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Objective Studies are rarely reported on how to avoid complement system attack during the transplantation of bone marrow mesenchymal stem cells (BMSCs).We explored the effect of the overexpression of CD59 on complement membrane attack-induced damage to rat BMSCs (rBMSCs) during autologous transplantation.Methods BMSCs from SD rats were cultured and treated with CD59 overexpression plasmid or rBMSC empty vector or left untreated,followed by detection of the expression of CD59 in the rBMSCs by flow cytometry.Then the rBMSCs were incubated with autologous rat serum (ARS),inactivated ARS (iARS),CD59+ARS,or CD59+iARS or not incubated.The cytotoxicity of the serum complement on the rBMSCs was observed by PI staining and the apoptosis of the rBMSCs determined by flow cytometry.Results The expression of CD59 was significantly higher in the rBMSCs treated with CD59 than in those untreated (7,4.9％ vs 50.5％,P＜0.05).The apop tosis rate was remarkably lower in the rBMSCs not incubated ([8.4± 1.1] ％) and those incubated with CD59+ARS ([19.1 ±3.1] ％) than in those incubated with ARS ([40.3±4.3] ％) (P＜0.05).The deposition of the complement membrane attack complex was significantly decreased in the rBMSCs not incubated (50.1％) and those incubated with CD59+ARS (71.0％) as compared with those incubated with ARS (99.7％) (P＜0.05).The apoptosis rate of the rBMSCs treated with CD59 was markedly lower than that of those left untreated (P＜0.05).Conclusion The overexpression of CD59 inhibits the damage induced by complement to rBMSCs by reducing the formation of the complement membrane attack complex during autologous transplantation.
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Objective To explore the effect of complement activation on bone marrow mesenchymal stem cells (BMSCs)and evaluate the effect after transfection of complement regulatory proteins.Methods Bone marrow aspirate was harvested from 10 cases of patients suffered from fractures.Mesenchymal stem ceils were isolated,indentified cultured and then experimented in vitro.The complement cytotoxicity on the mesenchymal stem cells in autologous serum was measured by Europium cytotoxicity assay.The samples were divided into BMSCs group,BMSCs+ autologous human serum (AHS) group and BMSCs+ inactivated autologous human serum (iAHS) group.The complement membrane attack complex (MAC) deposited on the membranes was detected by flow cytometry.Finally,the cytotoxicity on BMSCs was measured after transfected with membrane complement regulatory proteins (mCRPs).All samples were divided into BMSCs with mCRPs untransfected group and BMSCs with mCRPs transfected group.Results More than 95％ of cells derived from bone marrow were identified to be mesenchymal stem cells through detection of cell surface markers by flow cytometry.The cytotoxicity of untreated cells was 0.41％± 1.48％.BMSCs harvested from the 10 patients all had cytotoxicity after incubated with autologous serum,and the cytotoxicity was 32.59％±2.73％,while cytotoxicity after incubated with complement inactivated autologous serum was 2.59％±3.08％,which was similar to control group.Complement attack complex (MAC) could be detected on the BMSCs incubated with autologous serum,which implied the complement activation.After transfection of mCRPs,the cytotoxicity of autologous serum on transfected cells was decreased.The cytotoxicity of untransfected cells (41.70％±4.47％) had significant difference compared to the cells transfected with CD55 (21.87％±2.19％),the cells transfected with CD59 (18.67％± 1.42％),and the cells transfected with CD46+CD55+CD59 (28.43％±2.14％).CD55,CD59 and CD46+CD55 +CD59 transfected groups could impair effectively the cytotoxicity from complement.However,the cytotoxicity impairment was less effective in CD46 transfected cells (39.30％±3.96％),which had no significant difference compared to untransfected cells.Conclusion Membrane complement regulatory proteins could effectively protect bone marrow mesenchymal stem cells from attacks by complement.
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ObjectiveTo investigate the clinical and pathological characteristics of dermatomyositis with muscular perifascicular atrophy (PFA).MethodsA series of 104 consecutive patients clinically and pathologically diagnosed as dermatomyositis by muscle biopsy in our laboratory from December,2003 to August,2011,were enrolled in this study. Muscle biopsy of all the enrolled patients had shown PFA of muscle fibers.ResultsAmong the 104 patients,34 were males and 70 were females with a mean age of 45 years old.Among them,8 cases had normal electromyogram;42 had normal serum creatine kinase level;11 were diagnosed as carcinoma;75 were found to be combined with interstitial lung disease (ILD).Based on morphologic changes of muscle biopsy,they were divided into pure PFA group with 54 cases and PFA plus focal damage group with 50 cases.Compared with the pure PFA group,there was prominent mononuclear cell infiltration into perimysial intermediate sized vessels and membrane attack complement (MAC) deposition in the intramuscular capillaries in the PFA plus group.Skin biopsy had been taken in 12 cases together with muscle biopsy and had shown the border effectof both PFA and interface dermatitis in muscle and skin.ConclusionsOur study suggests that chronic immune vascular damage and insufficiency in dermatomyositis may cause ischemia and focal myofiber damage in watershed regions. The incidence of ILD in our dermatomyositis patients with PFA is high.
