Debora Pehl

Nuclear actin aggregation is a hallmark of anti-synthetase syndrome–induced dysimmune myopathy

Objective: To analyze antisynthetase syndrome–associated myositis by modern myopathologic methods and to define its place in the spectrum of idiopathic inflammatory myopathies (IIMs). Methods: Skeletal muscle biopsies from antisynthetase syndrome–associated myositis and other IIMs from different institutions worldwide were analyzed by histopathology, quantitative PCR, and electron microscopy. Results: Myonuclear actin filament inclusions were identified as a unique morphologic hallmark of antisynthetase syndrome–associated myositis. Nuclear actin inclusions were never found in dermatomyositis, polymyositis, sporadic inclusion body myositis, autoimmune necrotizing myopathy associated with signal recognition particle or 3-hydroxy-3-methylglutaryl-coenzyme A reductase autoantibodies, or nonspecific myositis associated with other systemic diseases, harboring myositis-associated autoantibodies, and presenting myofiber necrosis. We show that molecules involved in actin filament formation and actin shuttling mechanisms are altered in antisynthetase syndrome, and may thus be involved in pathologic myonuclear actin aggregation. In addition, we have identified a typical topographic distribution of necrotic myofibers predominantly located at the periphery of muscle fascicles accompanied by inflammation and destruction of the perimysial connective tissue. Conclusion: Antisynthetase syndrome–associated myositis is characterized by distinctive myonuclear actin filament inclusions, including rod formations and a typical necrotizing perimysial myositis. This supports the hypothesis that antisynthetase syndrome–associated myositis is unique and should not be grouped among dermatomyositis, polymyositis, sporadic inclusion body myositis, necrotizing autoimmune myositis, or nonspecific myositis. Classification of evidence: This study provides Class II evidence that for patients with IIMs, the presence of myonuclear actin filament inclusions accurately identifies patients with antisynthetase syndrome–associated myositis (sensitivity 81%, specificity 100%).

Mast cells protect from post-traumatic brain inflammation by the mast cell-specific chymase mouse mast cell protease-4

Mast cells (MCs) are found abundantly in the brain and the meninges and play a complex role in neuroinflammatory diseases, such as stroke and multiple sclerosis. Here, we show that MC‐deficient KitW/KitWv mice display increased neurodegeneration in the lesion area after brain trauma. Furthermore, MC‐deficient mice display significantly more brain inflammation, namely an increased presence of macrophages/microglia, as well as dramatically increased T‐cell infiltration at days 4 and 14 after injury, combined with increased astrogliosis at day 14 following injury. The number of proliferating Ki67+ macrophages/microglia and astrocytes around the lesion area is more than doubled in these MC‐deficient mice. In parallel, MC‐deficient KitW‐sh/W‐sh mice display increased presence of macrophages/microglia at day 4, and persistent astrogliosis at day 4 and 14 after brain trauma. Further analysis of mice deficient in one of the most relevant MC proteases, i.e., mouse mast cell protease 4 (mMCP‐4), revealed that astrogliosis and T‐cell infiltration are significantly increased in mMCP‐4‐knockout mice. Finally, treatment with an inhibitor of mMCP‐4 significantly increased macrophage/microglia numbers and astrogliosis. These data suggest that MCs exert protective functions after trauma, at least in part via mMCP‐4, by suppressing exacerbated inflammation via their proteases.—Hendrix, S., Kramer, P., Pehl, D., Warnke, K., Boato, F., Nelissen, S., Lemmens, E., Pejler, G., Metz, M., Siebenhaar, F., Maurer, M. Mast cells protect from post‐traumatic brain inflammation by the mast cell‐specific chymase mouse mast cell protease‐4. FASEB J. 27, 920–929 (2013). www.fasebj.org

Mast cells protect from post-traumatic spinal cord damage in mice by degrading inflammation-associated cytokines via mouse mast cell protease 4.

