Daniela Franci

A truncation in the RYR1 gene associated with central core lesions in skeletal muscle fibres

A novel single-nucleotide deletion in exon 100 of the RYR1 gene, corresponding to deletion of nucleotide 14 510 in the human RyR1 mRNA (c14510delA), was identified in a man with malignant hyperthermia and in his two daughters who were normal for malignant hyperthermia. This deletion results in a RyR1 protein lacking the last 202 amino acid residues. All three subjects heterozygotic for the mutated allele presented with a prevalence of type 1 fibres with central cores, although none experienced clinical signs of myopathy. Expression of the truncated protein resulted in non-functional RYR1 calcium release channels. Expression of wild-type and RyR1R4836fsX4838 proteins resulted in heterozygotic release channels with overall functional properties similar to those of wild-type RyR1 channels. Nevertheless, small differences in sensitivity to calcium and caffeine were observed in heterotetrameric channels, which also presented an altered assembly/stability in sucrose-gradient centrifugation analysis. Altogether, these data suggest that altered RYR1 tetramer assembly/stability coupled with subtle chronic changes in Ca2+ homoeostasis over the long term may contribute to the development of core lesions and incomplete malignant hyperthermia susceptibility penetrance in individuals carrying this novel RYR1 mutation.

Is osteopontin involved in cutaneous fibroblast activation? Its hypothetical role in scleroderma pathogenesis

Osteopontin (OPN) is an extracellular matrix protein implicated in bone remodeling, but it presents also pro-inflammatory and pro-fibrotic properties. OPN expression also occurs upon exposure of cells to classical mediators of acute inflammation such as tumor necrosis growth factor alpha (TNF-α) and interleukin-1 beta (IL-1β), as well as fibrogenic cytokines such as transforming growth factor beta (TGF-β), although a detailed understanding of these regulatory pathways is still unknown. Plasma OPN levels in both limited and diffuse systemic sclerosis patients (ISSc and dSSc) were statistically higher compared to those of control subjects. Immunohistology demonstrated that high TGF-β levels, alpha smooth muscle actin (αSMA) levels and consequently high OPN levels were found in the affected skin of sclerodermic patients (ISSc and dSSc) compared to levels found in healthy skin. In order to better understand how OPN interferes with the fibrotic process, healthy skin fibroblasts were treated for 24 and 48 hours with bleomycin and with endothelin-1 (ET-1) plus TGF-β in order to induce the fibrogenesis. After 48 hours of stimulation, healthy treated fibroblasts showed statistically increased αSMA levels (index of differentiation into myofibroblasts) and simultaneously statistically increased OPN levels compared to healthy untreated ones. This study demonstrates that OPN levels increase simultaneously with the increasing of αSMA levels, therefore it is reasonable to hypothesize that OPN interferes in the pathogenesis of Systemic Sclerosis in the early stage of fibroblast differentiation process.

Expression of RXFP1 in skin of scleroderma patients and control subjects.

Objectives: Relaxin (RLX) is involved in extracellular matrix and collagen remodelling. The therapeutic role of the circulating isoform RLX-2 as an anti-fibrotic factor in systemic sclerosis (SSc) has been investigated. Several RLX family peptide receptors (RXFPs) are recognized in humans: RLX-2 is a ligand for RXFP1/LGR7 and RXFP2/LGR8. The aim of this study was to define the pattern of expression of LGR7 in different types of human skin cells and to compare normal skin with lesional and unaffected skin from patients with limited SSc (lSSc). Method: We analysed RXFP1 immunolocalization on skin biopsies and cultured fibroblasts from lSSc patients and control subjects. Western blot analysis was carried out on fibroblast lysates. Results: RXFP1 showed cytoplasmic localization on skin cells from control subjects and non-lesional skin from lSSc patients: keratinocytes, gland epithelial cells, endothelium, smooth muscle cells, and fibroblasts. Immunogold electron microscopy confirmed a diffuse epithelial cytoplasmic localization of RXFP1. A substantially lower RXFP1 expression was observed in scleroderma skin, with a lack of staining in most cells. Occasional weak reactivity was observed in cultured scleroderma fibroblasts, while control fibroblasts showed a diffuse cytoplasmic immunoreactivity of RXFP1, confirmed by Western blot analysis. Conclusions: The decreased cellular expression of RLX-2 receptor RXFP1 in scleroderma skin might represent a pro-fibrotic factor and contribute to the substantial inefficacy of RLX treatment in SSc, as reported in the literature. The pathophysiology of the decrease in RXFP1 may be linked to high RLX-2 serum levels previously detected in SSc, but it has yet to be elucidated.

