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Human parvovirus B19 (B19) is harmful during pregnancy since it can be vertically transmitted to the developing fetus. In addition, the anti-B19 antibodies induced by B19 infection are believed to have a cytopathic role in B19 transmission; however, knowledge regarding the effects of anti-B19 antibodies during pregnancy is limited. To investigate the possible roles of anti-B19 antibodies during pregnancy, the present study examined the effects of anti-B19-VP1 unique region (VP1u), anti-B19-VP2 and anti-B19-nonstructural protein 1 (NS1) immunoglobulin G (IgG) antibodies on BeWo trophoblasts. Briefly, BeWo trophoblasts were incubated with purified IgG against B19-VP1u, B19-VP2 and B19-NS1. Subsequently, the expression of surface proteins and apoptotic molecules were assessed in BeWo trophoblasts using flow cytometry, ELISA and western blotting. The expression levels of human leukocyte antigen (HLA)-G were significantly increased on BeWo trophoblasts treated with rabbit anti-B19-VP1u IgG, and were unchanged in those treated with rabbit anti-B19-NS1 and anti-B19-VP2 IgG, as compared with the control group. Furthermore, the expression levels of globoside (P blood group antigen) and cluster of differentiation (CD)29 (β1 integrin) were significantly increased in BeWo trophoblasts treated with rabbit anti-B19-NS1 and anti-B19-VP2 IgG, whereas only CD29 was also significantly increased in cells treated with anti-B19-VP1u IgG. In addition, the number of cells at sub-G1 phase; caspase-3 activity; and the expression of intrinsic and extrinsic apoptotic molecules, including Fas-associated death domain protein, activated caspase-8, activated caspase-3, B-cell lymphoma 2-associated X protein, cytochrome c, apoptotic peptidase activating factor 1 and activated caspase-9, were significantly increased in BeWo trophoblasts treated with anti-B19-VP1u and anti-B19-NS1 IgG. In conclusion, the present study demonstrated that antibodies against B19 may have a crucial role in pathological processes during pregnancy. These findings may help to elucidate the mechanisms underlying transmission of the B19 virus during pregnancy. [ABSTRACT FROM AUTHOR]

The molecular pathogenesis of infectious spleen and kidney necrosis virus (ISKNV) infections is important but has rarely been studied in connection to host organelle behavior. In the present study, we demonstrated that ISKNV can induce host cell death via a pro-apoptotic Bcl-2 and anti-apoptotic Bcl-2 family member imbalance in mitochondrial membrane potential (MMP or ΔΨm) regulation in GF-1 cells. The results of our study on ISKNV infection showed that it can induce host cell death by up to 80% at day 5 post-infection. Subsequently, in an apoptotic assay, ISKNV infection was seen to induce an increase in Annexin-V-positive signals by 20% and in propidium iodide (PI) staining-positive signals by up to 30% at day 5 (D5) in GF-1 cells. Then, through our studies on the mechanism of cell death in mitochondria function, we found that ISKNV can induce MMP loss by up to 58% and 78% at days 4 and 5 with a JC1 dye staining assay. Furthermore, we found that pro-apoptotic members Bax and Bak were upregulated from the early replication stage (day one) to the late stage (day 5), but the expression profiles were very dynamically different. On the other hand, by Western blotted analysis, the anti-apoptotic members Bcl-2 and Bcl-xL were upregulated very quickly at the same time from day one (two-fold) and continued to maintain this level at day five. Finally, we found that pro-apoptotic death signals strongly activated the downstream signals of caspase-9 and -3. Taken together, these results suggest that ISKNV infection can induce Bax/Bak-mediated cell death signaling downstream of caspase-9 and -3 activation. During the viral replication cycle with the cell death induction process, the anti-apoptotic members Bcl-2/Bcl-xL interacted with the pro-apoptotic members Bax/Bak to maintain the mitochondrial function in the dynamic interaction so as to maintain the MMP in GF-1 cells. These findings may provide insights into DNA-virus control and treatment.

