Abstract:
This disclosure describes methods and compositions for immunohistochemically detecting the presence of a SRP autoantibody in a biological sample.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims priority to U.S. Provisional Application Ser. No. 61/535,610, filed on Sep. 16, 2011. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application. 
     
    
     TECHNICAL FIELD  
       [0002]    This disclosure generally relates to autoantibodies. 
       BACKGROUND  
       [0003]    Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic RNA. An autoantibody against an SRP polypeptide was described initially in 1986 as a marker of polymyositis. In that report, Reeves et al demonstrated that the patient&#39;s serum IgG immunoprecipitated SRP extracted from human erythroleukemia cells. Since that time, numerous case reports and series have confirmed the association of SRP autoantibodies with both polymyositis and dermatomyositis. 
       SUMMARY  
       [0004]    This disclosure describes methods and compositions for immunohistochemically detecting the presence of a SRP autoantibody in a biological sample. 
         [0005]    In one aspect, a method of detecting the presence of a SRP autoantibody in a biological sample is provided. Such a method typically includes the steps of contacting a tissue section with a biological sample and a detectably-labeled secondary antibody under conditions in which a complex is formed between SRP polypeptides in the tissue section, and a corresponding SRP autoantibody in the biological sample, if present, and the detectably-labeled secondary antibody; and identifying a pattern of complex formation in the tissue sample by detecting the detectably-labeled secondary antibody. Generally, the presence of a pattern of complex formation is indicative of the presence of a SRP autoantibody in the biological sample, and the absence of a pattern of complex formation is indicative of the absence of a SRP autoantibody in the biological sample. 
         [0006]    In some embodiments, the tissue section is a tissue section from gut. As described herein, the pattern of complex formation in the gut is in the cytoplasm of proximal epithelial cells in mucosa and enteric neurons in the gut wall. In some embodiments, the tissue section is a tissue section from kidney. As described herein, the pattern of complex formation in the kidney is in renal tubules. In some embodiments, the tissue section is a tissue section from brain. As described herein, the pattern of complex formation in the brain is in cytoplasm of cerebellar Purkinje, Golgi neurons and granular neurons. 
         [0007]    Representative SRP polypeptides include, without limitation, SRP54 or SRP72. Representative biological samples include blood, serum, plasma, or spinal fluid. In some embodiments, the tissue section is from mammal tissue (e.g., mouse tissue). Typically, the presence of a SRP autoantibody in the biological sample is indicative of the presence of an autoimmune myopathy (e.g., inflammatory or non-inflammatory necrotizing myopathy), a neuropathy, a connective tissue disease, or cancer. 
         [0008]    In another aspect, a composition is provided that includes a tissue section that exhibits a staining pattern following immunohistochemistry with a biological sample that comprises SRP autoantibodies. Particularly, when the tissue section is gut, the staining pattern is in the cytoplasm of proximal epithelial cells in mucosa and enteric neurons in the gut wall; when the tissue section is kidney, the staining pattern is in renal tubules; and/or when the tissue section is brain, the staining pattern is in cytoplasm of cerebellar Purkinje, Golgi neurons and granular neurons. 
         [0009]    In still another aspect, a composition is provided that includes a tissue section and a description of a staining pattern that is indicative of the presence of a SRP autoantibody. 
         [0010]    Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. 
     
    
     
       DESCRIPTION OF DRAWINGS  
         [0011]      FIG. 1  shows immunohistochemical staining patterns for PCA-1 (anti-Yo) (A) or anti-SRP (B). In panel A, perikarya of Purkinje neurons (chunky pattern) and molecular layer neurons were immunoreactive. The granular layer is non-stained except for occasional Golgi neuronal perikaryon. In panel B, the anti-SRP IgG stained perikarya of Purkinje neurons and molecular layer neurons and yielded a “chicken wire” appearance in the perikarya of granular layer neurons. GL=granular layer, ML=molecular layer. 
           [0012]      FIG. 2  shows muscle biopsies. Panel A shows healthy muscle fibers stained with hematoxylin and eosin; Panel B shows few necrotic fibers, with one invaded by macrophages. There is no evidence of widespread inflammation and no autoaggressive inflammatory exudate. Panel C shows a higher power magnification of the same field, showing a necrotic fiber engulfed by macrophages and two pale necrotic fibers in the upper right corner. The black arrow indicates a basophilic muscle fiber with plump nuclei. This represents a regenerating fiber. Panel D shows, with acid phosphatase staining, red-colored macrophages invading two necrotic fibers. 
       
