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Reilly Mary - One of the best experts on this subject based on the ideXlab platform.

  • Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin
    'FASEB', 2019
    Co-Authors: Ali Tayyibah, Bednarska Joanna, Vassilopoulos Stéphane, Tran Martin, Diakonov Ivan, Ziyadeh-isleem Azza, Guicheney Pascale, Gorelik Julia, Korchev Yuri, Reilly Mary
    Abstract:

    International audienceDynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a whole class of dynamin-dependent endocytosis, including clathrin-mediated endocytosis by caveoli. It has been suggested that mutations in the DNM2 gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactly mutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used live correlative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study how disease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly following their dynamics of formation, maturation, and internalization in skin fibroblasts from patients with centronuclear myopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we have developed, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, and significantly increases the lifetime of CCPs. Differently, p.R522H slows down the formation of CCPs without affecting their internalization. We also found that CNM mutations in DNM2 affect the distribution of caveoli and reduce dorsal ruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electron microscopy data, argue for the impairment of several forms of endocytosis in DNM2-linked CNM.-Ali, T., Bednarska, J., Vassilopoulos, S., Tran, M., Diakonov, I. A., Ziyadeh-Isleem, A., Guicheney, P., Gorelik, J., Korchev, Y. E., Reilly, M. M., Bitoun, M., Shevchuk, A. Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin

Mary Reilly - One of the best experts on this subject based on the ideXlab platform.

  • Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin
    FASEB Journal, 2019
    Co-Authors: Tayyibah Ali, Joanna Bednarska, Stéphane Vassilopoulos, Martin Tran, Ivan Diakonov, Azza Ziyadeh-isleem, Pascale Guicheney, Julia Gorelik, Yuri Korchev, Mary Reilly
    Abstract:

    Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a whole class of dynamin-dependent endocytosis, including clathrin-mediated endocytosis by caveoli. It has been suggested that mutations in the DNM2 gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactly mutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used live correlative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study how disease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly following their dynamics of formation, maturation, and internalization in skin fibroblasts from patients with centronuclear myopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we have developed, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, and significantly increases the lifetime of CCPs. Differently, p.R522H slows down the formation of CCPs without affecting their internalization. We also found that CNM mutations in DNM2 affect the distribution of caveoli and reduce dorsal ruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electron microscopy data, argue for the impairment of several forms of endocytosis in DNM2-linked CNM.-Ali, T., Bednarska, J., Vassilopoulos, S., Tran, M., Diakonov, I. A., Ziyadeh-Isleem, A., Guicheney, P., Gorelik, J., Korchev, Y. E., Reilly, M. M., Bitoun, M., Shevchuk, A. Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin.

G.b. Gaeta - One of the best experts on this subject based on the ideXlab platform.

  • AB0493 Anti-TNFα treatment is associated with increased liver stiffness in HBV occult carrier patients
    Annals of the Rheumatic Diseases, 2013
    Co-Authors: R. Tirri, G. Stornaiuolo, P. Sessa, M. Orefice, Giuseppina Brancaccio, Gabriele Valentini, G.b. Gaeta
    Abstract:

    Background HBV occult carriers (i.e HBsAg-/HBcAb+) are at risk for viral reactivation under immunosuppressive treatment for cancer and transplantation. Even if rarely, HBV reactivation has been also reported in occult HBV infection patients with Rheumatoid Arthritis (RA) or Spondyloarthritis (SA) undergoing anti TNFα treatment. No data are available on the influence of such treatment on liver architecture. Objectives This study was devoted to address this topic. Methods Liver fibrosis, as evaluated by Transient Elastografy (TE), was investigated in 73 patients with RA or SA undergoing anti TNFα treatment. Out of them, 6 were HBcAb+/HBsAb-; 33 HBcAb +/HBsAb+; in 2 HBcAb+, HBsAb was not available; 32 were HBcAb-. The patients were observed for a mean period of 43,9±26,3 months, during which ALT was measured every 4 weeks. HBV-DNA was evaluated if ALT increased more than 2 x upper normal value in 2 consecutive determinations. For each patient, 10 measurement of liver stiffness were performed and reported as average kPa value. A cut off value of 7,3 was assumed to denote significant fibrosis. BMI was calculated in all patients. Results Out of the seventy three patients (37F, aged 56,9±11,3) investigated, 18 were treated with Infliximab (IFX), 24 with Etanercept (ETA), 24 with Adalimumab (ADA) and 7 with Golimumab (GOL); 20 patients were also treated with Methotrexate (MTX), 10 with Leflunomide (LFN), 26 with Sulfasalazine (SSZ) and 12 with Prednisone (PDN) ( 0.05). No case of HBV reactivation was recorded. A stiffness value >7,3 KPa was detected in 10/41 (24,4%) anti HBc + patients vs 1/32 (3%) patients anti HBc - (p=0.02). Conclusions In our series the presence of anti-HBc antibodies identifies a subgroup of patients with liver damage, despite normal ALT values. This suggests that anti-TNFα therapy might promote liver fibrosis in occult HBV carriers or that some anti-HBc positive patients had a past-history of liver damage due to previous active HBV infection. These results await to be assessed in a longitudinal study. References Calabrese LH, Zein NN, Vassilopoulos. Hepatitis B virus (HBV) with immunosuppressive therapy in rheumatic diseases: assessment and preventive strategies. Ann Rheum Dis 2006;65;983-989. Caporali R et al. Safety of tumor necrosis factor α blockers in hepatitis B virus occult carriers (hepatitis B surface antigen negative/anti-hepatitis B core antigen positive) with rheumatic diseases. Arthritis Care Res. 2010, 749-754. Castera L, Forns X, Alberti A. Non-invasive evaluation of liver fibrosis using transient elastography. J Hepatol. 2008 May;48(5):835-47. Bonino F, Arena U, Brunetto MR, Coco B, Fraquelli M, Oliveri F, Pinzani M, Prati D, Rigamonti C, Vizzuti F; Liver Stiffness Study Group “Elastica” of the Italian Association for the Study of the Liver. Liver stiffness, a non-invasive marker of liver disease: a core study group report. Antivir Ther. 2010;15 Suppl 3:69-78. Disclosure of Interest None Declared