Background: Antibodies against spliceosome Sm proteins (anti-Sm autoantibodies) are specific to the autoimmune disease systemic lupus erythematosus (SLE). Anti-Sm autosera have been reported to specifically recognize Sm D1 and D3 with symmetric di-methylarginines (sDMA). We investigated if anti-Sm sera from local SLE patients can differentially recognize Sm proteins or any other proteins due to their methylation states. Results: We prepared HeLa cell proteins at normal or hypomethylation states (treated with an indirect methyltransferase inhibitor adenosine dialdehyde, AdOx). A few signals detected by the anti-Sm positive sera from typical SLE patients decreased consistently in the immunoblots of hypomethylated cell extracts. The differentially detected signals by one serum (Sm1) were pinpointed by two-dimensional electrophoresis and identified by mass spectrometry. Three identified proteins: splicing factor, proline- and glutamine-rich (SFPQ), heterogeneous nuclear ribonucleoprotein D-like (hnRNP DL) and cellular nucleic acid binding protein (CNBP) are known to contain methylarginines in their glycine and arginine rich (GAR) sequences. We showed that recombinant hnRNP DL and CNBP expressed in Escherichia coli can be detected by all anti-Sm positive sera we tested. As CNBP appeared to be differentially detected by the SLE sera in the pilot study, differential recognition of arginine methylated CNBP protein by the anti-Sm positive sera were further examined. Hypomethylated FLAG-CNBP protein immunopurified from AdOx-treated HeLa cells was less recognized by Sm1 compared to the CNBP protein expressed in untreated cells. Two of 20 other anti-Sm positive sera specifically differentiated the FLAG-CNBP protein expressed in HeLa cells due to the methylation. We also observed deferential recognition of methylated recombinant CNBP proteins expressed from E. coli by some of the autosera. Conclusion: Our study showed that hnRNP DL and CNBP are novel antigens for SLE patients and the recognition of CNBP might be differentiated dependent on the level of arginine methylation [ABSTRACT FROM AUTHOR]

Ocimum gratissimum (OG) is known as a food spice and traditional herb, which has been recommended for the treatment of various diseases. To investigate the hepatoprotective effect of OG aqueous extract (OGAE), maleWistar rats challenged by carbon tetrachloride (CCl4) were used as the animal model of chronic hepatic injury. Significantly increased serum catalase and DPPH levels were detected in CCl4-administrated rats that were treated with OGAE or silymarin as compared to those rats that were treated with saline or CCl4. In contrast, significantly decreased stress proteins including HSP70 and iNOS were observed in livers of CCl4-administrated rats that were treated with OGAE or sylimarin as compared to those rats that were treated with saline or CCl4. Moreover, significant decreases of M MP-9/ M MP-2 ratio, uPA, phosphorylated ERK (p-ERK) and NF-κB (p-P65) were detected in livers of CCl4-administrated rats that were treated with OGAE or sylimarin as compared to those rats that were treated with saline or CCl4. These findings imply that OGAE can efficiently inhibit CCl4-induced liver injuries in rats and may therefore be a potential food or herb for preventing liver injuries. [ABSTRACT FROM AUTHOR]

Apoptosis involves in the pathogenesis of various cardiac abnormalities. This study intends to evaluate the effects of Li-Fu formula on cardiac apoptosis induced by hyper-cholesterol diet. Twenty-four male Golden Syrian hamsters were randomly divided into Control, Cholesterol and Li-Fu formula groups. Histopathological analysis, western blotting and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to measure the effects of Li-Fu formula on left ventricle. Significantly reduced TUNEL-positive cells andmitochondria- dependent apoptosis were observed in the left ventricle of hamsters from Li-Fu formula group compared to the Cholesterol group. Additionally, induced cardiac insulin like growth factor I receptor (IGFIR)-dependent survival pathway was detected in the Li-Fu formula group compared to the Cholesterol group. Besides, minor fibrosis, increased collagen deposition, and myofibril disarray was detected in the Cholesterol group, whereas the reductions of collagen deposition and myofibril disarray were observed in the Li-Fu formula group. This study demonstrated that Li-Fu formula not only reduced the mitochondria-dependent apoptosis and fibrosis, but also enhanced the IGF-I survival pathway in the left ventricle from high cholesterol-fed hamsters. We suggest the protective effects of Li-Fu formula on cardiac apoptosis and therapeutic potentials against cardiovascular disease. [ABSTRACT FROM AUTHOR]

Hypercholesterolemia diets are considered as major sources to cause cardiac hypertrophy. This study intends to evaluate the effects of Li-Fu formula on cardiac hypertrophy induced by hypercholesterolemia diet. Twenty-four male Golden Syrian hamsters were randomly divided into control, cholesterol and Li-Fu formula groups and fed with different experimental diets for 2 months. Histopathological analysis and western blotting were performed to measure the myocardial architecture, and various cardiac hypertrophy-associated molecules in the excised left ventricle from hamsters. The ratios of whole heart weight/body weight (BW) and left ventricle weight/BWwere significantly higher in the cholesterol group but significantly lower in the Li-Fu formula group. The protein levels of both atrial natriuretic peptide and brain natriuretic peptide were significantly increased in the cholesterol group but significantly reduced in the Li-Fu formula group. Additionally, significantly increased interleukin-6, STAT3, MEK5, p- ERK5 and non-cardiomyocyte proliferate signal molecules such as p-MEK and p-ERK, were detected in the cholesterol group but significantly reduced in the Li-Fu formula group. Notably, no significant variations of inflammatory signalingmolecules, including p-P38 and p-JNK, were detected in all groups. Our experimental results demonstrated the significant reductions of cardiac hypertrophy and related eccentric hypertrophy signaling, non-cardiomyocyte proliferate signaling in the excised left ventricle of hamsters from the Li-Fu formula.We suggested the protective effects of Li-Fu formula on cardiac hypertrophy that may be useful in prevention or treatment of hypertrophy-associated cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Background: Human 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. Methods: To 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. Results: Significantly 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. Conclusions: These findings suggested crucial association of B19-NS1 in development of autoimmunity by inducing apoptosis and specific cleavage of 70 kDa U1-snRNP. [ABSTRACT FROM AUTHOR]