    
    
       [0013]    Like reference symbols in the various drawings indicate like elements. 
       DETAILED DESCRIPTION  
       [0014]    Signal recognition particle (SRP) autoantibodies are an under-appreciated marker of autoimmune myopathies. As reported herein, SRP autoantibodies are not restricted to inflammatory myopathies. The experiments described herein have revealed the presence of IgGs specific for SRP polypeptides in a spectrum of myopathic disorders and in association with neoplasia. Remarkably, the preliminary serological findings disclosed herein demonstrate a strong association between IgGs specific for SRP polypeptide subunits 54 and 72 and the diagnosis of non-inflammatory necrotizing myopathy (14 of 22 seropositive patients; 63.6%). Thus, the methods and compositions described herein can aid in early recognition of these disorders and in early cancer detection, which, with prompt immunosuppressive treatment, may significantly improve the clinical outcomes of these patients. 
       Methods and Compositions  
       [0015]    Methods and compositions are described herein that can be used for detecting, by immunohistochemistry, the presence of a SRP autoantibody in a biological sample. Immunohistochemical methods are well known in the art. Simply by way of non-limiting examples, see U.S. Pat. Nos. 5,073,504; 5,225,325; and 6,855,552. See, also, Dabbs,  Diagnostic Immunohistochemistry,  2 nd  Ed., 2006, Churchill Livingstone; and Chu &amp; Weiss,  Modern Immunohistochemistry,  2009, Cambridge University Press. In the present methods, a tissue section is typically contacted with a biological sample in the presence of a secondary antibody that is detectably-labeled. 
         [0016]    The tissue sections used in the present methods are from mammals. For example, mouse tissue is routinely used in immunohistochemistry, but tissue from other rodents (e.g., rats) or other mammals (e.g., rabbits, primates, or humans) also can be used in the present methods. The tissue sections used in the methods described herein typically include gut tissue, kidney tissue, and/or brain tissue. An additional tissue section may include colon tissue. 
         [0017]    Representative biological samples that can be used in the methods described herein include, without limitation, blood, serum, plasma, or spinal fluid. As is well known in the art, the secondary antibody is an antibody raised against the IgG of the animal species in which the primary antibody originated. In addition, detectable labels are well known in the art and include, without limitation, fluorescent labels (e.g., FITC) and enzymatic labels (e.g., alkaline phosphatase (AP) or horseradish peroxidase (HP)). The three components (i.e., the tissue section(s), the biological sample, and the detectably-labeled secondary antibody) are combined under conditions in which a complex is formed between SRP polypeptides in the tissue section, and a corresponding SRP autoantibody in the biological sample, if present, and the detectably-labeled secondary antibody. It would be understood by those skilled in the art that immunohistochemistry routinely includes steps that are not necessarily discussed herein in detail such as washing the tissue samples to remove unbound secondary antibodies and the parallel staining experiments with proper controls. 
         [0018]    Using the detectable label and appropriate detection means, the pattern of complex formation within the tissue sections is identified. The pattern of complex formation within the tissue sections is directly related to the cellular location(s) of the antigen (e.g., an antigenic SRP polypeptide) bound by an autoantibody, when present, in the biological sample. As described herein, the presence of a particular pattern of complex formation in one or more types of tissue indicates the presence of a SRP autoantibody in the biological sample. 
         [0019]    As reported herein, when tissue sections from the gut are used with a biological sample that includes one or more SRP autoantibodies, staining, which represents complex formation, is observed in the cytoplasm of proximal epithelial cells in mucosa and enteric neurons in the gut wall. Also as reported herein, when tissue sections from the brain are used with a biological sample containing one or more SRP autoantibodies, staining is observed in cytoplasm of cerebellar Purkinje, Golgi neurons and granular neurons. In addition, when tissue sections from kidney are used with a biological sample that contains one or more SRP autoantibodies, staining occurs in the renal tubules. Those skilled in the art would understand that the absence of a particular staining pattern (or the presence of non-specific staining) typically indicates the absence of any SRP autoantibodies in the biological sample, provided the proper controls have been performed. 
         [0020]    To date, SRP polypeptides have been associated with polymyositis and dermatomyositis. As reported herein, the presence of a SRP autoantibody in biological samples from a number of individuals was associated with the presence of an autoimmune myopathy. The primary autoimmune myopathy observed was non-inflammatory necrotizing myopathy, but inflammatory necrotizing myopathy also was observed. The presence of a SRP autoantibody in a number of biological samples also was associated with a neuropathy, a connective tissue disease, or cancer. As used herein, SRP polypeptides refer to any of the six SRP polypeptides, identified by their molecular weight, SRP9 (kDa), SRP14, SRP19, SRP54, SRP68, and SRP72, as well as the small cytoplasmic 7SL-RNA. 