S.-k. Kwok - One of the best experts on this subject based on the ideXlab platform.

  • FRI0273 Reactivation of Hepatitis B Virus in Patients Treated with Anti-TNF Therapy
    Annals of the Rheumatic Diseases, 2014
    Co-Authors: Seung-hyun Jung, J.y. Kang, H.k. Min, Jung Hee Koh, Young Sun Suh, J.-h. Lee, Junguee Lee, J.y. Lee, J.-m. Kim, S.-k. Kwok
    Abstract:

    Background Anti-TNF therapy was known to increase the risk of certain infection. There are no sufficient data about the reactivation of hepatitis B virus (HBV) after anti-TNF therapy. Objectives The study was aimed to investigate the clinical course of hepatitis B in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS) after the introduction of anti-TNF therapy. Methods We retrospectively reviewed to identify patients infected with HBV treated with TNF inhibitors between October, 2004 and September, 2013. For patients with HBV infection, the liver enzyme and the viral status were monitored. The HBV reactivation was defined according to the Korean guideline by hepatology. Results Of 983 patients (RA, n=536; AS, n=447) who were treated with TNF inhibitors, 23 patients (RA, n=17; AS, n=6) had comorbidities of HBV infection. Eighteen patients were treated with etanercept, three with adalimumab, and two with infliximab. Seven patients received pre-emptive antiviral prophylaxis before anti-TNF therapy: 4 with entecavir, 1 with telbivudine, 1 with tenofovir, and 1 with lamivudine. Among 23 patients with HBV infection, 4 (17.4%) patients experienced the reactivation of HBV, which occurred in 3 patients without prophylaxis and 1 patient with lamivudine prophylaxis. In the latter case, YMDD mutation was identified and addition of adefovir resulted in virological response. The other three patients were treated successfully with entecavir or tenofovir. There was no subsequent events associated with HBV infection. Conclusions This study indicates the risk of HBV reactivation after anti-TNF therapy. Careful management is mandatory for patients who planned to be treated with TNF inhibitors. References Vassilopoulos D, Calabrese LH. Management of rheumatic disease with comorbid HBV or HCV infection. Nature reviews Rheumatology. 2012 Jun;8(6):348-57 Oketani M, Ido A, Uto H, Tsubouchi H. Prevention of hepatitis B virus reactivation in patients receiving immunosuppressive therapy or chemotherapy. Hepatology research: the official journal of the Japan Society of Hepatology. 2012 Jul;42(7):627-36 Korean Association for the Study of the L. KASL Clinical Practice Guidelines: Management of chronic hepatitis B. Clinical and molecular hepatology. 2012 Jun;18(2):109-62 Acknowledgements This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI10C2020). Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5251

Dimitrios Vassilopoulos - One of the best experts on this subject based on the ideXlab platform.

  • AB0108 Increased Expression of GM-CSF Secreting Peripheral B Cells in Patients with Rheumatoid Arthritis
    Annals of the Rheumatic Diseases, 2014
    Co-Authors: A. Makris, Sofia Adamidi, C. Koutsianas, Christina Tsalapaki, Emilia Hadziyannis, Dimitrios Vassilopoulos
    Abstract:

    Background B cells secreting GM-CSF have been recently identified to participate in innate immunity against bacteria. GM-CSF is known also to play a key role in rheumatoid arthritis (RA) pathogenesis and is currently a target of investigational therapies. The existence of a B cell population secreting GM-CSF and its potential role in RA pathogenesis has not been studied so far. Objectives To determine the expression of GM-CSF secreting peripheral B cells in patients with RA. Methods 20 treatment-naive patients with active RA (females/males=17/3, mean age =56±11.6 years, median disease duration =1.5 years, RF and/or anti-CCP+=50%), 10 disease controls (psoriatic arthritis n=4, ANCA-associated vasculitides n=2, osteoarthritis n=2, Sjogren9s syndrome n=1, giant cell aortitis n=1) and 9 healthy controls were included in the study. Peripheral blood mononuclear cells (PBMC) isolated from heparinized whole blood after density gradient centrifugation were stimulated overnight with Phorbol Myristate Acetate (PMA) and ionomycin in the presence of Brefeldin. Cells were then stained with antibodies directed against CD19 and intracellular cytokine GM-CSF. Positive cells were quantified by flow cytometry and compared between the different groups. Results The % of B cells (CD19+) did not differ between the three groups (7.14±4.01 vs. 6.63±3.52 vs. 8.54±1.99, p=NS). Nevertheless, an expanded population of CD19GM-CSF + cells was detected in RA patients (4.44±2.64%) compared to disease (0.89±1.44%, p=0.0004) and healthy (0.18±00.24%, p=0.00002) controls. There was no difference in GM-CSF expression between disease and healthy controls. There was no difference in CD19-GM-CSF expression between seropositive (RF and/or anti-CCP+, 4.41±2.68%) and seronegative (4.46±2.8%, p=0.86) RA patients. No correlation was observed between the % of GM-CSF+ B cells and CRP levels (p=0.86, Spearman correlation). Conclusions Our study shows for the first time an expanded population of peripheral GM-CSF secreting B cells in untreated RA patients with active disease, not present in patients with other inflammatory or non-inflammatory rheumatic diseases as well as in healthy controls. More studies are needed in order to determine the functional role of these cells in RA pathogenesis. References Cornish AL et al. G-CSF and GM-CSF as therapeutic targets in rheumatoid arthritis. Nat Rev Rheumatol 2009; 5:554-9 Rauch P et al. Innate Response Activator B Cells Protect Against Microbial Sepsis. Science 2012;335: 597-601 Acknowledgements This work was supported in part by research grants from the Hellenic Society for Rheumatology and the Special Account for Research Grants (S.A.R.G.), National and Kapodistrian University of Athens, Athens, Greece. Disclosure of Interest A. Makris: None declared, S. Adamidi: None declared, C. Koutsianas: None declared, C. Tsalapaki: None declared, E. Hadziyannis: None declared, D. Vassilopoulos Grant/research support: Roche, Abbott, Merck, UCB, Pfizer DOI 10.1136/annrheumdis-2014-eular.4566

  • SAT0127 Long-Term Safety and Efficacy Following the Administration of Multiple Rituximab Cycles in Rheumatoid Arthritis (RA) Patients: The Multicenter, Prospective Launch Study
    Annals of the Rheumatic Diseases, 2013
    Co-Authors: L. Settas, A Andrianakos, Spyros Aslanidis, Panagiota Boura, M Katsounaros, Dimitrios Vassilopoulos, P. Athanassiou, K. Tempos, G. Skarantavos, C. Antoniadis
    Abstract:

    Background About one third of RA patients do not initially respond to treatment with an anti-TNF agent whereas a similar rate demonstrates lack of efficacy over time. Rituximab/Mabthera administration (temporary B-lymphocyte depletion) is one of the therapeutic options for them. Objectives The LAUNCH prospective study aimed at the evaluation of long-term efficacy and safety data following rituximab administration in standard clinical practice Methods 17 Rheumatology sites in Greece enrolled 234 adult patients (63.0±12.4 years, 79.5% women) with severe RA and an inadequate response or non-tolerance to anti-TNF treatment. Rituximab (1gr) was administered IV on days 1 and 14 of each cycle, repeated every 6-12 months, for up to 7 cycles. Of these patients 41.2% and 56.2% had received one, or more, anti-TNF agent(s), respectively. Adverse events, DAS28, and the quality of life evaluation indices (Euroqol) were collected every 2 to 6 months for 5 years according to each site’s standard clinical practice Results During 496 patient/years, 28 adverse events /100 pt-yrs (including9.9 serious adverse events and 7.7 serious infectious per 100pt-yrs, respectively) were observed. Of the total number of adverse events a 46.7% was not related to rituximab. The mean number of adverse events per patient remained stable during repeated treatment cycles. Disease activity at baseline (mean±SD DAS28 of 5.36±1.40) was significantly reduced in cycles 1,2, 3, 4, 5, and 6 by 1.34, 2.12, 2.25, 2,56, 2.42 and 2.79, respectively (p Conclusions Rituximab administration in clinical practice for up to 5 years demonstrated an acceptable safety profile which was maintained over time. Likewise, maintenance and/or improvement of efficacy with repeated treatment cycles in patients with severe RA not responding to anti-TNF were evident Disclosure of Interest L. Settas: None Declared, A. Andrianakos: None Declared, S. Aslanidis: None Declared, P. Boura: None Declared, M. Katsounaros: None Declared, D. Vassilopoulos: None Declared, P. Athanassiou: None Declared, K. Tempos: None Declared, G. Skarantavos: None Declared, C. Antoniadis : None Declared, L. Sakkas: None Declared, A. Andonopoulos: None Declared, V. Galanopoulou: None Declared, F. Solioti: None Declared, K. Boki: None Declared, E. Vritzali Employee of: Roche Hellas SA, P. Sfikakis: None Declared