Mast cells (MCs) are found abundantly in the central nervous system and play a complex role in neuroinflammatory diseases such as multiple sclerosis and stroke. In the present study, we show that MC-deficient Kit(W-sh/W-sh) mice display significantly increased astrogliosis and T cell infiltration as well as significantly reduced functional recovery after spinal cord injury compared to wildtype mice. In addition, MC-deficient mice show significantly increased levels of MCP-1, TNF-α, IL-10 and IL-13 protein levels in the spinal cord. Mice deficient in mouse mast cell protease 4 (mMCP4), an MC-specific chymase, also showed increased MCP-1, IL-6 and IL-13 protein levels in spinal cord samples and a decreased functional outcome after spinal cord injury. A degradation assay using supernatant from MCs derived from either mMCP4(-/-) mice or controls revealed that mMCP4 cleaves MCP-1, IL-6, and IL-13 suggesting a protective role for MC proteases in neuroinflammation. These data show for the first time that MCs may be protective after spinal cord injury and that they may reduce CNS damage by degrading inflammation-associated cytokines via the MC-specific chymase mMCP4.

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.

Th2-M2 immunity in lesions of muscular sarcoidosis and macrophagic myofasciitis.

To analyse the paradox of a lack of giant cell formation and fibrosis in chronic lesions of macrophagic myofasciitis (MMF) in comparison with muscular sarcoidosis (MuS).

The lymphoid follicle variant of dermatomyositis

Objective: To investigate the clinical and morphologic spectrum of early adult–onset dermatomyositis (DM), an inflammatory disease that affects small vessels of the muscle and the skin. Methods: Histologic evaluation of frozen muscle samples was employed to visualize the cellular organization of ectopic lymphoid structures in muscle biopsies obtained from 2 patients diagnosed with DM. Clinical presentation and morphologic features, as well as treatment and follow-up, were assessed and documented. Electron microscopy was used to confirm the light microscopic diagnosis of DM. Clonality analysis of B-cell populations using PCR was performed. Results: Muscle biopsy of both patients fulfilled the morphologic European Neuromuscular Centre criteria of DM. Analyses of muscle biopsy samples revealed ectopic lymphoid follicle-like structures that showed a remarkable similarity to secondary lymphoid organs (SLOs) with distinct T- and B-cell compartmentalization. Our 2 patients exhibited an atypical and mild clinical presentation and responded favorably to therapy. Conclusions: The clinical and histopathologic features of DM can be atypical, and the presence of SLOs is not inevitably linked to an unfavorable prognosis.

LGI-1-positive limbic encephalitis: a clinicopathological study.

Encephalitis with antibodies against the leucine-rich, glioma-inactivated subunit of the VGKC-complex, recently described as anti-LGI-1 limbic encephalitis, is considered a sub-form of limbic encephalitis usually occurring without any detectable paraneoplastic cause [1, 8]. In this rare condition, pathogenetically relevant autoantibodies against the leucine-rich, glioma-inactivated 1 (LGI-1), a secreted neuronal protein, have been discovered [3]. The paucity of published cases does not allow for a prognostic prediction or knowledge of ideal treatment conditions, but immunosuppressive treatment has been successful in some patients [2]. An 80-year-old woman was transferred after a first seizure. Her husband reported that while sitting in an armchair in front of the TV screen, his wife exhibited sudden pronounced breathing, followed by sitting up stiffly and falling to the ground with consecutive loss of consciousness. Myoclonic jerks were absent, but she showed severe sweating and unconsciousness lasted about 10 min. A recent history of memory loss over several months was reported. Her past medical history included diabetes, arterial hypertension, cardiac and renal insufficiency, and chronic bronchopulmonary disease. Laboratory studies showed serum hyponatremia, and cerebrospinal fluid (CSF) analysis revealed entirely normal results, except for detection of three oligoclonal strands isolated in the CSF (glucose, lactate, and protein levels as well as Reiber indices proved to be normal). Serological analysis of CSF and serum showed negative results for Herpes simplex virus 1 & 2, Borrelia burgdorferi, and Treponema pallidum. Further PCR investigation of the CSF was not done because of normal cell count. Cerebral MRI demonstrated swelling of the left hippocampus with T2 hyperintensity of the left hippocampus (Fig. 1), but without contrast enhancement. Interictal EEG disclosed a frontomesial disturbance without epileptic discharges. At that time, diagnosis of de novo temporal lobe epilepsy was made and symptomatic anticonvulsive therapy with valproic acid was started. Five weeks later, the patient was transferred again in severely reduced general condition after two further epileptic seizures. She showed clinical and EEG signs of nonconvulsive status epilepticus (NCSE), faciobrachial dystonic seizures were absent. Serum hyponatremia (125 mmol/l) was detected, but further MRI studies revealed no significant progression of the lesions (Fig. 1). Repeated CSF analysis displayed unremarkable routine parameters, but revealed high serum anti-LGI-1-antibody titers (1:100) and moderate CSF titers (1:10). Serum and CSF were negative for anti-Yo, -Ri, -Hu, -Ma, -NMDA, -AMPA, -CV2, and -PCA2 autoantibodies. Death occurred 5 days after the second admission. Postmortem histopathology of the brain demonstrated CD45 and CD8 T cells in intimate proximity with hippocampal neurons, mainly in the CA4 segment, however with only mild neuronal loss. In addition, CD45 and CD8 T cells were identified in the meninges of the temporal lobe as well as in intraparenchymal vessels, predominantly on the left side. CD20, CD79a B cells were absent. Activated microglial cells and activated GFAP J. Schultze-Amberger (&) Department of Neurology, Klinikum Ernst von Bergmann, Charlottenstr. 72, 14469 Potsdam, Germany e-mail: jschultze-amberger@klinikumevb.de

