Macrophage migration inhibitory factor has a permissive role in concanavalin A-induced cell death of human hepatoma cells through autophagy.

Concanavalin A (ConA) is a lectin and T-cell mitogen that can activate immune responses. In recent times, ConA-induced cell death of hepatoma cells through autophagy has been reported and its therapeutic effect was confirmed in a murine in situ hepatoma model. However, the molecular mechanism of ConA-induced autophagy is still unclear. As macrophage migration inhibitory factor (MIF), which is a proinflammatory cytokine, can trigger autophagy in human hepatoma cells, the possible involvement of MIF in ConA-induced autophagy was investigated in this study. We demonstrated that cell death is followed by an increment in MIF expression and secretion in the ConA-stimulated human hepatoma cell lines, HuH-7 and Hep G2. In addition, ConA-induced autophagy and cell death of hepatoma cells were blocked in the presence of an MIF inhibitor. Knockdown of endogenous MIF by small hairpin RNA confirmed that MIF is required for both ConA-induced autophagy and death of hepatoma cells. Furthermore, signal pathway studies demonstrated that ConA induces signal transducer and activator of transcription 3 (STAT3) phosphorylation to trigger MIF upregulation, which in turn promotes Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3)-dependent autophagy. By using a murine in situ hepatoma model, we further demonstrated that MIF contributes to anti-hepatoma activity of ConA by regulating STAT3-MIF-BNIP3-dependent autophagy. In summary, our findings uncover a novel role of MIF in lectin-mediated anti-hepatoma activities by regulating autophagy.

Peptide mimicrying between SARS coronavirus spike protein and human proteins reacts with SARS patient serum.

Molecular mimicry, defined as similar structures shared by molecules from dissimilar genes or proteins, is a general strategy used by pathogens to infect host cells. Severe acute respiratory syndrome (SARS) is a new human respiratory infectious disease caused by SARS coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV plays an important role in the virus entry into a cell. In this study, eleven synthetic peptides from the S protein were selected based on its sequence homology with human proteins. Two of the peptides D07 (residues 927-937) and D08 (residues 942-951) were recognized by the sera of SARS patients. Murine hyperimmune sera against these peptides bound to proteins of human lung epithelial cells A549. Another peptide D10 (residues 490-502) stimulated A549 to proliferate and secrete IL-8. The present results suggest that the selected S protein regions, which share sequence homology with human proteins, may play important roles in SARS-CoV infection.

Antibody to severe acute respiratory syndrome (SARS)-associated coronavirus spike protein domain 2 cross-reacts with lung epithelial cells and causes cytotoxicity.

Both viral effect and immune-mediated mechanism are involved in the pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. In this study, we showed that in SARS patient sera there were autoantibodies (autoAbs) that reacted with A549 cells, the type-2 pneumocytes, and that these autoAbs were mainly IgG. The autoAbs were detectable 20 days after fever onset. Tests of non-SARS-pneumonia patients did not show the same autoAb production as in SARS patients. After sera IgG bound to A549 cells, cytotoxicity was induced. Cell cytotoxicity and the anti-epithelial cell IgG level were positively correlated. Preabsorption and binding assays indicated the existence of cross-reactive epitopes on SARS-CoV spike protein domain 2 (S2). Furthermore, treatment of A549 cells with anti-S2 Abs and IFN-gamma resulted in an increase in the adherence of human peripheral blood mononuclear cells to these epithelial cells. Taken together, we have demonstrated that the anti-S2 Abs in SARS patient sera cause cytotoxic injury as well as enhance immune cell adhesion to epithelial cells. The onset of autoimmune responses in SARS-CoV infection may be implicated in SARS pathogenesis.

Immunopathogenesis of dengue virus infection.

