Chun-Chou Tsai

Increased cardiac injury in NZB/W F1 mice received antibody against human parvovirus B19 VP1 unique region protein.

Human parvovirus B19 (B19) infection has been postulated to both myocardial injury and development of systemic lupus erythematosus (SLE). However, the influence of anti-B19-VP1u antibodies on cardiac disorders in SLE is still obscure. To elucidate the effects of anti-B19-VP1u IgG in SLE, passive transfer of PBS, normal rabbit IgG or rabbit anti-B19-VP1u IgG was injected intravenously into NZB/W F1 mice, respectively. Significant expression of IL-1β, IL-6 and TNF-α were detected in NZB/W F1 mice receiving rabbit anti-B19-VP1u IgG. Markedly cardiomyocyte disarray and lymphocyte infiltration were observed in left ventricle of hearts from NZB/W F1 mice receiving rabbit anti-B19-VP1u IgG. Additionally, significant increases of matrix metalloproteinase-9 (MMP9) activity and protein expression were detected in left ventricle of hearts from NZB/W F1 mice receiving B19-VP1u IgG. Accordingly, significant increase of phosphorylated p-38 and NF-κB proteins were observed in left ventricle of hearts from NZB/W F1 mice receiving B19-VP1u IgG. However, no significant variation of cardiac atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), heart-type fatty acid-binding protein (h-FABP) and creatine kinase MB (CK-MB) were detected among all experimental groups. These findings firstly demonstrated the aggravated effects of anti-B19 VP1u IgG on cardiac injury by induction of inflammatory but not myocardial infarction-associated proteins through activation of phosphorylated p-38 and NF-κB signaling.

The association of anti-parvovirus B19-VP1 unique region antibodies with antiphospholipid antibodies in patients with antiphospholipid syndrome.

BACKGROUND Human parvovirus B19 (B19) infection has been identified as a trigger of antiphospholipid syndrome (APS). However, the precise role of B19-VP1 unique region (VP1u) in patients with antiphospholipid syndrome remains unclear. METHODS IgM and IgG against B19-VP, and serum levels of antibodies directed against cardiolipin (CL), beta2-glycoprotein-I (beta2GPI) and phospholipid (PhL) were determined using ELISA in 45 APS patients. Humoral responses of anti-B19-VP1u were assessed by Western blot and B19 DNA was detected by nested PCR. Absorption experiments were performed using B19-VP1u protein to determine the binding specificity of antiphospholipid antibodies (aPL). RESULTS One and 18 of 45 APS patients had detectable levels of anti-B19-VP IgM and anti-B19-VP IgG, indicating recent and past infection respectively. All serum samples from APS patients with diagnostic pattern DNA(-)/IgM(-)/IgG(+) had anti-B19-VP1u activity. APS patients with anti-B19-VP1u antibody had a 4-fold increased risk for recurrent vascular thrombosis compared with those without anti-B19-VP1u antibody. The binding inhibition of CL, beta2GPI, and PhL by absorption with B19-VP1u ranged from 31.4% to 91.1%, 0.8% to 59.8% and 20.2% to 72.1% respectively. Significantly higher inhibition to beta2GPI by B19-VP1u absorption was observed in APS patients with anti-B19-VP1u antibody than in those without anti-B19-VP1u antibody. CONCLUSIONS We show a close association of B19 infection with aPL production and suggest B19-VP1u may be of pathogenetic importance in some patients with APS.