         [0021]    In addition to the immunohistochemistry methods described herein, one or more further immunoassays can be performed, either before or after the immunohistochemistry methods. For example, in one embodiment, a Western blot may be performed using, for example, a panel of antigens known to be associated with autoantibodies, the results of which may warrant further evaluation using, for example, the immunohistochemistry methods described herein. In another embodiment, an immunohistochemistry method as described herein may be performed, followed by a Western blot in order to, for example, further confirm the specific antigens recognized by the autoantibodies in the biological sample. 
         [0022]    Tissue sections used in immunohistochemistry are well known in the art and are commercially available from a number of companies (e.g., Asterand, Inc. (Detroit, Mich.); Euroimmun (Morris Plains, N.J.); or Imgenex (San Diego, Calif.)). Since the staining pattern described herein is novel, particularly with respect to SRP-associated autoimmune diseases, compositions including tissue sections (e.g., gut, brain, and/or kidney) are provided herein. Such tissue samples, following immunohistochemistry with a biological sample that contains one or more SRP autoantibodies, exhibit the staining pattern described herein. Alternatively, a composition is provided that includes tissue sections (e.g., gut, brain, and/or kidney) and a description of the staining pattern described herein such that, a person of ordinary skill, after performing the immunohistochemistry methods, could identify the presence of the staining pattern described herein and, thus, the likely presence of a SRP autoantibody. 
         [0023]    In accordance with the present invention, there may be employed conventional molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims. 
       EXAMPLES  
     Example 1  
     Patients  
       [0024]    The method of patient ascertainment in this study distinguishes it from any previous study because the patients herein were not selected on the basis of having a rheumatologic or muscle disease, inflammatory or otherwise. They were identified incidentally in the course of blinded immunohistochemical testing of a large number of patients for neural-restricted IgG autoantibodies. The tested patient population had diverse subacute neurological problems and the differential diagnosis included paraneoplastic autoimmunity. In no case was SRP autoantibody testing requested. For patients seen at Mayo Clinic, clinical information including laboratory, radiological and oncological data was obtained from Mayo Clinic records. For patients seen at other institutions, information was obtained by telephone consultation and correspondence with the caring physician. 
       Example 2  
     Immunofluorescence Screening  
       [0025]    The substrate for this integral component of the service serological evaluation was a frozen composite section of mouse cerebellum/midbrain, gut and kidney tissues (4 μm), prefixed 10 minutes in 10% formalin. Sera were diluted (1:240) in PBS containing 1% BSA, absorbed with bovine liver powder, applied to the substrate and washed after 40 minutes. Bound IgG was detected by applying FITC-conjugated goat-IgG reactive with all human IgG subclasses (Southern Biotechnology). 
       Example 3  
     Western Blot  
       [0026]    A crude preparation of rough microsomes was isolated from mouse pancreas by the method of Walter and Blobel (Walter et al., 1983,  Methods Enzymol.,  96:84-93; Walter et al., 1983,  Methods Enzymol.,  96:682-691) and denatured by boiling (5 minutes) in sample buffer containing 2% SDS and 10% 2-mercaptoethanol. Molecular weight standards included broad range markers (Precision Plus Protein™ Standards Dual Color, BioRad). The proteins were separated electrophoretically in 10% polyacrylamide, then transferred electrophoretically to nitrocellulose (verified by 0.1% Ponceau S (Sigma) staining) Residual binding sites on nitrocellulose were blocked with 10% milk powder. Patients&#39; sera were diluted (1:400) with blocking buffer, and applied to the transblotted proteins. After 1 hour at room temperature followed by washing, bound IgG was detected using HRP-conjugated goat anti-human IgG (Biosource). An enhanced chemiluminescence substrate (SuperSignal West Pico Chemiluminescence Substrate, Thermo Scientific (Product # 34080)) was used to detect HRP. 
       Example 4  
     ELISA  
       [0027]    The presence of SRP54-IgG was evaluated by applying sera (diluted in PBS containing 10% normal goat serum, in doubling steps from 1:200) to Imulon II plates coated with recombinant human SRP54 protein (Diarect AG; 0.5 μg/mL). After holding for 2 hours at 37° C., the wells were washed three times with PBS containing sodium azide (0.02%) and goat serum (2%), and alkaline phosphatase-conjugated goat IgG specific for human IgG was added. Then, after 1 hour at 37° C., the wells were washed three times with PBS and phosphatase substrate (1 mg/ml in diethanolamine buffer) was added. The reaction product was measured photometrically 1 hour later (ELx800; Bio-Tek Instruments Inc; wavelength at 405 nm). Values greater than 150% of the mean optical density yielded by corresponding dilutions with healthy control human sera were considered positive. Positive sera were retested and titrated further as necessary to determine the endpoint dilution. Results were expressed as titer (i.e., reciprocal of the final positive dilution). 
       Example 5  
     Patient Antibody Purification  
       [0028]    Mouse pancreatic lysate proteins separated by electrophoresis were trans-blotted to nitrocellulose. Bound antigenic protein was located by Western Blot staining of excised vertical edge strips. Horizontal intervening strips bearing antigens, and a control horizontal strip lacking the antigen of interest, were exposed to patients&#39; sera (1:400 dilution absorbed with bovine liver powder, 2 hours). After washing the strips to remove non-specifically bound antibody (six times in 20 mM Tris-HCl (pH 7.6) containing 300 mM NaCl and 0.1% Tween-20), bound IgG was eluted in 0.1 M acetic acid, neutralized with 2 M Tris (pH 8.0), dialyzed 16 hr against PBS with 0.02% sodium azide, and concentrated by Amicon Ultra tube centrifugation (to a final volume &lt;100 μL). This IgG was applied to the composite mouse tissue substrate to evaluate its immunofluorescence staining pattern. 