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.

G.P.77

Juvenile dermatomyositis (jDM) is an inflammatory disease that affects small vessels of the muscle, the skin, and other organs, which may lead to severe systemic disease. Muscle biopsy can help to secure a firm diagnosis and thus serve as an important adjunct to atypical clinical presentation. As previously suggested, ectopic lymphoid follicle-like structures (LFLS) in inflamed muscle could indicate a severe course of disease. Histological evaluation of frozen muscle samples was employed to visualize the cellular organisation of ectopic lymphoid structures in muscle biopsies obtained from 2 patients diagnosed with juvenile dermatomyositis (jDM). Clinical presentation, morphological features, as well as treatment and follow-up were assessed and documented. Electron microscopy was used to confirm the light microscopic diagnosis of jDM. Clonality analysis of B-cell populations using polymerase chain reaction (PCR) was performed. Muscle biopsy of both patients fulfilled the morphological ENMC criteria of DM. Analyses of muscle biopsy samples revealed LFLS that showed a remarkable similarity to secondary lymphoid organs (SLOs) with distinct T- and B-cell compartmentalization. In contrast to previous reports our two patients exhibited a mild clinical presentation and responded favourably to therapy. Together, our data suggest that the clinical spectrum of jDM appears to be more diverse than so far documented, and that the presence of SLOs is not inevitably linked to unfavourable prognosis.

P.21.3 Skeletal muscle provides a permissive environment for Th2-M2 polarisation in neuromuscular sarcoidosis

Neuromuscular involvement may affect more then 60% of patients suffering from sarcoidosis. We have recently described that macrophages and giant cells in skeletal muscle exhibit an unexpected status of alternative activation (M2). Objective: The intrinsic immune signature of the granulomas, was compared to the cytokine profile of adjacent non-inflamed muscle tissue and to healthy control muscle. Granulomas and contiguous muscle from 9 patients with biopsy-proven neuromuscular sarcoidosis were cut out by laser capture microdissection (LCM). Markers and activators of the T helper cell 1 (Th1) – classical macrophage activation (M1) and Th2 – alternatively activated (M2) immune response as well as molecules involved in giant cell development were assessed by real-time PCR. STAT-6-induced Th2 immunity leads to upregulated expression of CD206 and SOCS1 in the granuloma in comparison to adjacent tissue. DAP12 and RAC1, genes that regulate giant cell formation, are significantly induced in the granulomas. Conversely, STAT-1-induced Th1 immunity, IFN γ and CXCR3 are expressed in the granulomas and the surrounding tissue at elevated levels, however without statistical differences. While Th1-mediated immunity is upregulated in the whole inflamed muscle specimen, Th2-M2 markers are expressed at significantly higher levels in the granulomata. These results indicate that muscle tissue per se may provide a permissive environment for M2 polarisation in neuromuscular sarcoidosis.