Up-regulation of adhesion molecule expression and induction of TNF-α on vascular endothelial cells by antibody against human parvovirus B19 VP1 unique region protein

BACKGROUND Human parvovirus B19 infection has been frequently described as a cause or trigger of various autoimmune diseases. In previous studies, we have postulated the association among human parvovirus B19 (B19)-VP1 unique region (VP1u), production of anti-beta2-glycoprotein I (anti-beta2GPI) antibody and anti-phospholipid syndrome (APS)-like autoimmunity. However, the precise role of B19-VP1u in induction of APS is still obscure. METHODS To further elucidate the pathogenic roles of VP1u in B19 infection and autoimmunity, we examined the effect of anti-B19-VP1u IgG antibodies on endothelial cells that is recognized to play crucial roles in APS. Human vascular endothelial cells, ECV-304, were incubated with various preparations of purified human or rabbit IgG. The activation of endothelial cells and production of cytokines were assessed by flow cytometry and ELISA, respectively. RESULTS Purified IgG from rabbits immunized with recombinant B19-VP1u proteins can up-regulate ICAM-1 (CD54), VCAM-1 (CD106), E-selectin (CD62E), MHC class II (HLA-DR, DP, DQ) molecule expression, and TNF-alpha production in endothelial cells as compared to those endothelial cells cultured with control IgG. Additionally, significantly increased phosphorylated-P38 mitogen-activated protein kinase (P38 MAPK) and iNOS were observed in both human anti-beta2GPI IgG and rabbit anti-B19-VP1u IgG treated-ECV-304 cells, respectively. CONCLUSIONS These experimental results imply that antibodies against B19-VP1u play important roles in the immunopathological processes as well as human anti-beta2GPI IgG that leads to development of APS by involving p38 phosphorylation and iNOS activation. It could provide a clue in understanding the role of anti-B19-VP1u antibodies in APS manifestations.

Effects of human parvovirus B19 VP1 unique region protein on macrophage responses

BackgroundActivity of secreted phospholipase A (sPLA2) has been implicated in a wide range of cellular responses. However, little is known about the function of human parvovirus B19-VP1 unique region (VP1u) with sPLA2 activity on macrophage.MethodsTo investigate the roles of B19-VP1u in response to macrophage, phospholipase A2 activity, cell migration assay, phagocytosis activity, metalloproteinase assay, RT-PCR and immunoblotting were performed.ResultsIn the present study, we report that migration, phagocytosis, IL-6, IL-1β mRNA, and MMP9 activity are significantly increased in RAW264.7 cells by B19-VP1u protein with sPLA2 activity, but not by B19-VP1uD175A protein that is mutated and lacks sPLA2 activity. Additionally, significant increases of phosphorylated ERK1/2 and JNK proteins were detected in macrophages that were treated with B19-VP1u protein, but not when they were treated with B19-VP1uD175A protein.ConclusionTaken together, our experimental results suggest that B19-VP1u with sPLA2 activity affects production of IL-6, IL-1β mRNA, and MMP9 activity, possibly through the involvement of ERK1/2 and JNK signaling pathways. These findings could provide clues in understanding the role of B19-VP1u and its sPLA2 enzymatic activity in B19 infection and B19-related diseases.

Treatment with cystamine reduces apoptosis in liver from NZB/W F1 mice.

Increased population with hepatic diseases and apoptosis were found in patients with SLE and implicated in the pathogenesis of SLE. Since cystamine has been demonstrated to be beneficial to NZB/W F1 mice in our previous report, this study intends to investigate the effects of cystamine in liver from NZB/W F1 mice. Decreased apoptosis was detected in liver from NZB/W F1 mice given cystamine as compared to those given PBS by TUNEL and caspase-3 activity assay. Fas-dependent apoptotic proteins including Fas, cleaved caspase-8 and tBid were reduced in liver from NZB/W F1 mice given cystamine as compared to those given PBS. Additionally, the mitochondria-dependent apoptotic proteins including cytochrome c and Apaf-1 were reduced in liver from NZB/W F1 mice given cystamine as compared to those given PBS. Moreover, increased BCL-2 protein was observed in liver from both mice. Notably, increased NF-kappaB protein was detected in liver from NZB/W F1 mice given cystamine as compared to those given PBS. These experimental results suggest the effect of cystamine in reducing apoptosis in liver from NZB/W F1 mice through Fas-dependent and mitochondrial-dependent pathways. The phosphorylation of NF-kappaB (p65) could be a possible mechanism involving anti-apoptotic effects of cystamine in liver from NZB/W F1 mice.

