Myositis Highlights

Lisa Christopher-Stine, M.D.

Abstract #1151: A Novel Autoantibody against 200/100 Kd Proteins Is Associated with Necrotizing Myopathy

Authors:

Lisa Christopher-Stine, Grace Hong, Livia Casciola-Rosen, Andrea M. Corse and Andrew Mammen,
Johns Hopkins University School of Medicine, Baltimore, MD

Background:

The inflammatory myopathies are a family of conditions characterized by proximal muscle weakness, elevated serum muscle enzymes, and inflammatory infiltrates on muscle biopsy. Nearly 1/5 of patients with the clinical features of myositis do not have a significant degree of inflammation on muscle biopsy. Rather, biopsies from these patients demonstrate numerous degenerating, necrotic, and regenerating myofibers. Although necrosis may be seen in patients with muscular dystrophies or toxic exposure as well as in those patients with myositis specific autoantibodies (MSAs), a substantial proportion of patients with necrotizing myopathies have none of these known associations.

The purpose of this study was to identify novel autoantibody associations in patients with predominantly necrotic features in the absence of substantial inflammatory infiltrates on muscle biopsy

Methods:

225 patients in the Johns Hopkins Myositis Cohort had both a muscle biopsy available for review at our institution and banked serum. Biopsies were evaluated for the presence of inflammation, regeneration, degeneration, necrosis, and vacuolar change. Antibody specificities in patient sera were assessed by performing immunoprecipitations from 35S-methionine labeled HeLa cell lysates.

Results:

38 patients in the JHU Myositis Cohort had predominant necrosis on muscle biopsy without histologic findings of perifascicular atrophy or red-rimmed vacuoles. Sera from these patients were screened by immunoprecipitation for the presence of novel autoantibodies. 12 patients had known autoantibody association or other diagnoses [SRP (6), anti-Jo-1(1), 1 anti-PL12 (2), anti-PL-7 (1), profound hypothyroidism(1), and dysferlinopathy(1)]. The remaining 26 patients had no known myositis-specific antibodies or other diagnosis to explain the necrotizing myopathy. A novel autoantibody specificity was found in 16 of these 26 patients (62%). These sera immunoprecipitated a pair of proteins with molecular weights of 200 kDa and 100 kDa respectively. In contrast, control human sera did not precipitate these or other proteins. Only one additional patient with this autoantibody specificity was identified among 197 patients without prominent features of necrosis on muscle biopsy.

Conclusions:

A novel autoantibody specificity which is associated with characteristic muscle biopsy findings and a variable clinical phenotype has been identified. Since there were no instances of sera which precipitated only one of these proteins, we hypothesize that these may be subunits of a protein complex. These findings demonstrate that patients with anti-200/100 represent a distinct subgroup of necrotizing myopathy patients that were previously considered to be “autoantibody negative.”

Editorial Comment:

This study comes from our cohort of myositis patients at Johns Hopkins. It helps further elucidate a potential autoimmune mechanism responsible for a large portion of necrotizing myopathies previously thought to be autoantibody negative. Interestingly, 63% of these patients had been exposed to statins. It is unclear if this exposure potentiates this myopathy. Because a third of the patients had no myotoxic exposures, this clearly cannot be the only mechanism. Given the autoantibody association and the response to immunosuppression in these patients, it is likely that this newly discovered myosiis subset is indeed immune-mediated

Future studies will be directed towards identifying the autoantigens recognized by these autoantibodies.

Abstract #585: - Expanded Proinflammatory T Cells in Inclusion Body Myositis

Authors:

Jayesh Pandya, Andreas Fasth, Snjolaug Arnardottir, Eva Lindroos, Vivianne Malmström and Ingrid E. Lundberg Karolinska Institute, Stockholm, Sweden

Background:

Inclusion body Myositis (IBM) is a chronic, inflammatory myopathy of unknown cause characterized clinically by asymmetric muscle weakness and muscle atrophy, particularly of the quadriceps and finger flexor muscles. It is thought to be resistant to conventional immunosuppressive drugs. On muscle biopsy, IBM patients have leukocyte infiltrates, preferentially T cells and macrophages. This group of investigators has previously demonstrated the accumulation of a specific phenotype of T cells, (CD28null T cells) in muscles of patients with dermatomyositis and polymyositis. The CD28null T cells are apoptosis resistant, pro-inflammatory and cytolytic cells that hypothetically that are thought to have a role in disease mechanisms of IBM. The aim of this study was to investigate the frequency and also the effector functions of CD28null T cells in IBM and whether this subset of T cells is clonally expanded.

