICAL FEATURES

11. Typical florid cutaneous manifestations Gottron’s sign or papules Heliotrope rashes Involvement of the neck and upper back (“V” and “shawl” rashes) Cuticular overgrowth Flagellate erythema Lower frequency of mi-2 autoantibodies in amyopathic DM Japanese patients  punctate perionychium hemorrhages

12. CLINICAL FEATURES

13. MALIGNANCY IN Mi2 ?! A Dutch study also suggested an increased risk of cancer in patients with antibodies to the N-terminal fragment of the anti-Mi-2-beta antigen THIS case report shows colon cancer in a patient of DM with Mi2-alpha antibody

14. S erum anti-Mi-2 antibody levels positively correlation: PGA VAS scores Serum CK levels Reversely correlation: MMT8 scores N o significant correlation cutaneous VAS scores joint VAS scores gastrointestinal VAS scores pulmonary VAS scores cardiac VAS scores constitutional VAS scores

15. Interestingly, anti-Mi-2b antibody levels declined in all of these patients but never disappeared completely in any of the patients during the follow-up period.

16. Mi2 & Muscle Bx most muscle biopsies showed positive staining for MHC I (76.9%) membrane attack complex (76.9%) myocyte necrosis (65.4%) inflammatory cell infiltration (80.8%).

17. mild myopathy despite high creatine kinase levels without lung involvement and/or cancer Overall  good prognosis favourable response to corticosteroids

18. PROGNOSIS in Mi2 Among the 33 surviving patients 32 (97.0%) showed clinical remission: 24 patients (72.7%) achieved complete remission eight patients (24.3%) had partial remission one patient (3.0%) had no response to treatment.

19. SURVIVAL DM patients who were anti-Mi-2b-positive had a significantly lower mortality rate compared to patients who were anti-Mi-2 􏰀 -negative The five-year survival rates for these two groups were 97.1% and 80.8%, respectively

20. ANA IF

21. Mi2 ON IIF Indirect immunofluorescence (IIF) on HEp-2 cells fine, tiny speckled pattern in interphase nuclei in meta- phase cells chromatin is not stained medium-high titers (320 – 5120) No cytoplasmic staining is visible

22. Methods of detection Immunoprecipitation IP line immunoassay (LIA) particle-based multi-analyte technology (PMAT) ELISA tests are more sensitive but less specific than either gel precipitation or immunoblot tests. optimal testing is achieved by ELISA screening and confirmation of positive results on immunoblot. diagnostic sensitivity and specificity of approximately 4–18% and 98–100% By using recently developed ELISA tests, 9–10% of patients with PM and 8% of those with IBM have been found anti-Mi-2 positive

23. METHODS ELISAs standardization, large-scale reproducibility quantitative results. disadvantage possible loss of conformational epitopes formation of neo-epitopes Immunoblot (IB) assays simultaneously test for many autoantibodies recognition of linear epitopes only

24. IP is the gold standard evaluates the binding of the autoantibodies to the RNA and protein complexes in their native conformation, yielding the best sensitivity and specificity The major limitations technical difficulties costs use of radioactive reagents

25. TREATMENT Rituximab in Myositis (RIM) trial revealing a significant improvement in interferon chemokine score and clinical illness

26. MDA-5 Anti-melanoma differentiation-associated gene 5

27. MDA5 was initially identified in 2002, as a type I IFN inducible gene in human melanoma cells, and the first function described for this protein was to induce the death of cancer cells Mda5 is encoded by the gene ifih1 (interferon-induced with helicase c domain 1) and is part of the retinoic acid inducible gene- i -like receptor family (RIG-1). Detect molecular patterns of viruses that have gained access to the cytosol of the infected cell.