Increased expression of Matrix Metalloproteinase 9 in liver from NZB/W F1 mice received antibody against human parvovirus B19 VP1 unique region protein

BackgroundHuman parvovirus B19 infection has been postulated to the anti-phospholipid syndrome (APS) in autoimmunity. However, the influence of anti-B19-VP1u antibody in autoimmune diseases is still obscure.MethodsTo elucidate the effect of anti-B19-VP1u antibodies in systemic lupus erythematosus (SLE), passive transfer of rabbit anti-B19-VP1u IgG was injected intravenously into NZB/W F1 mice.ResultsSignificant reduction of platelet count and prolonged thrombocytopenia time were detected in anti-B19-VP1u IgG group as compared to other groups, whereas significant increases of anti-B19-VP1u, anti-phospholipid (APhL), and anti-double strand DNA (dsDNA) antibody binding activity were detected in anti-B19-VP1u group. Additionally, significant increases of matrix metalloproteinase-9 (MMP9) activity and protein expression were detected in B19-VP1u IgG group. Notably, phosphatidylinositol 3-phosphate kinase (PI3K) and phosphorylated extracellular signal-regulated kinase (ERK) proteins were involved in the induction of MMP9.ConclusionThese experimental results firstly demonstrated the aggravated effects of anti-B19-VP1u antibody in disease activity of SLE.

Exacerbating effects of human parvovirus B19 NS1 on liver fibrosis in NZB/W F1 mice.

Systemic lupus erythematosus (SLE) is an autoimmune disorder with unknown etiology that impacts various organs including liver. Recently, human parvovirus B19 (B19) is recognized to exacerbate SLE. However, the effects of B19 on liver in SLE are still unclear. Herein we aimed to investigate the effects of B19 on liver in NZB/W F1 mice by injecting subcutaneously with PBS, recombinant B19 NS1, VP1u or VP2, respectively. Our experimental results revealed that B19 NS1 protein significantly enhanced the TGF-β/Smad fibrotic signaling by increasing the expressions of TGF-β, Smad2/3, phosphorylated Smad2/3, Smad4 and Sp1. The consequent fibrosis-related proteins, PAI-1 and α-SMA, were also significantly induced in livers of NZB/W F1 mice receiving B19 NS1 protein. Accordingly, markedly increased collagen deposition was also observed in livers of NZB/W F1 mice receiving B19 NS1 protein. However, no significant difference was observed in livers of NZB/W F1 mice receiving B19 VP1u or VP2 as compared to the controls. These findings indicate that B19 NS1 plays a crucial role in exacerbating liver fibrosis in NZB/W F1 mice through enhancing the TGF-â/Smad fibrotic signaling.

Human Parvovirus B19 NS1 Protein Aggravates Liver Injury in NZB/W F1 Mice

Human parvovirus B19 (B19) has been associated with a variety of diseases. However, the influence of B19 viral proteins on hepatic injury in SLE is still obscure. To elucidate the effects of B19 viral proteins on livers in SLE, recombinant B19 NS1, VP1u or VP2 proteins were injected subcutaneously into NZB/W F1 mice, respectively. Significant expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected in NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Markedly hepatocyte disarray and lymphocyte infiltration were observed in livers from NZB/WF 1 mice receiving B19 NS1 as compared to those mice receiving PBS. Additionally, significant increases of Tumor Necrosis Factor –α (TNF-α), TNF-α receptor, IκB kinase –α (IKK-α), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) and nuclear factor-kappa B (NF-κB) were detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Accordingly, significant increases of matrix metalloproteinase-9 (MMP9) and U-plasminogen activator (uPA) were also detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Contrarily, no significant variation on livers from NZB/W F1 mice receiving B19 VP1u or VP2 was observed as compared to those mice receiving PBS. These findings firstly demonstrated the aggravated effects of B19 NS1 but not VP1u or VP2 protein on hepatic injury and provide a clue in understanding the role of B19 NS1 on hepatic injury in SLE.