Cellular responses are determined by a number of signaling cues in the local microenvironment, such as growth factors and extracellular matrix (ECM). In cultures of mammary epithelial cells (MECs), functional differentiation requires at least two types of signal, lactogenic hormones (i.e., prolactin, insulin, and hydrocortisone) and the specialized ECM, basement membrane (BM). Our previous work has shown that ECM affects insulin signaling in mammary cells. Cell adhesion to BM promotes insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and association of PI3K with IRS-1, whereas cells cultured on stromal ECM are inefficient in transducing these post-receptor events. Here we examine the mechanisms underlying ECM control of IRS phosphorylation. Compared to cells cultured on BM, cells on plastic exhibit higher level of RhoA activity. The amount and the activity of Rho kinase (Rok) associated with IRS-1 are greater in these cells, leading to serine phosphorylation of IRS-1. Expression of dominant negative RhoA and the application of Rok inhibitor Y27632 in cells cultured on plastic augment tyrosine phosphorylation of IRS-1. Conversely, expression of constitutively active RhoA in cells cultured on BM impedes insulin signaling. These data indicate that RhoA/Rok is involved in substratum-mediated regulation of insulin signaling in MECs, and under the conditions where proper adhesion to BM is missing, such as after wounding and during mammary gland involution, insulin-mediated cellular differentiation and survival would be defective. J. Cell. Physiol. 220: 476–484, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Background: Human 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. Methods: To 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. Results: Significant 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. Conclusion: These experimental results firstly demonstrated the aggravated effects of anti-B19- VP1u antibody in disease activity of SLE. [ABSTRACT FROM AUTHOR]

Background: Activity 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. Methods: To 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. Results: In 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. Conclusion: Taken 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. [ABSTRACT FROM AUTHOR]

Abstract  Japanese encephalitis virus (JEV) is known as an important mosquito-borne human pathogen that causes Japanese encephalitis and may lead to lethal effect. Since monocyte has been demonstrated to play transmissible role for JEV, rare study is reported to clarify the effect of JEV envelope (JEVE) protein on monocyte. This study intends to investigate the effects of JEVE protein inside monocyte. Notably, significant decreased expression of tumour necrosis factor (TNF)-α mRNA in RAW264.7 cells transfected with pEGFP-JEVE was observed as compared to those cells transfected with pEGFP. Increased p21Waf1/Cip1 protein was observed in both pEGFP and pEGFP-JEVE transfected RAW264.7 cells. However, increased p53 protein was only detected in pEGFP-transfected cells but not pEGFP-JEVE transfected cells as well as the result that no increased expression of nuclear factor-kB was observed in pEGFP-JEVE transfected cells. These experimental results indicate the effects of JEVE protein in alleviating TNF-α mRNA expression that is associated with the increased p53-independent p21Waf1/Cip1 expression and provide an explanation in the role of JEV transmission through monocyte. [ABSTRACT FROM AUTHOR]

Anti-centromere antibody (ACA) has been reported to be associated with Sjogren’s syndrome (SS) and the clinical significance of anti-CENP-H antibody remains unknown. To determine the clinical significance of anti-CENP-H and anti-centromere antibodies in primary SS, sera from 62 patients with primary SS and 40 normal controls were examined for anti-SS-A/SS-B antibodies, ACA and anti-CENP-H antibodies, by enzyme-linked immunosorbent assay and indirect immunofluorescence (IIF), respectively. Of the 62 serum samples with primary SS, 17 were positive with ACA and anti-CENP-H antibodies. Sera from SS patients with anti-CENP-H and ACA antibodies do not contain anti-SS-A/Ro and/or anti-SS-B/La antibodies. No anti-CENP-H antibody was found in sera of normal controls. An increased frequency of ACA and anti-CENP-H antibodies was found for the first time in patients with SS. Anti-CENP-H antibodies and anti-SS-A/Ro or anti-SS-B/La antibodies are present mutually exclusive. Patients with anti-CENP-H antibodies had a lower frequency of rheumatoid factor (RF). SS can be subdivided serologically into two groups; group one with anti-SS-A/Ro and/or anti-SS-B/La antibody, group two with ACA and/or anti-CENP-H antibodies. We recommend that ACA or anti-CENP-H antibodies should be considered as one of the serological markers for SS. [ABSTRACT FROM AUTHOR]

