Investigation Of Rheumatoid Arthritis Pathogenesis And Research Of RA-ILD

Background Rheumatoid arthritis(RA)is a systemic autoimmune disorder that is characterized by chronic joint inflammation.Extreme angiogenesis and abnormal cell proliferation are two main events of RA synovium.Peptidylarginine deiminase (PAD)converts arginine residue to citrulline residue and five isoforms of the enzymes (PADI1,2,3,4 and 6)have been identified in diverse human tissues.Genome-wide single nucleotide polymorphism(SNP)analysis has shown that PADI4(also termed PAD4 and pad5 in some references)gene is closely associated with RA among the Japanese population and Koreans.Immunohistochemistry,Western blotting and Northern hybridization have revealed abundant expression of PADI4 in RA synovial tissue.Recent studies indicated that citrullinated proteins that are produced by enzymatic deimination of PAD are particularly important for RA pathogenesis. Antithrombin,fibronectin,fibrin,collagen and vimentin in RA synovium change their structural properties after the post-translational modification and can consequently promote autoimmunity as well as tissue destruction of the diseased joint.Keratin comprises a group of water-insoluble proteins that form intermediate filaments in most epithelial tissues.So far,over 20 human keratins have been identified and they are divided into 2 subfamilies:acidic(typeⅠ)(Mol.Wt:40,45,46, 48,50,51,55 and 56.6 kDa)and basic(typeⅡ)(Mol.Wt:52,56,58,59,64 and 65-67 kDa)subfamilies.Keratin plays important roles in cell differentiation and apoptosis as well as in disease states.Post-translational modifications of keratin such as glycosylation,phosphorylation and transglutamination can affect the physiological processes of cells and mediate cell development.Serum from patients with RA contains a spectrum of autoantibodies including rheumatoid factor(RF),anti filaggrin autoantibody(AFA),anti keratin antibody (AKA),anti perinuclear factor(APF)and anti cyclic citrullinated peptide antibody (anti CCP)that together constitute one primary outcome of disturbed immunoregulation.In 1979,Young et al.found using indirect immunofluorescence that the serum of RA patients could label the stratum corneum of the rat esophageal epithelium and named the responsible antibody antikeratin,because keratin constitutes the major protein component of the tissue.AKA is highly specific to RA. The antibody is detectable in sera of 30-80%of RA patients and closely correlates with the severity of joint tissue destruction and clinical RA activity.However,a direct biochemical and immunological correlation between cytokeratin and AKA has been obscure.Quismorio et al.reported that pre-absorbing the RA serum with human epidermal keratin did not abolish AKA activity.Vincent et al.also demonstrated that AKA differs from naturally occurring autoantibody to human epidermal keratin. Although the above studies suggested that keratin is not the actual target of AKA, their data did not reject presence of cytokeratin or its novel form(s)in RA synovium. Therefore,we decided to investigate the possible expression of keratin as well as its citrullination in RA synovial tissue.In this paper,we also discussed the potential contribution of the cytokeatin for pathogenesis of the disease.Objective Keratin is the main component of cellular intermediate filament.The post-translational modification of keratin plays important role for cell differentiation and apoptosis as well as disease states.The conversion of peptidylarginine to citrulline,termed citrullination,is profoundly involved in the pathogenic process of rheumatoid arthritis(RA).Our object is to explore the expression and citrullination of keratin in synovial tissue of rheumatoid arthritisMethods We collected 12 synovial membrane samples from RA patients,28 from osteoarthritis(OA)during arthroplasty.We also obtained 9 normal synovial tissues from patients after accident during operation.These samples were kept. Immunohistochemistry,double immunofluorescent labeling and Immunoprecipitation and Western blot analysis were used in our tests.Results The results discovered that various keratin forms have been detected cytokeratin in many cells at the lining area of RA synovial membrane.The cells expressing the cytokeratin also emitted citrulline signals when they were in the vicinity of extracellular deposits and approached the exterior of the synovial membrane.Western blot confirmed citrullination of the keratin that was purified from RA synovial tissues.The above results show the presence of citrulllinated cytokeratin in RA syovial membrane.Conclusion The expression and citrullination of cytokeratin in synovial tissue of rheumatoid arthritis were found and the cytokeatin may play an important role in the pathogenesis of the RA. Background Rheumatoid arthritis(RA)is a common,functionally disabling disease with genetic