       Example 6  
     cDNA Cloning  
       [0029]    RNA from human pancreas was reverse-transcribed to provide first-strand complementary DNA (cDNA). Gene specific primer (SRP72 R3; 5′—CCA TAT CTC ACT AGG CAG AC—3′ (SEQ ID NO:1)) and Superscript III RT (Invitrogen) were used. A gene specific primer pair (SRP72 F1; 5′—ATG GCG AGC GGC GGC AGC GG—3′ (SEQ ID NO:2), SRP72 R3; 5′—CCA TAT CTC ACT AGG CAG AC—3′ (SEQ ID NO:3)) was used to amplify cDNA. A 2 kb product was purified using Wizard SV Gel and PCR Clean-Up System (Promega), and ligated into pcDNA4/HisMax-TOPO vector that was used to transform TOP 10 competent cells. One clone identified by restriction mapping with HindIII followed by DNA sequencing was identical to human SRP72 with the exception of one frameshift mutation and a silent mutation at codon 7 (GGG to GGT). The frameshift mutation was corrected using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Plasmid DNA was used to transform XL-10 Gold Ultracompetent cells (Stratagene). Four clones identified by HindIII restriction mapping and subsequent DNA sequencing demonstrated that the frameshift mutation had been corrected. 
       Example 7  
     Recombinant Protein  
       [0030]    HEK293 cell lines were transfected (FuGENE 6 Transfection Reagent; Roche) with plasmid DNA encoding SRP72 (obtained using Qiafilter Maxi Kit; Promega). 
       Example 8  
     Immunohistochemical Characteristics  
       [0031]    In performing the standard clinical immunofluorescence screening assay, 32 patients were identified whose serum IgG yielded striking cytoplasmic staining of cerebellar Purkinje and Golgi neurons. In this respect, the pattern resembled the PCA-1 (anti-Yo) pattern ( FIG. 1 ) but, unlike PCA-1, this IgG also stained the cytoplasm of granular neurons (e.g., in a “chicken-wire” pattern). Furthermore, the reactivity of this IgG was not restricted to neurons; it also bound to proximal epithelial cells in the gastric mucosa and to renal tubules. The resemblance of the cytoplasmic staining in large neurons to that of PCA-1 immunoreactivity ( FIG. 2 ) suggested that the autoantigen detected by this novel IgG might be a component of endoplasmic reticulum, as has been shown for PCA-1. 
       Example 9  
     Western Blot Characteristics  
       [0032]    In further testing, IgG in these 32 patients&#39; sera bound to microsomal antigens of molecular size consistent with known SRP subunits. Twelve bound to a 54 kD molecule, seven bound to a 72 kD molecule, and thirteen bound to both 54 and 72 kD molecules. 
       Example 10  
     Clinical, Serological and Neoplastic Associations, Co-Existing Autoantibodies and Demographic Data  
       [0033]    Table 1 in Appendix A provides information on the patients evaluated herein. Twenty-eight of the 32 patients identified by immunofluorescence presented with a subacute, progressive myopathy characterized by proximal muscle weakness and, in some cases, axial muscle weakness. Bulbar musculature was involved in ten patients (36%). Four patients (14%) had Raynaud phenomenon. Among the 10 patients whose IgG bound only to SRP54, six had necrotizing myopathy, three had polymyositis, and one had non-specified myopathy. Among the five patients whose IgG bound only to SRP72, three had necrotizing myopathy and two had inflammatory myopathy. Among the thirteen patients whose IgG bound to both SRP54 and SRP72, seven had necrotizing myopathy, three had inflammatory myopathy, and three had indeterminate myopathy. 
         [0034]    Of the remaining four identified patients, other autoimmune disorder diagnoses were neuropathy, 2; Sjögren syndrome, 1; and motor neuronopathy, 1. 
       Example 11  
     Histopathology  
       [0035]    Six of the 8 Mayo Clinic patients whose muscle biopsies were reviewed had common pathological findings of necrotic and regenerating fibers with scant or no inflammatory cells ( FIG. 2 ). The seventh patient had fiber necrosis with marked mononuclear cellular infiltrates surrounding perimysial vessels as well as endomysial connective tissue. The eighth patient had inflammatory exudates at both perimysial and endomysial sites. 
       Example 12  
     Oncological Findings and Other Autoimmune Disorders  
       [0036]    Ten patients (36%) had evidence of a malignant or benign neoplasm: colon carcinoma, 1; colonic polyps, 1; endometrial carcinoma, 1; rectal tubular adenoma and thyroid adenoma, 1; Hodgkin lymphoma, 1; lung and mediastinal nodule with smoking history, 1; lung nodule, 2; and hilar plus axillary lymphadenopathy, 1; pituitary microadenoma, 1; papillary thyroid carcinoma, 1). In most patients, the diagnosis of neoplasia was made prior to myopathy onset. Thirteen patients had one or more coexisting autoantibodies: (thyroglobulin, 1; thyroperoxidase, 2; both 4); a voltage-gated potassium channel complex antibody, 3 (median 0.08 nmol/L; normal range 0.00-0.02 nmol/L); GAD65 autoantibody, 1 (0.08 nmol/L; normal range 0.00-0.02 nmol/L); ANA, 3; aquaporin-4-IgG, 1 (25 nmol/L, detected by immunoprecipitation; normal range 0.00-9.99 nmol/L). 
       Example 13  
     Treatment Responses  
       [0037]    Clinical follow-up information was available for 18 patients. All but one received corticosteroid and immunosuppressant drugs. Thirteen improved after treatment, and five did not improve. Of those that did not improve, one received just corticosteroid, one received intravenous steroid and IV-Ig, one received cyclophosphamide and steroid, one received only IV-Ig, and treatment (steroid and methotrexate) was delayed in one. See Table 1 in Appendix A. 
         [0038]    It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims. 
         [0039]    Disclosed are methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 APPENDIX A 
               
