Hans-Hilmar Goebel

Review: Immune-mediated necrotizing myopathies – a heterogeneous group of diseases with specific myopathological features

Immune‐mediated necrotizing myopathies (IMNMs) are now well recognized among the so‐called idiopathic inflammatory myopathies (IIMs), which also comprise dermatomyositis (DM), polymyositis (PM), sporadic inclusion body myositis (sIBM) and non‐specific myositis. All of these conditions are defined on the basis of distinct clinical symptoms, in combination with results derived from muscle biopsy and additional data, such as measurement of the serum creatine kinase (CK) level as well as myositis‐associated and myositis‐specific autoantibodies, electromyography (EMG) and modern imaging techniques. Importantly, diagnosis of one of the above mentioned myositis forms implies a specific clinical syndrome or a distinct disease. However, there is considerable clinical heterogeneity, and overlap requiring further diagnostic precision. Classification and subclassification of IIMs are highly debated and the subjects of intense research, especially as clinical trials with anti‐inflammatory agents should follow universally defined and accepted criteria. This review focuses on the description of the spectrum of immune‐mediated necrotizing myopathies with an emphasis on their myopathological features.

Differential roles of hypoxia and innate immunity in juvenile and adult dermatomyositis

Dermatomyositis (DM) can occur in both adults and juveniles with considerable clinical differences. The links between immune-mediated mechanisms and vasculopathy with respect to development of perifascicular pathology are incompletely understood. We investigated skeletal muscle from newly diagnosed, treatment-naïve juvenile (jDM) and adult dermatomyositis (aDM) patients focusing on hypoxia-related pathomechanisms, vessel pathology, and immune mechanisms especially in the perifascicular region. Therefore, we assessed the skeletal muscle biopsies from 21 aDM, and 15 jDM patients by immunohistochemistry and electron microscopy. Transcriptional analyses of genes involved in hypoxia, as well as in innate and adaptive immunity were performed by quantitative Polymerase chain reaction (qPCR) of whole tissue cross sections including perifascicular muscle fibers.Through these analysis, we found that basic features of DM, like perifascicular atrophy and inflammatory infiltrates, were present at similar levels in jDM and aDM patients. However, jDM was characterized by predominantly hypoxia-driven pathology in perifascicular small fibers and by macrophages expressing markers of hypoxia. A more pronounced regional loss of capillaries, but no relevant activation of type-1 Interferon (IFN)-associated pathways was noted. Conversely, in aDM, IFN-related genes were expressed at significantly elevated levels, and Interferon-stimulated gene (ISG)15 was strongly positive in small perifascicular fibers whereas hypoxia-related mechanisms did not play a significant role.In our study we could provide new molecular data suggesting a conspicuous pathophysiological ‘dichotomy’ between jDM and aDM: In jDM, perifascicular atrophy is tightly linked to hypoxia-related pathology, and poorly to innate immunity. In aDM, perifascicular atrophy is prominently associated with molecules driving innate immunity, while hypoxia-related mechanisms seem to be less relevant.

Necrosis in anti-SRP+ and anti-HMGCR+myopathies: Role of autoantibodies and complement

Objective To characterize muscle fiber necrosis in immune-mediated necrotizing myopathies (IMNM) with anti–signal recognition particle (SRP) or anti–3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGCR) antibodies and to explore its underlying molecular immune mechanisms. Methods Muscle biopsies from patients with IMNM were analyzed and compared to biopsies from control patients with myositis. In addition to immunostaining and reverse transcription PCR on muscle samples, in vitro immunostaining on primary muscle cells was performed. Results Creatine kinase levels and muscle regeneration correlated with the proportion of necrotic fibers (r = 0.6, p < 0.001). CD68+iNOS+ macrophages and a Th-1 immune environment were chiefly involved in ongoing myophagocytosis of necrotic fibers. T-cell densities correlated with necrosis but no signs of cytotoxicity were detected. Activation of the classical pathway of the complement cascade, accompanied by deposition of sarcolemmal immunoglobulins, featured involvement of humoral immunity. Presence of SRP and HMGCR proteins on altered myofibers was reproduced on myotubes exposed to purified patient-derived autoantibodies. Finally, a correlation between sarcolemmal complement deposits and fiber necrosis was observed (r = 0.4 and p = 0.004). Based on these observations, we propose to update the pathologic criteria of IMNM. Conclusion These data further corroborate the pathogenic role of anti-SRP and anti-HMGCR autoantibodies in IMNM, highlighting humoral mechanisms as key players in immunity and myofiber necrosis.