Methods:

A cohort of 20 patients with IBM were analyzed for the frequency of circulating CD4+CD28null and CD8+CD28null T cells in peripheral blood and muscle biopsies that were taken at different time points during disease course. The TCR-Vβ usage was determined by the flow cytometry (n=6). For functional analysis, peripheral blood mononuclear cells (PBMC) were polyclonally stimulated with plate bound anti-CD3 for 6 and 72 hrs (n=5) and the frequencies of intracellular IFNγ and CD107a (a marker of degranulation and cytotoxicity) containing T cells were recorded by multicolor flow cytometry.

Results:

CD28null T cell populations were clearly expanded in peripheral blood of IBM patients and both the CD4+ CD28null and CD8+ CD28null T cell populations were highly TCR Vβ restricted compared to the CD28+ subsets from the same patient. Different patients displayed different TCR Vβ restrictions, and the expansions were consistent over time. Anti-CD3 stimulation of peripheral blood mononuclear cells resulted in a fast and high frequency of CD28null T cells of both the CD4+ and CD8+ subsets significantly more positive for IFNγ and CD107a compared to CD28+ subsets. Both the TCR Vβ dominant and non- dominant CD28null subsets were equally activated under these conditions.

Conclusions:

The TCR Vβ restriction of CD28null T cells in IBM patients suggest that a limited numbers of antigens are involved in driving the expansion and high TCR Vβ restriction of CD28null T cells in IBM patients. Functional evaluation experiments revealed that CD28null T cells in both the CD4+ and CD8+ compartment are proinflammatory and cytotoxic effector cells. The TCR Vβ dominant and non-dominant CD28null T cell subsets displayed equal quality and quantity of effector function. Further analysis is ongoing to evaluate antigen specificity of these CD28null T cells and their presence in skeletal muscle infiltrate of IBM patients.

Editorial Comment:

The Authors rightfully point out that functional characterization of proinflammatory T cells will provide important clues for designing new T cell-targeted therapeutics for inflammatory myopathies. IBM is traditionally resistant to conventional immunosuppressive therapies. Greater understanding of the role of such proinflammatory T cells suggest that they are a potential, and long-awaited therapeutic target in inclusion body myositis.

Abstract #586: Evidence for the Implication of Th-1 and Treg Cells but Not Th-17 in Inclusion Body Myositis

Authors:

Yves Allenbach Jr.1, Julia Wanschitz Sr.2, Michelle Rosenzwajg Sr.1, Coralie Bloch-Queyrat Sr.3, Serge Herson4, David Klatzmann Sr.1 and Olivier Benveniste41UMR 7211, CNRS,Université Pierre et Marie Curie, University of Paris 06, Paris, France, 2UMR 974, CNRS, Université Pierre et Marie Curie, University of Paris 06, Paris, France, 3Hôpital Pitié-Salpêtrière, 4Hôpital Pitié-Salpêtrière, Paris, France

Background:

Inclusion Body Myositis (IBM) is the most common acquired myopathy in patients above the age of 50 years. It is an inflammatory myopathy characterized by CD8+ cytotoxic infiltrates and beta amyloid deposits. IBM remains resistant to conventional treatment. To date, only severe T cell depletion by Anti-thymocyte Globulin or Alemtuzumab have been shown to slow down the course of the disease suggesting a role for the adaptative immune system. Regulatory T cells (Treg) are key regulators of this system. These investigators described Treg in 22 IBM patients, but also Th1/Th2/Th17 and inflammatorys responses.