Parvovirus B19 has been reported to be detected in the sera of patients with acute or chronic hepatitis. The prevalence and clinical significance of B19 DNA in serum samples from patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection were investigated.Serum samples from 54 patients with HBV infection, 51 with HCV infection and 53 normal controls were examined for anti-B19 antibodies and B19 DNA by enzyme-linked immunosorbent assay (ELISA), the nested polymerase chain reaction (PCR), Southern blotting and direct nucleotide sequencing, respectively.Anti-B19 IgM and IgG antibodies were detected in 19 (35.2%) and 46 (85.2%) of 54 serum samples from patients with HBV infection, and eight (15.7%) and 36 (70.6%) of 51 serum samples from patients with HCV infection. B19 DNA was detected in serum samples of 20 (37%) of 54 patients with HBV infection and 12 (23.5%) of 51 patients with HCV infection, but not in 53 serum samples from normal controls. The occurrence of liver dysfunction was not affected by B19 infection in patients with HBV and HCV infection (P > 0.05). All of the 20 serum samples with B19 DNA from patients with chronic HBV infection and all of the 12 serum samples with B19 DNA from patients with chronic HCV infection exhibited TW-3 subtype and TW-9 subtype, respectively. The variant subtypes of B19 were found to be distinctive in patients with HBV or HCV infection.These data revealed that human parvovirus B19 infection was frequently found in patients with chronic HBV or HCV infection. The variant genotypes were present in patients with different chronic hepatitis. The coinfection of B19 with HBV or HCV did not increase the frequency of liver dysfunction in patients with chronic hepatitis. Long-term longitudinal studies are required to determine the natural course of parvovirus B19 infection and whether its coinfection affects the natural history of chronic hepatitis B or hepatitis C. [ABSTRACT FROM AUTHOR]

Human parvovirus B19 has been found in various tissues in addition to erythroid lineage cells, and non-structural protein (NSI) is reported to induce cytotoxicity and apoptosis in erythroid lineage cells, but the mechanism in non-permissive cells is still unclear. To address this issue, we have constructed the NSI gene in a cytomegalovirus episomal vector, pEGFP-C1 and transfected it into monkey epithelial cells, COS-7. EGFP-NSI expression in transfected cells was monitored and assessed by fluorescence microscopy, RT-PCR and Western blot. The flow cytometric analysis showed that the NSI-transfected cells were arrested at G1 phase by paclitaxel treatment and there was increased apoptosis. The expression of p53, an important molecule in apoptosis and cell cycle regulation, and its downstream cell cycle kinase inhibitors p16INK4 and p21WAF1/CIP1 were up-regulated in the NS1-transfected cells. Also, increased expression of the pro-apoptotic Bcl-2 members Bax, Bad and activation of caspase 3 and caspase 9, but not the activation of caspase 8 or Fas were detected in the NS1-transfected cells, p53-induced Bax expression and subsequent activation of caspase 9 is probably the apoptotic pathway in NS1-transfected cells since activation of the caspase 9 was suppressed by the p53 inhibitor and apoptosis was significantly inhibited by the caspase 9 inhibitor. Our results suggest that the cell death of the NS1-transfected cells is associated with mitochondria related apoptosis. These findings might provide alternative information for further study and characterization of B19 NS1 protein in B19 non-permissive cells. [ABSTRACT FROM AUTHOR]

Purpose: Evaluate if etomidate modulates adrenal apoptosis and if this influences the development of critical illness-related corticosteroid insufficiency (CIRCI) in hemorrhagic shock (HS). Material and methods: Four groups of 16 male Wistar rats: G0 (control group anesthetized with isoflurane and mechanical ventilation), G1 (like G0, but with buprenorphine), G2 (like G1 with HS), and G3 (like G2 with etomidate 1 mg/kg, IV, before HS). HS induced by collecting 30% of blood volume. Resuscitation performed 90 min later with the collected blood and normal saline. Hemodynamic parameters, blood gas analysis, adrenocorticotropic hormone (ACTH), corticosterone (CS), and TNF-α, IL6, IL10 were determined at 0, 90, 150 and 240 min post-HS induction (at the corresponding time points in G0 and G1). Apoptosis and necrosis were determined by TUNEL and caspase-3 immunofluorescence and a necrosis score, respectively. Results: HS groups had significantly higher levels of apoptosis and necrosis than G1 and G0. Compared with G2, etomidate-treated animals had significantly lower levels of CS (compatible with CIRCI), PO2, PO2/FiO2, BE, HCO3, apoptosis and necrosis and significantly higher cytokine levels. Conclusions: Etomidate was associated with CIRCI. HS was associated with adrenal gland apoptosis and necrosis. The latter were decreased by etomidate, possibly by both direct and indirect mechanisms. [ABSTRACT FROM AUTHOR]