Human parvovirus B19 nonstructural protein NS1 enhanced the expression of cleavage of 70 kDa U1-snRNP autoantigen

BackgroundHuman parvovirus B19 (B19) is known to induce apoptosis that has been associated with a variety of autoimmune disorders. Although we have previously reported that B19 non-structural protein (NS1) induces mitochondrial-dependent apoptosis in COS-7 cells, the precise mechanism of B19-NS1 in developing autoimmunity is still obscure.MethodsTo further examine the effect of B19-NS1 in presence of autoantigens, COS-7 cells were transfected with pEGFP, pEGFP-B19-NS1 and pEGFP-NS1K334E, a mutant form of B19-NS1, and detected the expressions of autoantigens by various autoantibodies against Sm, U1 small nuclear ribonucleoprotein (U1-snRNP), SSA/Ro, SSB/La, Scl-70, Jo-1, Ku, and centromere protein (CENP) A/B by using Immunoblotting.ResultsSignificantly increased apoptosis was detected in COS-7 cells transfected with pEGFP-B19-NS1 compared to those transfected with pEGFP. Meanwhile, the apoptotic 70 kDa U1-snRNP protein in COS-7 cells transfected with pEGFP-B19-NS1 is cleaved by caspase-3 and converted into a specific 40 kDa product, which were recognized by anti-U1-snRNP autoantibody. In contrast, significantly decreased apoptosis and cleaved 40 kDa product were observed in COS-7 cells transfected with pEGFP-NS1K334E compared to those transfected with pEGFP-B19-NS1.ConclusionsThese findings suggested crucial association of B19-NS1 in development of autoimmunity by inducing apoptosis and specific cleavage of 70 kDa U1-snRNP.

Antigenicity analysis of human parvovirus B19-VP1u protein in the induction of anti-phospholipid syndrome

ABSTRACT Mounting evidence suggests a connection between human parvovirus B19 (B19) and autoimmune diseases, and especially an association between the B19-VP1 unique region (VP1u) and anti-phospholipid syndrome (APS). However, little is known about the antigenicity of B19-VP1u in the induction of APS-like syndrome. To elucidate the antigenicity of B19-VP1u in the induction of APS, N-terminal truncated B19-VP1u (tVP1u) proteins were prepared to immunize Balb/c mice to generate antibodies against B19-tVP1u proteins. The secreted phospholipase A2 (sPLA2) activities and binding specificity of mice anti-B19-tVP1u antibodies with cardiolipin (CL) and beta-2-glycoprotein I (β2GPI) were evaluated by performing immunoblot, ELISA and absorption experiments. A mice model of passively induced APS was adopted. Although sPLA2 activities were identified in all B19-tVP1u proteins, only amino acid residues 61–227 B19-tVP1u exhibited a higher sPLA2 activity. Autoantibodies against CL and β2GPI exhibited binding activities with all B19-tVP1u proteins. IgG that was purified from mice that had been immunized with amino acid residues 21–227 to 121–227 B19-tVP1u proteins exhibited significantly higher binding activity with CL. IgG that was purified from mice that had been immunized with amino acid residues 21–227, 31–227, 82–227 and 91–227 B19-tVP1u proteins exhibited significantly higher binding activity with β2GPI. Accordingly, significantly higher binding inhibition of CL was detected in the presence of amino acid residues 61–227 and 101–227 B19-tVP1u. Significantly higher binding inhibition of β2GPI was detected in the presence of amino acid residues 21–227, 31–227, 82–227 and 91–227 B19-tVP1u. The mice that received amino acid residues 31–227 or 61–227 anti-tB19-VP1u IgG revealed significant thrombocytopenia and those that received amino acid residues 21–227, 31–227, 61–227, 71–227, 82–227, 91–227, 101–227 or 114–227 anti-tB19-VP1u IgG exhibited significantly prolonged aPTT. These findings provide further information concerning the role of B19-VP1u antigenicity in APS-like autoimmunity.