Histopathological findings in systemic sclerosis-related myopathy: fibrosis and microangiopathy with lack of cellular inflammation

Objectives: The objective of this study was to identify specific histopathological features of skeletal muscle involvement in systemic sclerosis (SSc) patients. Methods: A total of 35 out of 112 SSc-patients (32%, including 81% female and 68% diffuse scleroderma) presenting clinical, biological and electromyographic (EMG) features of muscle weakness, were included. Patients underwent vastus lateralis biopsy, assessed for individual pathologic features including fibrosis [type I collagen (Coll-I), transforming growth factor β (TGF-β)], microangiopathy [cluster of differentiation 31 (CD31), pro-angiogenic vascular endothelial growth factor A (VEGF-A), anti-angiogenic VEGF-A165b], immune/ inflammatory response [CD4, CD8, CD20, human leucocyte antigens ABC (HLA-ABC)], and membranolytic attack complex (MAC). SSc biopsies were compared with biopsies of (n = 35) idiopathic inflammatory myopathies (IIMs) and to (n = 35) noninflammatory myopathies (NIMs). Ultrastructural abnormalities of SSc myopathy were also analyzed by transmission electron microscopy (TEM). Results: Fibrosis in SSc myopathy (81%) is higher compared with IIM (32%, p < 0.05) and with NIM (18%, p < 0.05). Vascular involvement is dominant in SSc muscle (92%), and in IIM (78%) compared with NIM (21%, p < 0.05). In particular, CD31 shows loss of endomysial vessels in SSc myopathy compared with IIM (p < 0.05) and with NIM (p < 0.01). VEGF-A is downregulated in SSc myopathy compared with IIM (p < 0.05) and NIM (p < 0.05). Conversely, VEGF-A165b is upregulated in SSc myopathy. The SSc immune/inflammatory response suggested humoral process with majority (85%) HLA-ABC fibral neoexpression and complement deposits on endomysial capillaries MAC, compared with IIM (p < 0.05), characterized by CD4+/CD8+/B-cell infiltrate, and NIM (p < 0.05). TEM analysis showed SSc vascular alterations consisting of thickening and lamination of basement membrane and endothelial cell ‘swelling’ coupled to endomysial/perimysial fibrosis. Conclusions: Fibrosis, microangiopathy and humoral immunity are predominant in SSc myopathy, even if it is difficult to identify specific histopathological hallmarks of muscle involvement in SSc, since they could be present also in other (IIM/NIM) myopathies.

Characterization of lymphatic vessels in human peripheral neuropathies

Immunohistological studies on lymphatics’ topography in human peripheral nerve are few and recent. By D2-40 immunohistochemistry, we previously described lymphatics in epineurium of human sural nerve. Lymphangiogenesis is described in inflammation. In angiopathic and vasculitic neuropathies proliferation of epineurial blood capillaries is reported. The aim of our study is therefore to investigate the topography and the density of lymphatics in human peripheral nerve and to search possible correlation with blood capillary neovascularization in different neuropathies. We examined biopsied sural nerves of patients suffering from CIDP (chronic inflammatory demyelinating polyneuropathy), vasculitic neuropathy and non inflammatory axonal neuropathy. Immunohistochemistry for detection of lymphatic marker podoplanin (D2-40 antibody) and for general endothelial marker CD31, as well as for Schwann cells protein S-100, was carried out on serial cryostat sections. Morphometric analysis was performed. Lymphatic capillaries were detected in epineurium, most consistently in adjacency of main blood vessels. Occasionally lymphatics were dilated and repleted with mononuclear cells. Podoplanin was also expressed by perineurium and by Schwann cells. No lymphatics were observed endoneurially. Lymphatics showed a far lesser density than blood capillaries and increase of epineurial vascularization resulted significantly associated with higher density of lymphatics. Density variations of epineurial lymphatics, accompanying blood capillaries proliferation, suggest that lymphangiogenesis may occur in neuropathies, in response to inflammation/ischaemia. Lymphatics’ responsiveness to molecular microenvironment is indicated by their expression of growth factor receptors, such as VEGFR3. Lack of lymphatics in closed endoneurial environment is in agreement with analogous findings in brain. Non lymphatic expression of podoplanin is reported in several cell types of nervous system: normal and tumoral ependymal cells, perineurium and Schwann cells. As biological functions of podoplanin, involved in cell migration and cytoskeletal reorganization, are uncompletely understood, its localization on non lymphatic structures of peripheral nerve needs to be defined.