28. MDA5 is able to detect and limit the replication of several RNA viruses Picornavirus fl avivirus coronavirus families DNA viruses of the herpesvirus family MDA5 is also able to sense endogenous dsRNA mitochondrial dsRNA generated by bilateral transcription of mitochondrial DNA. This biological mechanism is tightly regulated : mitochondrial degradasome enzymes  polynucleotide phosphorylase ( PNPase ), avoiding the accumulation of dsRNA and thus deleterious activation of MDA5

29. PNPase de fi ciency massive accumulation of long mitochondrial dsRNA escaping into the cytosol uncontrolled activation of MDA5 upregulation of interferon stimulated genes In line with this, patients with hypomorphic mutations in PNPT1 , the gene that encodes for PNPase , have type I interferonopathies characterized by a constitutive activation of the IFN-I axis

30. The strongest disease association was found with alleles of the type II HLA alleles HLA-DRB1 One non-HLA locus, an intronic variant of WDFY4, has been recently associated with the anti-MDA5 DM in Japanese patients

31. Infection as trigger Antiviral function of the MDA5 antigen. MDA5 is an IFN-I Inducible gene IFN-I could be the starting point for most of the Pathophysiological pathways.

32. Vasculopathy hypothesis Patients frequently show typical cutaneous features such as skin ulcers due to severe vasculopathy Histopathology of palmar papules, as well as lung tissues highlights vasculopathy Biomarkers of endothelial dysfunctions have been identi fi ed in the sera of patients Patients exhibited a strong IFN-I signature distributed in the vasculature of the skin

33. It is important to emphasize that these particular features are observed in the skin and the lungs of patients with anti-MDA5 DM no or few signs of vasculopathy nor of an enhanced IFN-I signature, compared to other DM, in muscle tissue of anti-MDA5 DM patients serum levels of endothelin and vWF are higher in anti-MDA5 DM patients who have cutaneous ulcers and ILD and IFN-I signature correlates positively with endothelin levels

34. blood vessels exposure to IFN-I in anti-MDA5 DM  endothelial injury,  cutaneous and pulmonary lesions

35. Vasculopathy & pulmonary lesions Anti-MDA5 DM is associated with an accumulation of activated macrophages (M2) in alveoli Several markers of macrophage activation are elevated in the serum of anti-MDA5 DM patients, such as a soluble form of CD206, which is highly expressed by alveolar macrophages Other soluble markers CD163 chitotriosidase ferritin

36. Neopterin levels another marker of macrophage activation a/w RP-ILD reduced survival ferritin markers of disease severity Negative correlation with pulmonary function

37. Multiple chemokines can induce the recruitment of M2 macrophages CX3CL1 (Fractalkine) secretion induced by IFN-I in pulmonary vascular endothelial cells induces the recruitment of CX3CR1+M2 macrophages in the lungs M2 macrophages produce TGF- b  pulmonary fi brosis

38. The severity of the disease correlates with the titers of anti- MDA5 Abs The use of therapies that target the humoral immune response has shown its effectiveness in patients with anti-MDA5 DM MDA5 expression has been shown to be enhanced in skin biopsies of patients

39. anti-MDA5 IgA and IgG were the predominant isotypes. anti-MDA5 IgG1 potent activators of the complement pathway Higher serum ferritin levels severe interstitial pneumonia higher mortality rate. IgG1 + IgG4 poor prognosis

40. MDA-5

41. EPIDEMIOLOGY rare disease < 2% of IIM in Europe Among the subgroup of DM Prevalence  7 to 60% higher prevalence in Asian (11-60%) female/male ratio  0.6 to 7.3 (F/M >1 in 14 out of 16 studies)

42. PHENOTYPES

43. Anti-MDA5 DM was fi rst described by Sato et al. in 2005, in a Japanese cohort with CADM and RP-ILD CADM is not suf fi cient to de fi ne anti-MDA5 DM not all CADM  anti-MDA5 Abs. (23% to 100%) prevalence of CADM among anti-MDA5 Abs (17 to 100%)

44. MDA5- Clinical presentation clinically amyopathic DM (CADM). skin rash signs of skin vasculopathy ulcerating skin lesions (palmar surface of the hands) polyarthritis rapid ILD with a high mortality rate (between 33–67%)

45. Cutaneous manifestations periorbital heliotrope (blue – purple) rash erythematous rash on the face, or the anterior chest (V-sign) shawl sign Violaceous papules or plaques MCP/PIP  Gottron ’ s papules cracked palmar fi ngertips ( “ mechanic ’ s hands ” ).