and environmental contributors.It occurs in approximately 0.32～0.36%of the Chinese and adversely affects quality of life,functional status,and survival.Beyond its impact on the joints,pulmonary involvement occurs regularly and is responsible for a significant portion of the morbidity and mortality.Although pulmonary infection and/or drug toxicity are frequent complications,lung disease directly associated with the underlying RA is more common.The airways,vasculature, parenchyma,and pleura can all be involved,with variable amounts of pathologic inflammation and fibrosis.The true adverse clinical impact of the most important of these directly associated disorders,RA-associated interstitial lung disease(RA-ILD), has only recently begun to reveal itself.Our knowledge of the underlying pathobiology and the impact of our current immunomodulatory and biologic therapies on the lung disease are less than incomplete.However,what is clear is the importance of progressive lung fibrosis in shortening survival and impairing quality of life in RA as well as in other connective tissue diseases.In fact,lung disease is the second most common cause of death,following infection,for patients with RA and has been reported to effect between 1-40%of patients.RA associated interstitial lung disease is often subtle in onset,slowly progressive and of unclear etiology and response to treatment.The impact of historically available and newer biologic therapies in altering the outcome of RA-ILD is unknown;translational studies focused on the pathobiology and clinical studies focused on RA-ILD are needed.Objective To observe the difference of manifestation,lung HRCT,pulmonary function tests,arterial blood gas and change of cytokines TGF-β1,TNF-α,IGF-1, PDGF-AB between RA and RA-ILD.Methods 65 cases came from our rheumatology department.Among them 30 cases were diagnosised as RA-ILD,35 cases were diagnosised as RA.Another 30 volunteers were invited to joint our investigation.We observed the manifestations, relatived lab examination,lung HRCT,lung functions,blood gas and determined the cytokines of TGF-β1;TNF-α;IGF-1;PDGF-AB by ELASA.Results The manifestation of RA-ILD were serious than that of RA.These are still difference in ESR,CRP,RF,serum globulin,pulmonary arterial pressure between RA and RA-ILD.(P<0.01)The main resparitary manifestations of RA-ILD are cough,expectoration,chest distress,short breath,chest pain,change of breath sounds,Velcro rales,dyspnea.The main manifestations of lung HRCT are thickening of interlobular septum,thickening of pachynsis pleurae,thickening of bronch tubal wall,mosaic's sign,bronchiectasis,emphysema,patching shadow,honeycombing, fiber scar。Pulmonary function test show that there are difference between RA and RA-ILD in VC,FVC,MMF and DLCO(P<0.01)。The arterial gas test show that PO2 of RA-ILD was lower than that of RA(P<0.01).The serum cytokines show than both cytokines of RA and RA-ILD are higher than that of volunteer(P<0.01),and the cytokines of RA-ILD were higher than that of RA (P<0.01).Conclution The manifestations of RA-ILD are serious than that of RA. Cytokines of TGF-β1;TNF-α;IGF-1 may play an important role on the pathogenesis of RA-ILD. Background Lung fibrosis is a kind of serious complication of many rheumatic diseases such as rheumatoid arthritis(RA),systemic lupus erythematosus(SLE), multiple myositis(DM)and dermatomyositis(PM),and mixed connective tissue disease(MCTD),but the pathogenesy is still unclear.With the development of new therapy to rheumatic diseases,more and more patients can live longer than before.Lung fibrosis is becoming a major problem for rheumatic diseases and autoallergic disease patients.Recentlly more and more works discovered that some cytokines may play an important role in the process of lung fibrosis.A number of animal models were used to study the pathgenesis interstitial pulmonary fibrosis (IPF).Several of these models provide evidence that certain cytokines were key in the disease process.Paraquat is one of the most widely used herbicides in the world.It has been approved for use by authorities in more than 120 countries.Paraquat was discovered in 1955 and introduced to the market place in 1962.During the 40 years between introduction and the present day numerous successful practical uses of the herbicide have been developed.In addition the characteristics of the chemical have allowed significant changes to be made in the ways that some crops are grown. Paraquat is a relatively non-selective foliage-applied contact herbicide.It is inactivated on contact with almost all naturally occurring soils and it was this property, perhaps above all others,that provided the greatest breakthrough in chemical weed control at the time of its discovery.The active ingredient paraquat is a non-volatile white crystalline solid,chemical namel,1-dimethy-4,4-bipyridinium melting and decomposing