Progressive external ophthalmoplegia as initial manifestation of sporadic late-onset nemaline myopathy.

Sporadic late-onset nemaline myopathy (SLONM) is a rare disease and typically presents in patients older than 40 years with slowly progressive weakness in a limb-girdle distribution and unremarkable family history [1, 2]. Serum creatine kinase is usually normal, and electromyography may show myopathic changes. Nemaline rods in affected muscle fibers are considered the histopathological hallmark of nemaline myopathy and are mainly found within the sarcoplasm, inside of myonuclei, or both [3]. Ocular involvement has never been described in a patient with SLONM. We report a patient with SLONM suffering from prominent involvement of external ocular muscles. A 60-year-old female patient was reexamined for a chronic progressive oculopharyngeal syndrome that was present for 6 years. Initial symptoms were double vision and ptosis, followed by weakness of eye-closure. Two years after the onset of symptoms, she presented an exclusive picture of external ophthalmoplegia. During the following years she was unable to open or close her eyes voluntarily, lost her ability to whistle or smile, developed a hoarse voice and swallowing difficulties. There was no diurnal variation of symptoms, or fatigability. Family history for neuromuscular diseases was unremarkable. On examination she had complete bilateral ptosis and palsy of extraocular muscles, while pupillar size and pupillar reactions to light and accommodation were normal. She had a myopathic face with atrophy of masseter and temporalis muscles, severe bilateral facial palsy, and severe dysphonia. Strength was MRC grade 4 for neck flexion, head rotation, and neck extension. Further neurological examination was normal, notably fundoscopy and proximal and distal limb strength. Laboratory examinations provided normal values for standard parameters, serum creatine kinase, a comprehensive panel of autoimmune antibodies, including antibodies against acetylcholine receptor or muscle-specific kinase, anti-HIV 1/2 antibodies, and p24 antigen. Serum ACE, soluble Interleukin-2 receptor levels, serum and urine calcium levels, and repeated imaging studies ruled out sarcoidosis. Protein electrophoresis and immunofixation did not show any paraprotein, findings that rule out an association with HIV, sarcoidosis, or monoclonal gammopathy, as described in some patients in the literature [4–7]. MRI revealed symmetric atrophy of ocular muscles and fatty degeneration of pterygoideus medialis and masseter muscles, no signs of edema or contrast enhancement. Thorough neuromuscular transmission studies as well as spinal cord and brain imaging were unremarkable. No other organs were involved. Muscle biopsy of the sternocleidomastoideus revealed numerous rods, which appeared to be O. Wengert A. Meisel Department of Neurology, Charite-Universitatsmedizin Berlin, Chariteplatz 1, 10117 Berlin, Germany

Inflammatory myopathy with abundant macrophages (IMAM): The immunology revisited

We describe a patient with a clinically atypical presentation of inflammatory myopathy with abundant macrophages (IMAM) but with convincing muscle biopsy features of this subform of inflammatory myopathy. IMAM is characterized mainly by a conspicuous infiltration of muscle and connective tissue by numerous macrophages remote from necrotic and basophilic regenerating muscle fibers. Typically few, mostly CD4(+) T helper (Th) cells are also present. Here, we report a patient with IMAM and demonstrate, that most macrophages express the macrophage mannose receptor 1 (CD206) corresponding to alternatively activated (M2) polarization. Accordingly, signal transducer and activator of transcription 6 (STAT6), involved in Th2-M2 immunity, was expressed at high levels in skeletal muscle. However, TNFα, IFNγ and STAT1, mediators of the T helper 1-classically activated (M1) response were elevated in skeletal muscle and in blood, while expression of CD206 was elevated in skeletal muscle only. Our results argue that IMAM could be a distinct entity between the inflammatory myopathies rather than a subform of dermatomyositis.