46. SPECIFIC CUTANEOUS : MDA-5 palmar papules palmar surface or lateral sides of the fi ngers especially over MCP / PIP. central ivory coloration frequently painful. A/W hyperkeratosis complicated of ulcerations

47. Skin ulcerations deep painful ulcers Localition : Gottron ’ s papule, digital pulp and nail folds over Gottron ’ s sign on the knees, elbows The histology of skin ulcerations: Vasculopathy pauci-in fl ammatory medium vessel wall in fi ltration with mononuclear cells intravascular thrombus Major complication: gangrene Osteomyelitis digital amputation

48. Auricular skin lesions Less speci fi c antihelix/helix violaceous macules erythematous auricular papules

49. Wong-type DM iexceedingly rare DM subtype PRP(pityriasis rubra pilaris)-like morphology Hyperkeratosis overlying follicular infundibulum, risk for myositis and interstitial lung disease (ILD). not associated with internal malignancy.

50. Other lesions

51. Lungs & MDA-5

52. ILD can be absent at diagnosis and develop many years later, or completely absent in some patients over years of follow-up. Conversely, ILD can sometimes reveals anti-MDA5 DM.

53. The mortality rate (RP-ILD)  50% most deaths occurring during the very early stages of the illness Some studies suggest that beyond 6 months after onset, disease progression tends to settle down, and relapse seems uncommon recurrences have also been described in the form of cutaneous and/or severe respiratory relapse many years after onset and after months of clinical remission

54. MDA-5 & Malignancy Not exclusive few cases of cancer have been reported in patients with anti-MDA5 DM Metastatic small cell carcinoma with liver involvement was detected 12 months after anti-MDA5 DM diagnosis in a 60 year-old French woman Another study presented the case of a man diagnosed simultaneously with both anti-MDA5 DM and thyroid cancer

55. Other clinical features constitutional symptoms  fever (74%)

56. Ferritin Elevated ferritinemia , with no signi fi cant elevation of c-reactive protein. Ferritin levels correlate with the severity of the disease and ILD

57. Liver dysfunction Elevated levels of ALT / GGT without elevated creatine kinase Liver biopsies show steatosis and hepatocyte ballooning

58. CD4 & CD8 Decreased CD4+ and CD8+ T cell counts raised CD4+/CD8+ ratio increase of CD4+ and CD8+ T cell counts  parallel with pulmonary lesions improvement after treatment. Refractory ILD CD4+ and CD8+ T cell counts decrease the CD4+/CD8+ ratio increases

59. HRCT IN MDA-5 Common findings: GGA (83.3%) non-septal linear or plate-like opacity (83.3%) interlobular septal thickening (66.7%)

60. MDA-5 v/S RA arthritis  closely resembles rheumatoid arthritis, morning stiffness symmetric small joints of the hands, wrists and the ankles Although rarely  erosions on MRI RF maybe positive, ACCP is negative

61. MDA-5 v/s PsA arthritis is a common feature associated with psoriasis anti-MDA5 DM may initially manifest as psoriasiform skin lesions

62. A 20-year-old male fever, arthritis, rash Proximal muscle weakness for 7months. scaly, hyperkeratotic, hyperpigmented psoriasiform plaques on the scalp, ear and nasolabial folds Gottron’s papules were present over the hand and elbow joints, along with a heliotrope rash. Manual muscle testing score was 56/80

63. MDA-5 v/s Anti Synthetase syndrome some patients have a phenotype close to the antisynthetase syndrome Arthritis Raynaud ’ s syndrome mechanic ’ s hands ILD

64. Anti-MDA-5 antibodies titers correlate with the severity of disease higher levels are associated with rapidly progressive ILD in juvenile DM.