at 300C.Extremely soluble in water,it is practically insoluble in most organic solvents.Excessive production of oxygen free radicals has been proposed to play an important role in the pulmonary pathology.It is highly toxic for humans,and many cases of acute poisoning.We know little about the pathogenesy of pulmonary fibrosis caused by paraquat.It is imperative for us to understand the reason. Pulmonary fibrosis due to lipid peroxidation is a major symptom of paraquat intoxication.Most cases of paraquat poisoning result from intentional ingestion,with death resulting from hypoxemia secondary to lung fibrosis in moderate to severe poisonings.Paraquat-induced pulmonary fibrosis involves two factors,direct injury by oxygen free radicals and indirect injury by inflammatory cells and fibroblasts.We use paraquat as a tool to estahish a new kind of animal model of pulmonary fibrosis and investigate the effection of TGF-β1.Objective To establish a kind of new lung fibrosis rat model by paraquat and to observe the role of TGF-β1 on the pathogenesis of pulmonary fibrosis.Methods All 50 healthy adult Wistar rats were randomizedly assigned into normal control groups and paraquat poisoning group(60mg/kg,3d),paraquat poisoning group(60mg/kg,7d),paraquat poisoning group(60mg/kg,14d)and paraquat poisoning group(60mg/kg,21d).At the end of test the rats were killed and the serum and tissue were kept.The detected of serum TGF-β1 according to ABD-ELISA methods and the TGF-β1 mRNA were detected by Real-time PCR methods.Pathological changed were also observed.Result We found the increasing of serum,BALF and lung tissue TGF-β1 after paraquat poisoning at 3d,7d,14d and 21,and we also found that the increasing of TGF-β1 mRNA in lung tissue after paraquat poisoning 3d,7d,14d,but TGF-β1 mRNA in lung tissue almost recover at 21d.Pathological examination show that the fibrosis occured in lung tissue.Conclusion We established a typital lung fibrosis rat model by paraquat and discover that TGF-β1 may play an important role in the pathogenesis of pulmonary fibrosis. Background Tumor necrosis factor(TNFalpha)is a main mediator in the inflammatory answer of many diseases.The anti-TNFalpha antibodies etanercept block their action,preventing the inflammatory answer and the damage it produces. Tumor necrosis factor-alpha(TNF-alpha)plays an important role in the pathogenesis of such diseases as rheumatoid arthritis,ankylosing spondylitis,psoriatic arthritis,and juvenile chronic arthritis.It is also play an important role in acute lung injury.Recent years have brought improvement in the understanding of the pathogeneses of these diseases,resulting in the production of new groups of biological drugs,including, among others,anti-TNF-alpha antibodies.The use of TNF inhibitors has been a great advance in the treatment of patients with these inflammatory diseases.Etanercept is a dimeric fusion protein that joins the human p75 TNF receptor to the Fc domain of human IgG1.Pulmonary fibrosis due to lipid peroxidation is a major symptom of paraquat intoxication.Most cases of paraquat poisoning result from intentional ingestion,with death resulting from hypoxemia secondary to lung fibrosis in moderate to severe poisonings.Paraquat-induced pulmonary fibrosis involves two factors,direct injury by oxygen free radicals and indirect injury by inflammatory cells and fibroblasts.We use paraquat as a tool to estahish a new kind of animal model of pulmonary fibrosis and investigate the effection of TGF-β1.In this paper we will discuss the treatment effecetive of etanercept combined with methylprednisolone on acute lung injury caused by paraquat.Objective To discuss the treatment effecetive of etanercept combined with methylprednisolone on acute lung injury caused by paraquat. Methods All 50 healthy male adult Wistar rats were randomizedly assigned into paraquat induction group,paraquat induction+methylprednisolone group,paraquat induction group+Etanercept,paraquat induction+Etanercept+methylprednisolone group,normal conrol group.The rats were killed and the serum,BALF,lung tissue were kept.The detected of serum,BALF,lung tissue TNF-α;TGF-β1 according to ABD-ELISA methods and the lung tissue TNF-α;TGF-β1mRNA were detected by Real-time PCR methods.Pathological changed were also observed.Result The paraquat induction can increase the level of TNF-α;TGF-β1 of serum,BALF,lung tissue(P<0.01).Etanercept+methylprednisolone, methylprednisolone,Etanercept could inhabit this increasing.The effect sequence is etanercept+methylprednisolone>methylprednisolone>etanercept(P<0.05 or 0.01). Pathology show that etanercept and methylprednisolone could decease the lung injury caused by paraquat.Conclusion:Etanercept combined with methylprednisolone could decrease the dose of TNF-αand TGF-β1 in serum,BALF and lung tissue;They also decrease the expression of lung tissue TNF-αmRNA and TGF-β1 mRNA.