             
             
               
                   
               
               
                 Table 1 Demographic data, clinical presentations, evidence for neoplasia and treatment responses 
               
               
                 in 22 SRP-IgG-positive patients 
               
             
          
           
               
                 Pa- 
                   
                   
                 Smok- 
                   
                   
                   
                   
                 SRP-IgG 
                   
               
               
                 tient 
                 Age/ 
                   
                 ing 
                   
                 Neoplasm 
                 Muscle 
                 Treatment/ 
                 subunit 
                 Other 
               
               
                 No. 
                 Sex 
                 Race* 
                 History §   
                 Clinical Presentation 
                 Found 
                 biopsy 
                 Response 
                 specificity 
                 AutoAbs 
               
               
                   
               
             
          
           
               
                 1 
                 41/F 
                 C 
                 + 
                 Proximal muscle weakness 
                 Colon 
                 NM 
                 Corticosteroid/ 
                 54 
                 None 
               
               
                   
                   
                   
                   
                 of lower and upper 
                 carcinoma 
                   
                 poor response (Rx 
               
               
                   
                   
                   
                   
                 extremities, Raynaud 
                 (7 years 
                   
                 delayed) 
               
               
                   
                   
                   
                   
                 phenomenon, no bulbar 
                 before 
               
               
                   
                   
                   
                   
                 involvement, CPK &gt;10000 
                 myopathy) 
               
               
                 2 
                 44/F 
                 C 
                 + 
                 Progressive upper and 
                 Colon 
                 NM 
                 Prednisolone/no 
                 54 
                 None 
               
               
                   
                   
                   
                   
                 lower extremities 
                 polyps (5 
                   
                 follow-up 
               
               
                   
                   
                   
                   
                 weakness, arthritis, 
                 years 
               
               
                   
                   
                   
                   
                 dysphagia, No Raynaud 
                 before 
               
               
                   
                   
                   
                   
                 phenomenon, CPK 2824 
                 myopathy) 
               
               
                 3 
                 48/M 
                 C 
                 (+) 
                 Rapidly progressive limbs 
                 Hodgkin 
                 NM 
                 IVIg and 
                 54, 72 
                 None 
               
               
                   
                   
                   
                   
                 and axial weakness, 
                 lymphoma 
                   
                 corticosteroid/no 
               
               
                   
                   
                   
                   
                 dysphagia, CPK 3485 
                 (3 years 
                   
                 follow-up 
               
               
                   
                   
                   
                   
                   
                 before 
               
               
                   
                   
                   
                   
                   
                 myopathy) 
               
               
                 4 
                 50/M 
                 C 
                 + 
                 Progressive bilateral lower 
                 Mediastinal 
                 NM 
                 Prednisolone/ 
                 54 
                 None 
               
               
                   
                   
                   
                   
                 extremities weakness, 
                 and hilar 
                   
                 significant 
               
               
                   
                   
                   
                   
                 Raynaud&#39;s phenomenon, 
                 nodule 
                   
                 improvement 
               
               
                   
                   
                   
                   
                 CPK 8832 
               
               
                 5 
                 46/F 
                 C 
                 + 
                 Proximal muscle weakness 
                 None 
                 NM 
                 Prednisolone/ 
                 54, 72 
                 Thyroid 
               
               
                   
                   
                   
                   
                 of lower then upper 
                   
                   
                 improved 
               
               
                   
                   
                   
                   
                 extremities weakness, No 
               
               
                   
                   
                   
                   
                 dysphagia nor dyspnea. 
               