Dengue virus infection causes dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), whose pathogeneses are not clearly understood. Current hypotheses of antibody-dependent enhancement, virus virulence, and IFN-gamma/TNFalpha-mediated immunopathogenesis are insufficient to explain clinical manifestations of DHF/DSS such as thrombocytopenia and hemoconcentration. Dengue virus infection induces transient immune aberrant activation of CD4/CD8 ratio inversion and cytokine overproduction, and infection of endothelial cells and hepatocytes causes apoptosis and dysfunction of these cells. The coagulation and fibrinolysis systems are also activated after dengue virus infection. We propose a new hypothesis for the immunopathogenesis for dengue virus infection. The aberrant immune responses not only impair the immune response to clear the virus, but also result in overproduction of cytokines that affect monocytes, endothelial cells, and hepatocytes. Platelets are destroyed by crossreactive anti-platelet autoantibodies. Dengue-virus-induced vasculopathy and coagulopathy must be involved in the pathogenesis of hemorrhage, and the unbalance between coagulation and fibrinolysis activation increases the likelihood of severe hemorrhage in DHF/DSS. Hemostasis is maintained unless the dysregulation of coagulation and fibrinolysis persists. The overproduced IL-6 might play a crucial role in the enhanced production of anti-platelet or anti-endothelial cell autoantibodies, elevated levels of tPA, as well as a deficiency in coagulation. Capillary leakage is triggered by the dengue virus itself or by antibodies to its antigens. This immunopathogenesis of DHF/DSS can account for specific characteristics of clinical, pathologic, and epidemiological observations in dengue virus infection.

Involvement of oxidative stress, NF-IL-6, and RANTES expression in dengue-2-virus-infected human liver cells.

The liver has been suspected to be one of the major targets of dengue virus infection. Here, we detected increasing secretion of the chemokine RANTES (regulated upon activation, normal T cell expressed and secreted), which functions to recruit the immune cells, in dengue-virus-infected liver cells and patients. Three luciferase reporter genes with various deletions at the 5'-end of the RANTES promoter were constructed to explore the RANTES activation mechanism in human liver cells. The reporter gene was optimally activated by dengue-2 virus when the RANTES promoter contains the region from the transcription starting site (+1) to the nucleotide at the -181 position. NF-IL-6 and an undefined factor forming DNA-protein complexes in the RANTES promoter E and A/B regions in the infected cells were demonstrated by electrophoretic mobility shift assay. Further analysis showed that oxidative stress was an upstream inducer of NF-IL-6 and RANTES signaling in dengue-virus-infected liver cells. This finding was demonstrated by three antioxidants (N-acetyl-l-cysteine, nitro-l-arginine methyl ester, and pyrrolidine dithiocarbamate) used to suppress the activation. In contrast, the DNA binding activity of the undefined factor was not affected by the antioxidant treatment, indicating the existence of an oxidant-independent pathway. We hypothesize that dengue virus infection of the liver cells may trigger both an oxidant-dependent and an oxidant-independent pathway to up-regulate RANTES mRNA expression through activating NF-IL-6 and an undefined factor, respectively. In conclusion, the present study suggests a new direction for the study of liver pathogenesis involving RANTES in host immune responses during dengue virus infection.

Infection of five human liver cell lines by dengue-2 virus.

Elevated serum transaminase levels of dengue patients indicate the possible impact of dengue virus infection on liver function. To elucidate the action of dengue virus infection in liver cells, an in vitro cell line system was established that mimicked the liver status of diverse clinical patients. Briefly, four hepatoma cell lines (HA22T, Huh7, Hep3B, and PLC) and one nonmalignant hepatocyte cell line (Chang liver) were included, representing various levels of tumorigenicity and differentiation. Our data showed that in these five cell lines, dengue-2 virus attached to each cell type equally well; however, this virus had higher replication rates and levels of virion production in differentiated Huh7, PLC, Hep3B, and Chang liver cells. Likewise, a lower replication rate was observed in the de-differentiated HA22T cells. Differentiation-related factors seem to play an important role in dengue virus replication. Further study showed that sodium butyrate (NaB, a differentiation inducer) treatment enhanced dengue virus replication in HA22T cells. Moreover, we found that the severity of morphologic aberration and the increase in aspartate aminotransferase (AST) levels correlated with the virus replication rate in the four infected hepatoma cells. In conclusion, we showed that dengue virus can infect diverse liver cells with differing replication efficiency, which causes cytopathic effects (CPEs) of diverse severity. Among the CPEs, the increased AST levels correlated with the clinical results from 24 dengue fever patients, who showed increased AST levels at the onset of fever. In summary, we find that dengue-2 virus replicates actively and causes severe CPEs in differentiated hepatoma cells. Factors related to differentiation as well as tumorigenicity seem to play critical roles, though the mechanisms of action remain unclear.

The potential of soybean foods as a chemoprevention approach for human urinary tract cancer.