Pipestem capillaries in necrotizing myopathy revisited

Pipestem-capillaries in necrotizing myopathy, have been reported as a feature of a distinct type of myopathy. Here, we analyze four muscle biopsy specimens from patients exhibiting endomysial fibrosis associated with pipestem capillaries using histological and electronmicroscopic techniques. However, only one case displayed all of the originally described features, including necrotic fibres, capillary thickening and lack of a significant lymphocytic inflammation, while one case exhibited striking capillary pathology with minimal necrosis and absence of inflammation, and the other two cases were accompanied by additional pathological features. These data support the existence of a microangiopathy with pipestem capillaries as a characteristic and distinct histopathological pattern, and indicate that it occurs in the context of a variety of muscular disorders broader than initially suspected. Furthermore, we show that the pipestem-capillary associated decrease in fibre size is at least in part a result of hypoxic changes.

Expanding the spectrum of livedoid vasculopathy: peculiar neuromuscular manifestations.

Livedoid vasculopathy (LV) is a rare and recurrent chronic disorder mainly restricted to the skin. LV is characterized by recurrent purpura, livedo reticularis of the legs associated with painful ulcerations, resulting in atrophic, porcelain scars [1]. This disease is considered a thrombo-occlusive vasculopathy of superficial dermal micro-vessels due to either idiopathic cause or secondary to a defined state of thrombophilia [2]. Diagnosis is based on the characteristic skin lesions and presence of intraluminal thrombosis, endothelial cell hyperplasia and sub-intimal hyaline degeneration in dermal vessels [3]. LV is not a vasculitis sensu-stricto, as generally no inflammation occurs within the wall of the vessels, but perivascular inflammation may be present secondarily [4]. Neurological involvement in LV is rare and may expand its nosological spectrum. Symptoms comprise mononeuritis multiplex, and the underlying pathophysiology has been postulated to be the result of ischaemia [5,6]. However, vasculitis has been observed, in an association between LV and periarteritis nodosa (PAN) [7]. From a physiopathological point of view, this entity is surprising, as a general state of hypercoagulability leads to damage confined to the skin and much less frequently to the peripheral nervous system, but not to other tissues. Here, we describe LV patients with a peripheral neuropathy attributed to thrombotic vasculopathy without signs of vasculitis. Furthermore, we illustrate the involvement of the skeletal muscle, characterized by an extreme loss of capillaries, associated with peculiar perifascicular pathology. In a retrospective observational study, primary LV patients (n = 18, 10 females and mean age of 50.1 years) diagnosed by international criteria [8] were enrolled. We identified three patients, who had undergone combined nerve–muscle biopsy to explore peripheral neurological symptoms. Patients were 58, 44 and 22 years at time of the first skin manifestation (patient A, B and C, respectively). Neurological signs appeared respectively 10 (patients A), 20 (patient B) and 23 years after (patient C) the first skin manifestation. One patient (patient A) suffered from mononeuritis multiplex. Electroneurography confirmed popliteal sciatic nerve damage and showed injuries of median and ulnar nerves. Patient B suffered from sensory polyneuropathy of lower limbs. Patient C had bilateral dysesthaesia and hypaesthesia of the back of the right foot, and his EMG showed a motor and sensory axonal neuropathy. No proximal weakness was observed in any of the three patients, and all of them had normal CK levels. Other causes of peripheral neuropathy were excluded by extensive laboratory tests, and there was no history of drugs, neurotoxin exposure or association with cancer. All patients showed remarkably similar morphological alterations. A severe loss of myelinated axons, with only few thinly myelinated axons (with minimal signs of axonal regeneration) and endoneurial replacement by fibrous tissue, was the most prominent finding (Figure 1A,B). Furthermore, a relevant formation of collagen pockets on ultra-structural examination illustrated loss of un-myelinated axons (Figure 1C). All fascicles were equally affected. The second observation consisted of enlarged vessels with conspicuous engorgement mainly in the epineurium (Figure 1D). Importantly, in all cases, inflammatory infiltrates basically consisting of CD3 lymphocytes were detected around enlarged vessels in the epineurium (Figure 1D), however without signs of vasculitis. In addition, we found a remarkable vascular pathology mainly involving capillaries of the endoneurium. Alterations consisted of enlarged endothelial cells, which appeared thickened, occasional basal membrane duplication, apposition of endothelial cells layers, as well as necrosis of capillaries (Figure 1A,E,F). Indirect signs of neuropathy were also observed in all muscle biopsies with neurogenic features consisting of nuclear clumps, grouped atrophic fibres and fibre-type grouping (Figure 1G). Additional myopathic changes included split fibres, and round and hypertrophic fibres with internalized nuclei (Figure 1H). Although neurogenic changes are frequently associated with myopathic features especially during a chronic course of disease, we also detected myopathic changes not attributable to neu-