               
                   
                   
                   
                   
                 Hypothyroidism, CPK 1052 
               
               
                 6 
                 66/F 
                 C 
                 − 
                 Proximal muscle weakness 
                 Not found 
                 NM 
                 Corticosteroid/ 
                 54, 72 
                 Thyroid 
               
               
                   
                   
                   
                   
                 of lower extremities, 
                   
                   
                 improved 
               
               
                   
                   
                   
                   
                 Raynaud&#39;s phenomenon, 
               
               
                   
                   
                   
                   
                 Interstitial lung disease, 
               
               
                   
                   
                   
                   
                 dysphagia, CPK 7200 
               
               
                 7 
                 40/M 
                 ? 
                 (+) 
                 Proximal muscle weakness 
                 None 
                 Inflmm 
                 Corticosteroid/no 
                 54, 72 
                 Thyroid 
               
               
                   
                   
                   
                   
                 of upper then lower 
                   
                 myositis 
                 follow-up 
               
               
                   
                   
                   
                   
                 extremities, dysphagia, 
               
               
                   
                   
                   
                   
                 CPK 6307 
               
               
                 8 
                 48/F 
                 ? 
                 ? 
                 Severe myopathy 
                 None 
                 NM 
                 Corticosteroid/ 
                 54, 72 
                 VGKC; 
               
               
                   
                   
                   
                   
                   
                   
                   
                 poor response 
                   
                 thyroid 
               
               
                 9 
                 47/M 
                 ? 
                 + 
                 Rapidly progressive 
                 None 
                 Severe 
                 IV corticosteroid/ 
                 54, 72 
                 None 
               
               
                   
                   
                   
                   
                 proximal &gt;distal limbs in 6 
                   
                 muscle 
                 no follow-up 
               
               
                   
                   
                   
                   
                 months 
                   
                 atrophic 
               
               
                   
                   
                   
                   
                   
                   
                 fiber 
               
               
                 10 
                 39/F 
                 ? 
                 ? 
                 Progressive proximal 
                 None 
                 NM 
                 Metrotrexate and 
                 54 
                 ANA 
               
               
                   
                   
                   
                   
                 muscle weakness over 4-6 
                   
                   
                 corticosteroid/not 
               
               
                   
                   
                   
                   
                 weeks, wheelchair within 4 
                   
                   
                 improved 
               
               
                   
                   
                   
                   
                 weeks 
               
               
                 11 
                 50/F 
                 C 
                 ? 
                 6 months painless 
                 None 
                 NM 
                 IVIg, IV 
                 54, 72 
                 GAD65; 
               
               
                   
                   
                   
                   
                 progressive proximal 
                   
                   
                 corticosteroid/not 
                   
                 thyroid 
               
               
                   
                   
                   
                   
                 muscle weakness, now 
                   
                   
                 improved 
               
               
                   
                   
                   
                   
                 bed-ridden. CK 18000 
               
               
                 12 
                 50/M 
                 C 
                 − 
                 Rapidly progressive 
                 None 
                 Extensive 
                 No information 
                 54, 72 
                 None 
               
               
                   
                   
                   
                   
                 proximal &gt;distal muscles 
                   
                 muscle 
               
               
                   
                   
                   
                   
                 weakness, Bulbar 
                   
                 loss with 
               
               
                   
                   
                   
                   
                 involvement with respiratory 
                   
                 fatty 
               
               
                   
                   
                   
                   
                 failure. Muscle Bx: 
                   
                 replacement 
               
               
                   
                   
                   
                   
                 extensive muscle loss with 
               
               
                   
                   
                   
                   
                 fatty replacement, cardiac 
               
               
                   
                   
                   
                   
                 involvement 
               
               
                 13 
                 70/M 
                 ? 
                 ? 
                 Myopathy 
                 No 
                 Not 
                 No information 
                 54, 72 
                 None 
               
               
                   
                   
                   
                   
                   
                 information 
                 otherwise 
               
               
                   
                   
                   
                   
                   
                   
                 specified 
               
               
                 14 
                 47/M 
                 ? 
                 ? 
                 Proximal upper and lower 
                 None 
                 Polymyositis 
                 Corticosteroid and 
                 54 
                 Thyroid 
               
               
                   
                   
                   
                   
                 extremities weakness. 
                   