Methods:

22 IBM patients with a mean age of 70.1) as well as 22 gender and age matched control were included. No patient was currently being treated with immunosuppressive drugs, nor presented active infection or other autoimmune diseases. Peripheral blood mononuclear cells (PBMC) were analyzed using flow cytometry. PBMC were tested in vitro for their ability to produce IFN gamma and IL-17 upon stimulation with PMA and Ionomycin. Using a multiplex assay, the concentrations of 25 cytokines and chemokines were determined in the supernatant of stimulated PBMC and in the sera. Muscle biopsies of 7 of 22 IBM patients were tested by immunohistochemistry for presence of CD4+, CD8+ T cells and Tregs.

Results:

In blood, the mean percentage of activated CD4+ T cells (CD3+CD4+DR+) was higher in IBM patients than in controls (16.2±13.7% vs 8.7±4.2%; p=0.04). Additionally, terminally differentiated CD8+CD28- T cells were increased in IBM patients compare to controls (61±23.9% vs 44±20%, p=0.023). The mean percentage of CD3+CD4+IFNg+, CD3+CD8+IFNg+ and CD3+CD4+IL-17+ was not statically different in both groups. In contrast, the investigators observed an increase of IL-12 concentration in the sera of the IBM patients (301.36±142.08 pg/ml vs 154.25±-28.41 pg/ml p=0.0002) and of the chemokine IP-10 (377.73±296.72 pg/ml vs 61.147±44.64 pg/ml; p<0.0001).
The percentage of Treg (CD3+CD4+CD25+CD127-FOX3+) among CD4+ T cells was lower in IBM group compare to controls (5.5 ± 0.3% vs 6.6 ± 0.4% p= 0.043). Treg cells (FOXP3+) were detected in 6/7 muscle biopsies at a low frequency among CD4+ T cell infiltrates.

Conclusions:

Together these results suggest that CD4+ and CD8+ T cells are more activated and engaged in a Th1 lineage - not a Th17 one. Effector Th1 and activated CD8 cells may home the muscle attracted by IP-10. Treg cells are decreased in blood whereas they are present in the muscle, where they seem unable to control effector cells.

Editorial Comment:

Like the work in the previous Abstract, the refining of the role of the T reg, and their exact role in the myocyte, cell could lead to therapies that target this T cell subset.

Abstract #806: High Burden of Cardiovascular Risk in Patients with Inflammatory Myositis

Authors:

Irene Z. Whitt and Steven R. Ytterberg, Mayo Clinic, Rochester, MN

Background :

To determine cardiovascular (CV) risk and its predictors in patients with inflammatory myositis.

 

Framingham Risk of CV Event in 10 years

Patients

Low Risk (<10%)

Moderate Risk
(10-20%)

High Risk (>20%)

Total, n (%)

19 (30.2)

24 (38.1)

20 (31.8)

Candidates for lipid-lowering agent, %

11

54

89

With hypertension, %

6

67

63

With SBP < 130, %

74

58

50

Methods: :

This study was a cross-section, observational study of adult patients seen in a single clinic at the Mayo Clinic in the past year with probable or definite dermatomyositis, polymyositis, or connective tissue disease associated myositis based on Bohan and Peter criteria. Patients with inclusion body myositis and cancer-associated myositis were excluded. Standard CV risk factors were assessed and a determination was made of low, moderate, or high CV risk based on the current guidelines outlined by the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Univariate relationships between each potential predictor and each risk group were examined. A multivariable linear regression model was then developed for each significant (p < 0.05) risk marker.

Results:

63 patients met the inclusion criteria and had CV risk assessment. The distribution of CV risk is tabulated below.
Framingham Risk of CV Event in 10 years Patients Low Risk (<10%) Moderate Risk (10-20%) High Risk (>20%)
Total, n (%) 19 (30.2) 24 (38.1) 20 (31.8) Candidates for lipid-lowering agent, % 11 54 89 With hypertension, % 6 67 63 With SBP < 130, % 74 58 50

Across all risk levels, 51% of patients would be placed on a lipid-lowering agent according to the current ATP III R guidelines.