65. DM MDA-5 INFLAMMATION Perivascular Sparse INFLITRATES T & B Cells Macrophages FEATURE Perifascicular atrophy Perimysial clusters infiltrates MHC I Diffuse Focal / absent COMPLEMENT ATTACK COMPLEX Present Rare IFN signatures ↑↑↑ ↑

66. MDA-5 & Ro 52 27% of the anti-MDA5 antibody positive patients in our cohort also had Ro52 autoantibodies; Both Ro52 and MDA5 are cytoplasmic proteins which are highly induced by IFN. MDA5 is a member of the RIG-I-like receptor (RLR) family of proteins which sense viral dsRNA structures in the cytoplasm of cells and induce the production of type I IFNs through activation of IRF3. RLR family members RIG-I and MDA5 can also be activated by ubiquitin-induced oligomerization (ubiquitin E3 ligase TRIM25) to produce type I IFN Ro52 (TRIM21) is a member of the TRIM family of proteins, and has documented ubiquitin E3 ligase activity Ro52 has been shown to regulate IFN-signaling through interactions with IRF3 Perhaps interactions between Ro52 and MDA5 induce the formation of novel complexes which are particularly immunogenic, possibly explaining the co-existence of these antibodies in a subset of DM patients.

67. JDM : MDA-5

68. Juvenile DM (JDM) + MDA-5  6 to 12% of cases in European and North American cohorts third most frequent DM-associated Abs after anti-TIF1 g and anti-NXP2 associated with Higher frequency of constitutional symptoms (weight loss, fever and adenopathy) milder muscle involvement

69. Similarly to the adult form, the speci fi c cutaneous phenotype found in anti-MDA5 DM palmar papules Skin ulcerations Arthritis These lesions distinguish anti-MDA5 JDM from the other forms of JDM.

70. anti-MDA5 JDM has comparable outcome with the other forms of JDM fl ares are less frequent in this subset of JDM and necessitates less medication

71. METHODOLOGY FOR MDA-5

72. IMMUNOPRECIPITATION Initially performed by immunoprecipitations (IP) of sera from patients incubated with 35S-methionine-labeled K562 cell extracts radiolabeled IP is considered as the gold standard testing method to detect anti-MDA5 Abs. substitution of radiolabeled antigenic extracts by biotin-labelled recombinant MDA5 constitute a good alternative to bypass the use of radioactive materials IP remains dif fi cult to use in everyday practice time-consuming expensive. comigration of the MDA5 antigen with other antigens (NXP2, TIF1 g and OJ) Great expertise required

73. IIF : QUALITATIVE indirect immuno fl uorescence (IIF) staining on HEp-2 cells cytoplasmic staining with a fi nely granular appearance, very touchy, and requires a trained eye. Moreover, this IIF pattern is inconstant. can also be negative by IIF

74. immunodot assays (so-called line blot or dot blot) Commercialized immunodot assays Line blot LIA, Euroimmun , Lübeck , Germany dot blot D-Tek BlueDiver , Mons, Belgium particle-based multi-analyte technology (PMAT, Inova, Diagnostics, US)

75. These speci fi c qualitative assays Line Blot, Dot blot, have been validated using the IP assay as gold standard, speci fi city of 96-99% or 98% sensitivity of 75- 93% or 76% High level of agreement was also found between IP and PMAT

76. ELISA : QUANTITATIVE An ELISA based on a recombinant MDA5 fusion protein produced in insect cells sensitivity  98 % speci fi city  100% A major bene fi t of anti-MDA5 Abs ELISA precise quanti fi cation of the level of anti-MDA5 Abs follow-up of its variation

77. DISEASE ACTIVITY : MDA-5 LEVELS P redict disease outcome. anti-MDA5 Abs levels are signi fi cantly lower in surviving patients. outcome of patients with low titers of anti-MDA5 Abs is approximately the same as patients without anti-MDA5 Abs

78. MDA- & Ro

79. Other antibodies : SFPQ Abs directed against a nuclear protein, the splicing factor proline/glutamine-rich (SFPQ) 27 out of 51 patients (53%) with anti- MDA5 DM SFPQ is a multifunctional nuclear protein of 110 kDa participates in diverse molecular functions transcription regulation regulation of host innate immune response to viruses