                   
                 cyclophosphamide/ 
               
               
                   
                   
                   
                   
                 Elevated CK 
                   
                   
                 not improved 
               
               
                 15 
                 39/F 
                 ? 
                 ? 
                 Insidious proximal upper 
                 None 
                 Necrotizing 
                 No information 
                 54, 72 
                 None 
               
               
                   
                   
                   
                   
                 and lower extremities 
                   
                 myopathy 
               
               
                   
                   
                   
                   
                 weakness. Elevated CK. 
               
               
                   
                   
                   
                   
                 Persistent pain in muscle 
               
               
                 16 
                 55/F 
                 C 
                 (+) 
                 Proximal muscle weakness 
                 Endometrial 
                 Dermatomyositis 
                 No information 
                 35 kD 
                 ANA, 
               
               
                   
                   
                   
                   
                 (lower limb) with skin rash 
                 carcinoma 
                   
                   
                   
                 dsDNA, 
               
               
                   
                   
                   
                   
                 Muscle bx: perivascular and 
                 (3 years 
                   
                   
                   
                 SSA, 
               
               
                   
                   
                   
                   
                 perimysial infiltrate with 
                 before 
                   
                   
                   
                 SSB 
               
               
                   
                   
                   
                   
                 CD20 positive B-cell and 
                 myopathy) 
               
               
                   
                   
                   
                   
                 CD3 positive T-cell with 
               
               
                   
                   
                   
                   
                 perivascular atrophy. SLE 
               
               
                   
                   
                   
                   
                 with ANA, dsDNA, SSA, 
               
               
                   
                   
                   
                   
                 SSB+ 
               
               
                 17 
                 54/F 
                 ? 
                 ? 
                 3 yrs hx of proximal muscle 
                 None 
                 NM 
                 Metrotrexate plus 
                 72 
                 None 
               
               
                   
                   
                   
                   
                 weakness, feeding tube 
                   
                   
                 low dose 
               
               
                   
                   
                   
                   
                 and wheelchair. High CK. 
                   
                   
                 prednisolone/ 
               
               
                   
                   
                   
                   
                 Rx with IV steroid, IVIG 
                   
                   
                 improved 
               
               
                   
                   
                   
                   
                 improved can walk again 
               
               
                   
                   
                   
                   
                 Experienced couldn&#39;t get up 
               
               
                   
                   
                   
                   
                 from bed. Rx with PLEX 
               
               
                   
                   
                   
                   
                 and q 3 months, MTX + low 
               
               
                   
                   
                   
                   
                 dose prednisolone --&gt; 
               
               
                   
                   
                   
                   
                 doing well 
               
               
                 18 
                 37/F 
                 C 
                 − 
                 2007 proximal upper and 
                 Thyroid 
                 inflammatory 
                 Corticosteroid 
                 72 
                 None 
               
               
                   
                   
                   
                   
                 lower extremities 
                 adenoma, 
                 myositis 
                 80 mg/day, MTX 
               
               
                   
                   
                   
                   
                 weakness, breathing 
                 rectal 
                   
                 2.5 mg q week/ 
               
               
                   
                   
                   
                   
                 difficulty and blurred vision. 
                 tubular 
                   
                 improved 
               
               
                   
                   
                   
                   
                   
                 adenoma 
               
               
                 19 
                 77/F 
                 ? 
                 ? 
                 1 yr rapidly progressive 
                 Lung 
                 NM 
                 No information 
                 72 
                 Thyroid 
               
               
                   
                   
                   
                   
                 dysphagia, painless and 
                 nodule 
               
               
                   
                   
                   
                   
                 severe atrophy 4 
               
               
                   
                   
                   
                   
                 extremities --&gt; wheelchair 
               
               
                   
                   
                   
                   
                 bound. Muscle Bx 
               
               
                   
                   
                   
                   
                 consistent with necrotizing 
               
               
                   
                   
                   
                   
                 myositis. Anti-SRP is 
               
               
                   
                   
                   
                   
                 negative. CT chest has lung 
               
               
                   
                   
                   
                   
                 nodule but PET scan was 
               
               
                   
                   
                   
                   
                 negative. Further 
               
               
                   
                   
                   
                   
                 investigation for lung 
               
               
                   
                   
                   
                   
                 nodule ?? 
               