Several variables were assessed in univariate analysis with ordered logistic regression. Significant correlations were found for: hypertension (HTN) (p=0.001) and age (p=0.005). There were no significant correlations for the following variables: gender, hyperlipidemia, BMI, disease duration, current prednisone dose, current smoking status, chronic renal insufficiency, and family history of coronary heart disease. In a multivariate analysis, HTN was the only independent predictor of higher CV risk (p=0.004, R^2=0.155).

CONCLUSION:

Patients with inflammatory myositis have a high burden of CV risk. HTN was the only independent predictor of CV risk. However, only about 50% of high-risk hypertensive patients achieved the recommended goal of SBP<130. Although 51% of patients in this cohort would be candidates for a lipid-lowering agent, there is concern for worsening myositis with lipid lowering therapy. Further research is needed to determine the safety of using statins in patients with inflammatory myositis.

Editorial Comment:

This study demonstrates that there is a high burden of potentially treatable cardiovascular risk factors. Not unlike the rest of the populations, patients with myositis are candidates for lipid-lowering therapy. The issue looming is whether statins either potentiate or worsen inflammatory myopathies. In my own experience, I have attempted the use of statins in patients who have a very high benefit:risk ratio. Some patients seem unable to tolerate them while others do just fine. The ability to differentiate these groups will be important as more patients require lipid-lowering therapy.

Abstract #811: Rituximab Is An Effective Therapy for Anti-Signal Recognition Particle (Anti-SRP) Myopathy

Authors:

Ritu Valiyil, Livia Casciola-Rosen, Grace Hong, Andrew Mammen and Lisa Christopher-Stine Johns Hopkins University School of Medicine, Baltimore, MD

Background:

The myopathy associated with anti-signal recognition peptide (SRP) is a severe necrotizing immune-mediated disease characterized by rapidly progressive proximal muscle weakness, markedly elevated serum creatine kinase (CK) levels, and poor responsiveness to traditional immunosuppressive therapies. Currently, reports on the efficacy of B cell depletion therapy for anti-SRP associated myopathy are mixed. In this study, we describe eight patients with anti-SRP associated myopathy and their clinical response to treatment with the anti-CD20 monoclonal antibody rituximab.

Methods:

We identified eight patients with myopathy who tested positive for anti-SRP antibodies by immunoprecipitation and had been treated with rituximab as part of clinical care between 2006 and 2009. We reviewed their medical records to assess clinical, serologic, and histologic characteristics of their muscle disease and response to therapy. In five of the eight patients, serum samples were also collected before and after rituximab treatment. In those patients, autoantibodies were detected by immunoprecipitation and quantitated by densitometry, and the percent decreases in anti-SRP autoantibody levels were calculated.

Results:

In this study, the mean age was 37 years, 75% were female, and 50% were African-American. All patients presented with severe, rapidly progressive proximal muscle weakness with myalgias, dysphagia, and high CK levels with a mean maximum CK of 18,900 IU/l (3,148-56,000 IU/L). Six of the eight patients refractory to standard immunosuppressive therapy demonstrated improved manual muscle strength and/or decline in CK levels as early as two months after receiving two doses of rituximab. Three patients sustained the response for twelve to eighteen months after initial dosing. All patients were continued on adjunctive corticosteroids, but dosages were substantially reduced after rituximab. In four of the five patients tested, quantitative levels of serum anti-SRP antibodies also decreased after rituximab treatment.

Conclusion:

B cell depletion therapy with rituximab is an effective therapy for patients with anti-SRP myopathy. The substantial decrease in anti-SRP antibody levels after rituximab treatment also suggests that B cells and anti-SRP antibodies may play a role in the pathogenesis of this myopathy.

 Pt.