Isoflavones are excreted in human urine and can be modulated by soy-rich diets. Recently, isoflavones were suggested to have protective effects against bladder cancer cells. We sought to determine the efficacy of the antitumorigenic effects of isoflavones at concentrations found in the range of human urine excretion and compare normal urothelium and bladder cancer cells for differential cytotoxicity. A total of seven human bladder cancer cell lines and an immortalized uroepithelial cell line were used to examine the effects of genistein, daidzein, and biochanin-A, either individually or as an equal-proportion mixture regimen, on cell growth, DNA synthesis, alterations of cell cycle distribution, and induction of apoptosis. The role of cyclin B1 and cdc2 kinase in cell cycle arrest was analyzed. In addition, severe combined immunodeficient mice were used to confirm the anti-cancer effects of isoflavones in vivo. Cooperative action of isoflavones was more effective in growth inhibition and apoptosis induction than any single compound. Genistein tends to cause a dose-dependent induction of G2-M cell cycle arrest and an inhibition of cdc2 kinase activity. However, both daidzein and biochanin-A directly induced apoptosis without altering cell cycle distribution. The IC50 values in non-transformed cells were higher than those in most cancer cell lines, and the IC50 of the mixture regimen was within reach of the levels observed in urine after a soy challenge. Furthermore, both genistein and combined isoflavones exhibited a significant tumor suppressor effect in vivo (P < 0.05). The results justify the potential use of soybean foods as a practical chemoprevention approach for patients with urinary tract cancer.

Dengue virus infects human endothelial cells and induces IL-6 and IL-8 production.

In this study dengue virus (DV) was found to infect primary endothelial cells derived from human umbilical cord veins (HUVEC) and alter their cytokine production. Dengue virus infection of HUVEC was confirmed by an increase in plaque-forming units in the culture supernatant and by immunofluorescence assay. HUVEC produced large amounts of interleukin (IL)-6 and IL-8 but not IL-1beta after DV infection. Both the replication of DV and the production of IL-6 and IL-8 by HUVEC after DV infection were inhibited by ribavirin, an antiviral synthetic guanosine analogue. Additionally, increased serum levels of IL-6 and IL-8 were observed in patients with dengue hemorrhagic fever but not dengue fever. Therefore, our results suggest that endothelial cells can be a target for DV infection, and that DV-induced IL-6 and IL-8 production by endothelial cells may contribute to the pathogenesis of dengue hemorrhagic fever.

Alpha 1-acid glycoprotein-induced tumor necrosis factor-alpha secretion of human monocytes is enhanced by serum binding proteins and depends on protein tyrosine kinase activation.

The acute phase protein, alpha1 acid glycoprotein (AGP), stimulated human mononuclear cells as well as monocytes to secrete tumor necrosis factor-alpha (TNFalpha) which was demonstrated by ELISA, RT-PCR and functional assays. AGP-induced TNFalpha secretion of monocytes was enhanced in the presence of human plasma and inhibited by protein kinase inhibitors, indicating it is serum and tyrosine kinase dependent. The activation of tyrosine kinase in AGP-stimulated monocytes was further confirmed by immunoblotting of tyrosine phosphorylated proteins of monocytes at different time after AGP stimulation. Furthermore, several serum proteins such as C3, sCD14 and IgG were able to bind to AGP and enhanced TNFalpha secretion of human monocytes induced by AGP. Taken together, these results suggest serum proteins binding to AGP enhance its ability to stimulate human monocytes to secrete pro-inflammatory cytokines through a tyrosine kinase dependent pathway.

The dynamic responses of pro-inflammatory and anti-inflammatory cytokines of human mononuclear cells induced by uromodulin.

This study was undertaken to examine the dynamic response of human peripheral blood mononuclear cells (PBMC) in the secretion of proinflammatory and anti-inflammatory cytokines induced by uromodulin (URO). Levels of tumor necrosis factor-alpha (TNFalpha), TNF soluble receptor (sTNFRI and II), interleukin 1-beta (IL-1beta), and IL-1 receptor antagonist (IL-1Ra) in the supernatants of URO-stimulated PBMC were measured by ELISA. URO stimulated the secretion of all these cytokines in a dose dependent manner except sTNFRI. Peak levels of TNFalpha and IL-1beta were reached at 6-12 h, while 5-10 fold higher in sTNFR II and IL-1Ra levels were observed at 24-48 h after URO stimulation. URO-induced secretion of TNFalpha, IL-1beta, sTNFRII and IL-1Ra could be enhanced by human plasma. Specifically, serum proteins including C3, sCD14 and IgG not only bound to URO but also enhanced URO-induced TNFalpha secretion of PBMC. Collectively, our data suggest that URO might have dual immunomodulating effect through regulating the secretion of proinflammatory and anti-inflammatory cytokines, and that serum binding proteins might enhance this activity.