Muscle pathology patterns in possibly adjuvant related autoimmune/inflammatory syndrome (ASIA)

Growing evidence shows a link for biologically inert molecules, such as vaccine adjuvants and silicone implants, with the occurrence of autoimmunity-related disorders, defined as autoimmune/inflammatory syndrome induced by adjuvant-ASIA (1). Clinical conditions encompass siliconosis, the Gulf war syndrome, the macrophagic myofasciitis syndrome (MMF), post-vaccination phenomena and the spectrum of related syndromes is expanding (2). Involvement of skeletal muscle in ASIA is acknowledged in MMF, defined by long-term persistence of vaccine alum adjuvants within macrophages at sites of previous immunization. A few reports describe vaccine and silicone implants related autoimmune inflammatory myopathies (3). We carried out an immunopathological analysis of skeletal muscle biopsy in a case of MMF and two cases of possible ASIA myositis, chronologically subsequent to breast silicone implant. MMF showed the typical fascial/ perimysial macrophagic invasion, with no endomysial mononuclear infiltrates and fibral neolocalization of MHC-I complex restricted to the adjacency of macrophage deposits. The first myositis case presented with a subacute onset twenty years after an uneventful additive breast silicone implant. Endomysial inflammation, microangiopathy and multifocal fibral localization of MHC-II complex were observed. In the second patient, the onset of proximal weakness, myalgiae and a tenfold increase of creatinkinase levels occurred seven years after an unsuccessful additive mastoplasty, with rupture of prostheses and re-implantation three years later. Muscle biopsy, besides inflammation changes, showed peculiar myofibrillar disruption, with MHC-I reactive sarcoplasmic inclusions expressing several structural muscle proteins. Molecular pathogenesis of ASIA is yet undefined: genetical susceptibility is currently investigated (1,2). Due to the role of vaccines in medicine and the wide use of silicon medical devices, identification of their cause/effect link with autoimmunity is of great interest.

Immunohistochemical detection of myosin heavy chain isoforms in human cremaster muscle

Cremaster muscle (CM) forms a thin network of fascicles, around the spermatic cord and testis, connected by loose areolar tissue forming the cremasteric fascia. CM has a non somitic embryologic origin, as it derives from mesenchymal differentiation of the gubernacular tip (1).Thus it is not to be considered a passive extension of internal oblique muscle. CM is composed both of striated and smooth muscle cells; it is innervated by genitofemoral nerve (2). Its striated fibres, in contrast with skeletal muscles, present with a multifocal innervation by multiple neuromuscular synapses (3). Myosin isoforms are the major determinant of the contractile and biochemical heterogeneity of skeletal muscle fibers. Non somitic muscles, such as extrinsic ocular muscles, show a distinct pattern of myosin heavy chains distribution. The aim of our study was to characterize the expression of myosin isoforms in CM fascicles; biopsy samples were obtained from cases of cryptorchidism, retractile testis and inguinal hernia, undergoing surgery. Immunohistochemistry confirmed the previously identified type 1 predominance (1) and showed a high occurrence of hybrid fibres, coexpressing two or more myosin isoforms. In contrast with age-matched limb muscles, persistence of developmental/neonatal myosin heavy chains was detected, beyond the determined timecourse of physiological shifting from immature isoforms (4). On the basis of shared peculiar embryological derivation, expression of superfast extraocular myosin MyH13 was also investigated on CM specimens, showing sarcoplasmic reactivity, undetectable in limb muscles. The high share of hybrid fibres, the persistence of immature myosin and MyH13/MyHCslow coexpression are peculiar features, suggesting a functional/biochemical individuality of CM, related with multiple innervation and distinct embryological development.