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This study was performed in order to characterize the types of the infiltrating cells, and the expression profiles of major histocompatibility complex (MHC) class I and membrane attack complex (MAC) in patients with inflammatory myopathies and dysferlinopathy. Immunohistochemical stains were performed using monoclonal antibodies against several inflammatory cell types, MHC class I, and MAC in muscles from inflammatory myopathies and dysferlinopathy. There was significant difference in the types of infiltrating cells between polymyositis (PM), dermatomyositis (DM), and dysferlinopathy, including significantly high CD4+/CD8+ T cell ratio and B/T cell ratio in DM. In dysferlinopathy, CD4+ T cells were the most abundant and the proportions of infiltrating cell types were similar to those of DM. MHC class I was expressed in muscle fibers of PM and DM regardless of the presence of inflammatory infiltrates. MAC was expressed in necrotic fibers and vessels of PM and DM. One patient with early stage DM had a MAC deposits on endomysial capillaries. In dysferlinopathy, MAC deposit was also observed on the sarcolemma of nonnecrotic fibers. The analysis of inflammatory cells, MHC class I expressions and MAC deposits may help to differentiate dysferlinopathy from idiopathic inflammatory myopathy.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Dermatomyositis/immunology , Genes, MHC Class I , Membrane Proteins/genetics , Muscle Fibers, Skeletal/cytology , Muscle Proteins/genetics , Muscular Dystrophies, Limb-Girdle/immunology , Myositis/immunology , Polymyositis/immunology , T-Lymphocytes/cytologyABSTRACT
The complement system is composed of more than 25 different proteins and is usually divided into classical and alternative pathways. Complement component 7(C7) is one of the five terminal complement proteins that, upon activation of either the classical or the alternative pathway, interacts sequentially to form a large protein-protein complex, called membrane attack complex(MAC). Assembly of the MAC on target cells results in the formation of transmembrane pores that can lead to the killing of the cells. C7 deficiency is an autosomal recessive disorder that is mostly reported in Caucasians. The gene for C7 has been assigned to chromosome 5p13. To date, 15 different molecular defects leading to total or subtotal C7 deficient defects have been reported. C7 deficiency is associated frequently with recurrently bacterial infections, especially meningitis caused by Neisseria meningitidis. We report a case of a hereditary C7 deficiency associated with meningococcal meningitis.
Subject(s)
Bacterial Infections , Complement C7 , Complement System Proteins , Homicide , Membranes , Meningitis , Meningitis, Meningococcal , Neisseria meningitidisABSTRACT
AIM:To study the effect of human complement C 5b～9 complex on nitric oxide(NO) synthesis of glomerular mesangial cells (MC). METHODS: First, the human complement C 5b～9 complexes were isolated and glomerular MC of rats were cultured. Second, the MC were stimulated with C 5b～9 complex and changes of metabolism products of NO(NO 3 and NO 2) in MC culture supernatant at 6,24 and 48 hours after C 5b～9 stimulating were detected. Moreover, cGMP levels in cultured MC were also measured. RESULTS: NO 3/NO 2 contents from culture supernatant and cGMP levels in MC were increased parallelly after C 5b～9 complex stimulation. Further, NO synthesis was inhibited by L-NG-nitro-arginine-methylester(L-NAME). CONCLUSION: NO synthesis of rat glomerular MC was incerased by human complement C 5b～9 stimulation.
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AIM: To explore the localization and semi-quantification o f the glo merular complement C5b-9 complexes and synthesis of some inflammatory mediators or cytokines such as nitric oxide (NO) and tumor necrosis factor ?(TNF?) in the ra ts with anti-thymocyte serum nephritis(ATSN). METHODS: The animal mo del of rat AT SN was reproduced by a single intravenous injection of anti-thymocyte serum (ATS ). Then, the deposits of glomerular C5b-9 complexes were localized and quantifie d by immunohistochemical staining and microscopic image scanning separately. And the glomerular mesangial cells (MC) surrounded by C5b-9 complexes were counted under microscope. In addition, the expression of glomerular MC inducible NO synt hase(iNOS) mRNA and excretion of urinary NO metabolite ( NO - 2/NO - 3 ) and TNF ? in the rats with ATSN were detected. RESULTS: The MC in t h e rats with ATSN emerged necrosis followed by a rapid proliferation. In the early time of MC injury, the C5b-9 complexes were mainly seen in glomerular mesangium and MC sur fac e. But with the progression of ATSN, the MC enclosed by C5b-9 appeared gr adual decrease. Moreover, the expression of MC iNOS mRNA in early stage of ATSN obviously increased and the excretion of urinary NO - 2/NO - 3 and TNF ? also significantly increased. However, the changes of parameters mentioned abov e in ATSN proliferative stage (after 7 days) alleviated gradually. CONCLUSION: The second ary lysis of MC has relation to the deposition of C5b-9 complexes and synthesis and release of NO and TNF ? in rats with ATSN.