               
                 20 
                 45/M 
                 ? 
                 ? 
                 Myopathy 
                 None 
                 NM 
                 Plasmapheresis, 
                 72 
                 None 
               
               
                   
                   
                   
                   
                   
                   
                   
                 steroid and 
               
               
                   
                   
                   
                   
                   
                   
                   
                 methotrexate/ 
               
               
                   
                   
                   
                   
                   
                   
                   
                 improved 
               
               
                 21 
                 67/F 
                 ? 
                 + 
                 Myopathy 
                 Lung 
                 IM 
                 No information 
                 72 
                 AQP4 
               
               
                   
                   
                   
                   
                   
                 nodule 
               
               
                 22 
                 37/M 
                 ? 
                 − 
                 2 yrs progressive proximal 
                 Hilar and 
                 IM 
                 IVIg/not improved 
                 54 
                 VGKC 
               
               
                   
                   
                   
                   
                 upper extremities and 
                 axillary 
                   
                   
                   
                 ANA, 
               
               
                   
                   
                   
                   
                 shoulder weakness, a year 
                 lymphadenopathy 
                   
                   
                   
                 Anti-Sm, 
               
               
                   
                   
                   
                   
                 later developed proximal 
                   
                   
                   
                   
                 Anti- 
               
               
                   
                   
                   
                   
                 lower extremities 
                   
                   
                   
                   
                 RNP, 
               
               
                   
                   
                   
                   
                 weakness, mild dysphagia 
                   
                   
                   
                   
                 Anti- 
               
               
                   
                   
                   
                   
                 for solid foods 
                   
                   
                   
                   
                 SSA 
               
               
                 23 
                 53/M 
                 C 
                 − 
                 2 years painless, proximal 
                 None 
                 IM 
                 prednisolone 60 mg/ 
                 54, 72 
                 None 
               
               
                   
                   
                   
                   
                 leg weakness w/ 20 lb wt 
                   
                   
                 day with once 
               
               
                   
                   
                   
                   
                 loss and CK 13,000 
                   
                   
                 weekly metrotexate 
               
               
                   
                   
                   
                   
                 treatment initially 
               
               
                 24 
                 70/M 
                 C 
                 − 
                 6 months progressive 
                 Pituitary 
                 IM 
                 Prednisolone and 
                 35, 54, 72 
                 VGKC 
               
               
                   
                   
                   
                   
                 proximal lower extremities 
                 macroadenoma 
                   
                 cellcept 
               
               
                   
                   
                   
                   
                 muscle weakness then 
               
               
                   
                   
                   
                   
                 upper extremities and neck 
               
               
                   
                   
                   
                   
                 flexor weakness, 
               
               
                   
                   
                   
                   
                 hoarseness and dysphagia. 
               
               
                   
                   
                   
                   
                 No rash, pain, Raynaud 
               
               
                   
                   
                   
                   
                 phenomenon. 
               
               
                   
                   
                   
                   
                 CK ~6000. EMG consistent 
               
               
                   
                   
                   
                   
                 with axial and proximal 
               
               
                   
                   
                   
                   
                 myopathy. 
               
               
                 25 
                 55/F 
                 AA 
                 NA 
                 Since early 2009 severe 
                 None 
                 NM 
                 Respond well to 
                 54 
                 None 
               
               
                   
                   
                   
                   
                 progressive proximal 
                   
                   
                 steroid and IVIG 
               
               
                   
                   
                   
                   
                 muscle weakness, 
               
               
                   
                   
                   
                   
                 dysphagia &amp; resp failure. 
               
               
                   
                   
                   
                   
                 CK 17000. 
               
               
                 26 
                 80?F 
                 ? 
                 NA 
                 Proximal lower extremities 
                 NA 
                 NA 
                 NA 
                 54 
                 Striational, 
               
               
                   
                   
                   
                   
                 weakness 
                   
                   
                   
                   
                 VGCC 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 (N), 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 GAD65 
               
               
                 27 
                 83/F 
                 ? 
                 (+) 
                 Progressive severe 
                 NA 
                 NM 
                 NA 
                 54 
                 None 
               
               
                   
                   
                   
                   
                 generalized muscle 
               
               
                   
                   
                   
                   
                 weakness 
               
               
                 28 
                 64/F 
                 C 
                 − 
                 Severe proximal muscle 
                 Papillary 
                 IM 
                 Prednisolone + 
                 54 
                 None 
               
               
                   
                   
                   
                   
                 weakness; CK 4500. 
                 thyroid 
                   
                 IVIG 
               
               
                   
                   
                   
                   
                   
                 carcinoma 
               
               
                 29 
                 50/M 
                 ? 
                 + 
                 Late 2008 insidiously onset 
                 None 
                 NM 
                 Metotrexate + 
                 54 
                 None 
               
               
                   
                   
                   
                   
                 progressive weakness, 
                   
                   
                 prednisolone 
               
               
                   
                   
                   
                   
                 dysphagia need PEJ tube 
               
               
                   
                   
                   
                   
                 and loss of voice 
               
               
                   
               
               
                 *Race: C = Caucasian, AA = African American, ? = no information, 
               
               
                   § Smoking History: + = smoker, (+) = ex-smoker, − = non-smoker