Juvenile autophagic vacuolar myopathy – a new entity or variant?

Autophagic vacuolar myopathies (AVMs) comprise a heterogeneous cluster of diseases. These include lysosomal storage disease with or without abnormal acid maltase activity such as glycogen storage disease (GSD) type II (OMIM 232300) and lysosomal membrane protein LAMP-2-deficient Danon disease (OMIM 300257) [1] caused by mutations in the LAMP-2 gene (OMIM 309060). X-linked myopathy with excessive autophagy (XMEA; OMIM 310440), infantile AVM (OMIM 609500) and adult onset AVM with multi-organ involvement are also included in the spectrum of AVMs [2–5]. These latter diseases demonstrate distinct pathomorphological features, including vacuoles with autophagy-associated proteins and immunoreactivity for a multitude of sarcolemmal proteins (e.g. dystrophin, b-spectrin, dysferlin, caveolin-3, sarcoglycans), prominent acetylcholine esterase (AChE) activity and complement deposition [3,6]. Particularly in XMEA, ultrastructural evidence of intravacuolar debris associated with multiplication of the basal lamina and fusion of the vacuoles with the sarcolemma have been described as specific hallmarks for the disease, suggesting an aberrant exocytotic process [2,6]. Furthermore, the VMA21 gene on chromosome Xq28, which encodes a chaperone for assembly of lysosomal vacuolar ATPase, has been established as responsible for XMEA [7]. It is likely that AVMs share a common pathomechanism related to a dysfunctional autophagosomal machinery [1,6]. After a normal post-natal and infantile development, a 14-year-old boy from Yemen presented with mild proximal weakness affecting his lower extremities for a duration of 1 year. He showed Gowers sign upon neurological examination. Strength in his arms and distal leg muscles was unremarkable as was the rest of the entire neurological examination. In particular, no evidence of muscle or tendon contractures was found. Creatine kinase was elevated up to 15-fold, and a cardiac work-up disclosed a Wolf-Parkinson-White syndrome by 24-h electrocardiography, but no signs of cardiomyopathy by echocardiography, and no involvement of further internal organs was detectable. He had no intellectual deficits, and his family history was reported to be unremarkable, notably with an absence of a history of muscle diseases. A biopsy specimen from the quadriceps muscle was subjected to routine enzyme histochemistry, immunohistochemistry and ultrastructural examination. In most fibres, numerous vacuoles inside the sarcoplasm exhibited sarcolemmal lining, demonstrated by the strong but variable expression of plasma membraneor basal laminaassociated proteins, including b-spectrin, caveolin-3, laminin-a2 (300 kDa) (Figure 1B–D), dystrophin and dysferlin (not shown). Interestingly, invagination of the sarcolemma could be detected in Gomori trichrome, b-spectrin, caveolin 3, laminin-a2 and major histocompatibility complex (MHC) class I stains (Figure 1A–D, F). Vacuoles contained both AChE (Figure 2A) and non-specific esterase (Figure 2C) but lacked acid phosphatase activity (Figure 2B). Vacuoles were also lined by complement (C5b9), but none of the fibres demonstrated C5b9 staining of the sarcolemma (Figure 1E). Furthermore, MHC class I was upregulated on the sarcolemma and on the vacuolar lining (Figure 1F), while neither stained for MHC class II (not shown). Large autophagosomes were strongly immunopositive for LC3 (Figure 2D). Interestingly, LAMP-2 was strongly positive on numerous lysosomes (Figure 2E), which were more densely packed and larger when compared to normal controls (Figure 2E, inset). Ultrastructural studies identified cellular debris and myelin-like features in the centre of vacuoles (Figure 2F) as well as duplicated basal laminae (Figure 2G) and accumulation of glycogen, which was not membrane-bound (Figure 2H). Fusion of vacuoles with the sarcolemma of muscle fibres was not a detectable feature of this biopsy specimen. Molecular sequencing was performed and ruled out GSD II (juvenile ‘Pompe’ disease), Danon disease and XMEA. Sequencing of the entire GAA gene on chromosome 17q25, LAMP-2 gene on chromosome Xq24 as well as the VMA21 gene on chromosome Xq28 failed to reveal deletions. Taken together, the present case may represent the manifestation of a new disease or a variant among the genetically unidentified congenital infantile and adult AVM displaying some similarities with XMEA, but also distinctive clinicopathological features. We describe a juvenile male subject with normal intelligence suffering