 CK prior to herapy

 Highest prednisone dose (mg/day)

 Prior treatments

Lowest prednisone dose post-therapy 

 Lowest CK post-therapy

Outcome post B cell depletion, duration of remission

 1

 2710

 60

 AZA, MTX

 20

622 

 CK decline, improved strength, 10 months

 2

 1000

 160

 AZA, MTX, IVIg, plasma exchange

 5

 163

 Normalized CK, improved strength, 18 months

 3

 550

 40

 AZA, MTX, IVIg, MMF

 15

 126

 Normalized CK, improved strength, 19 months

 4

 1063

 80

 IVIg, plasma exchange

 50

 22

Died 1 month later from pneumonia

 5

 2900

 80

 MTX, IVIg

 10

 963

 CK decline for 12 months, then re-dosed for increased CK

 6

 2100

 60

 MTX, MMF, IVIg

 30

 1144

 CK decline, improved strength, 9 months

 7

 1250

 60

 MTX, MMF

 N/A

 1080

 CK decline, 5 months

 8

 3110

 60

 AZA, MTX, IVIg, plasma exchange

 15

 2100

 CK decline for 6 months, persistent weakness

Editorial Comment:

This study demonstrates that anti-SRP myopathy, a very resistant antibody- associated necrotizing myopathy, can be treated successfully with rituximab. The response is often sustained. In our experience, treating early with rituxmab in this patient population appeared to help prevent associated complications often observed, including severe dysphagia and rapid muscle decline- often to a wheelchair.

Abstract #819: Isolated Elevation of Aldolase in the Serum of Myositis Patients: A Biomarker of Damaged Early Regenerating Muscle Cells

Authors:

Livia Casciola-Rosen, Lisa Christopher-Stine, Andrea Corse, Grace Hong, John Hall and Antony Rosen, Johns Hopkins University School of Medicine, Baltimore, MD

Background:

Increased serum aldolase levels in the absence of increased serum creatine kinase (CK) levels occurs in patients with myositis, but the mechanism underlying this phenomenon is unclear. Recent studies have demonstrated that regenerating muscle cells express the highest concentrations of myositis autoantigens, and are likely major targets of immune attack in this disease. We therefore examined the gene and protein expression of aldolase and CK in differentiating muscle cells.

Methods:

Cultured human myoblasts were induced to differentiate into myotubes over 10 days in vitro. Total RNA was isolated on each day of culture and microarray analysis was performed using human Illumina Refseq8 beadchip arrays. Present genes were Z-transformed and normalized, and significant changes in gene expression were calculated by performing Z tests. Gene expression data for aldolase and CK was verified by performing QPCR using the TaqMan assay system. Detergent containing lysates, prepared at various times during differentiation, were immunoblotted with antibodies against aldolase, CK, vinculin and other differentiation markers. Muscle paraffin sections from patients with elevated aldolase levels and normal CK levels were stained with a polyclonal antibody against aldolase.

Results:

Gene expression analysis performed on differentiated human myoblasts demonstrated that aldolase expression was highest in myoblasts, and decreased slightly during differentiation into myotubes; however, levels remained robust. In contrast, CK mRNA is expressed at very low levels in undifferentiated myoblasts, but is upregulated by day 2 of culture, with expression levels peaking by day 3. A marker of muscle differentiation, embryonic myosin heavy chain (MYH3), displayed a similar expression pattern. Consistent with this, we showed by immunoblotting that aldolase protein expression is highest in myoblasts, and, although it decreases during differentiation, it is prominent throughout. In contrast, CK expression is absent in myoblasts and during the early stages of differentiation (the period of highest aldolase expression), and is only detected from day 4 onwards. Immunohistochemical staining confirmed that muscle cells with the highest levels of aldolase have features consistent with regeneration.

Conclusions:

In undifferentiated muscle cells, and those early in the differentiation process, aldolase is expressed in the absence of CK. This is a period in muscle cell differentiation in which autoantigen expression is also highest. Thereafter, both aldolase and CK are expressed. We propose that isolated serum aldolase elevation in myositis patients reflects preferential immune-mediated damage of early regenerating muscle cells. Such targeted damage may have important implications in terms of prognosis and therapy.

Editorial Comment:

Often plagued by those patients that express isolated aldolase elevation, our group sought to investigate the etiology of this phenomenon. Building on previous work demonstrating that regenerating muscle cells are often the source of autoantigen production, this study extends this observation by postulating that the regenerating myocyte’s immne- mediated damage is evidenced is reflected in the phenomenon of isolated serum aldolase production.