Architectural B-cell organization in skeletal muscle identifies subtypes of dermatomyositis

Objective To study the B-cell content, organization, and existence of distinct B-cell subpopulations in relation to the expression of type 1 interferon signature related genes in dermatomyositis (DM). Methods Evaluation of skeletal muscle biopsies from patients with adult DM (aDM) and juvenile DM (jDM) by histology, immunohistochemistry, electron microscopy, and quantitative reverse-transcription PCR. Results We defined 3 aDM subgroups—classic (containing occasional B cells without clusters), B-cell–rich, and follicle-like aDM—further elucidating IM B-lymphocyte maturation and immunity. The quantity of B cells and formation of ectopic lymphoid structures in a subset of patients with aDM were associated with a specific profile of cytokines and chemokines involved in lymphoid neogenesis. Levels of type 1 interferon signature related gene expression paralleled B-cell content and architectural organization and link B-cell immunity to the interferon type I signature. Conclusion These data corroborate the important role of B cells in DM, highlighting the direct link between humoral mechanisms as key players in B-cell immunity and the role of type I interferon–related immunity.

New variant of necklace fibres display peculiar lysosomal structures and mitophagy

Here, we describe a new variant of necklace fibres with specific myopathological features that have not been described thus far. They were observed in two patients, from two independent families with identical DNM2 (dynamin 2) mutation (c.1106 G > A (p.Arg369Gln)), displaying mildly heterogeneous clinical phenotypes. The variant is characterized by lysosomal inclusions, arranged in a necklace pattern, containing homogenous material, devoid of myonuclei. The so-called necklace region has a certain characteristic distance to the sarcolemma. Electron microscopy, including three dimensional reconstructions of serial section images highlights their ultrastructural properties and relation to neighbouring organelles. This new pattern is compared to the previously reported patterns in muscle biopsies containing necklace fibres associated with MTM1- and DNM2-mutations.