Abstract #88: Discordance Between Cardiac Troponin T Elevation in Patients with Inflammatory Myopathies Vs. Other Muscle Disease

Authors:

John P. Case, Stroger Hospital of Cook County, Chicago, IL, Augustine M. Manadan, Rush Univ Med Ctr, Chicago, IL and Rohit Aggarwal John H. Stroger, Jr. Hospital of Cook County and Rush University Medical Center, Chicago, IL

Background:

Patients may present with malaise and fatigue associated with elevated creatine kinase (CK), or a CK is determined early in the course of evaluation for musculoskeletal disease. Some are ultimately diagnosed with inflammatory myopathies (IM). In many however, the CK elevation is unrelated to IM but rather to non-IM causes. N previous experiments, this group of investigators has previously determined that cardiac troponin T (cTnT), but not cardiac troponin I (cTnI) is frequently elevated in patients with IM and that cTnT and CK are correlated. They undertook the present study to determine whether cardiac troponins are elevated in non-IM and their relationship to CK.

Methods:

This study was a retrospective study using a computerized database to identify patients who were evaluated for non-IM CK elevation, myopathy, or rhabdomyolysis between January 2004 and December 2008 by the Rheumatology service of Cook County Hospital. They compared these patients to those with IM described earlier. In each group, patients who had chronic kidney disease (CKD) or acute coronary syndrome (ACS) were excluded. Statistical comparisons were done with Pearson’s chi-square test.

Results:

The records of 56 patients with a non-IM diagnosis were retrieved. The CK was assayed in 54 (96%) and was elevated in 47 (89%). The etiology of the CK elevation was statin myopathy (16); neuromyopathy (6), metabolic myopathy (2), HIV (2), and viral syndrome (2). Non-statin medication toxicity, muscular dystrophy, alcohol, cardiomyopathy, and hyperthyroidism accounted for one case each. In one case the cause was multifactorial. In 13 cases the diagnosis was not determined. In the 9 patients with normal CKs (17%), the final diagnoses were steroid myopathy (3); and in one case each, alcohol, statin myopathy, cardiomyopathy, and myopathy from a non-statin medication. In two cases (4%) the CK was not assayed. One had cardiomyopathy, another had steroid myopathy. In the 47 patients who had elevated CK and had cTnT or cTnI assayed, cTnT was elevated in 3 of 19 (16%) and cTnI in 0 of 18 (0%). Inclusion body myositis was suspected in two of the patients with elevated cTnT, but biopsy was not done and formal diagnosis not made. In the third patient, elevated cTnT was attributed to the combination of cyclosporine and ezetimibe. cTnT was assayed in four patients with rhabdomyolysis. It was elevated in only one patient (cyclosporine/ezetimibe combination). Overall, cTnT elevation was not associated with CK elevation (p=0.445). By comparison (and as previously reported), in patients with IM who had elevated CK the cTnT was elevated in 18 of 23 (78%) and was highly associated with the CK (p=0.005). The percent frequency of cTnT positivity in IM patients with elevated CK is significantly higher, almost 5 fold, than in patients with non-IM (p<0.001).

Conclusion:

This study demonstrated that an elevated cTnT is not associated with non-IM CK elevation. The Authors suggest that evaluating a patient with possible IM in the non-ACS, non-CKD setting, obtaining the cTnT level may help in the diagnosis, since it is usually elevated in IM but rarely elevated in non-IM. The reason is unclear. The cTnI is not elevated in IM or in non-IM.

Editorial Comment:

This study is quite intriguing. It appears that cTnT is a consequence of an immune-mediated process. It helps to demonstrate that there are non-cardiac sources of cTnT. Interestingly, cTnI seems to remain cardiac-specific. Although I am not sure that the Authors’ contention that this test can serve to help differentiate non-IM from IM will be utilized in a practical setting, I do feel that determining the mechanism for this dichotomy from an immunologic standpoint deserves further exploration.

AddThis Social Bookmark Button