Antibodies against dengue virus E protein peptide bind to human plasminogen and inhibit plasmin activity.

Both mice and rabbits immunized with dengue virus E protein peptide spanning amino acids 100-119 (D4E) produced antibodies that reacted not only with the D4E peptide itself but also with human plasminogen, as shown by ELISA and Western blot. Sera from dengue virus-hyperimmunized mice and dengue patients also contained antibodies against D4E and plasminogen. Furthermore, such sera all contained plasmin inhibitory activity. Using affinity-purified anti-D4E antibodies and free D4E peptide for competitive inhibition, we demonstrated that the inhibition of plasmin activity was due to anti-D4E antibodies rather than other substances in the sera. Taken together, these results suggest dengue virus E protein amino acids 100-119 are a cross-reactive immunogenic region, and antibodies against this region may interfere with human fibrinolysis.

Uromodulin and Tamm-Horsfall protein induce human monocytes to secrete TNF and express tissue factor.

Effects of uromodulin (URO) and Tamm-Horsfall protein (THP), the most abundant proteins in the urine of pregnant and normal women, respectively, on the induction of TNF-alpha secretion and tissue factor (TF) expression of human monocytes were studied. THP, URO, and its fragments stimulated human mononuclear cells to proliferate and secrete TNF-alpha. The release of URO and THP-induced TNF-alpha in monocytes was dependent upon protein tyrosine kinase activation that results in tyrosine phosphorylation. URO and THP also induced TF expression of human monocytes and monocytic cell line U937 in a dose-dependent manner. TF expression was transient, reached its peak at 6 h and declined toward basal levels by 24 h. Reverse transcriptase-PCR and dot-blot analysis confirmed the induction of TF mRNA synthesis. URO and THP-induced TF expression were inhibited by actinomycin D and pentoxifylline further supporting the requirement of de novo TF mRNA synthesis. The possibility of LPS contamination of URO and THP was excluded because: 1) URO and THP-induced TF expression were inhibited by specific Ab; 2) URO was less capable of inducing TF in HUVEC as compared with LPS; 3) polymyxin B blocked the induction of Limulus clotting by LPS but not by URO and THP; 4) both LPS-sensitive (C3H/HeN) and -resistant (C3H/HeJ) mice produced little or no TNF-alpha after URO challenge. Therefore, our findings suggest that URO and THP play a significant role in the innate immunity of the urinary system and that the immunostimulatory activity of URO is potentially useful for immunotherapy.

Effects of alpha 1-acid glycoprotein on tissue factor expression and tumor necrosis factor secretion in human monocytes.

Activated monocytes express tissue factor (TF) and secrete tumor necrosis factor alpha (TNF alpha), which are important in the initiation of blood coagulation and inflammation. We investigated the effect of alpha 1-acid glycoprotein (alpha 1-AGP), an acute phase protein, on the induction of the expression of TF and the secretion of TNF alpha in human monocytes in vitro. The TF activity of both fresh human monocytes and human monocytic cell line U937 significantly increased in a dose-dependent manner after a 6 h incubation with human or bovine alpha 1-AGP. The activity of TF gradually tailed off after 24 h. RT-PCR and Southern blot analysis revealed that TF mRNA synthesis was induced in monocytes. Inhibition of alpha 1-AGP induced TF expression by actinomycin D (ActD) further support that de novo TF mRNA synthesis was required. The specificity of the alpha 1-AGP-induced TF activity was demonstrated by anti-alpha 1-AGP antibody inhibition. TNF alpha secretion in alpha 1-AGP stimulated monocytes was also increased; this could be blocked by pentoxifylline (PTX). The possible contamination of lipopolysaccharide (LPS) in the alpha 1-AGP was excluded by limulus amoebocyte lysate. Therefore, these results indicate that alpha 1-AGP may contribute to the cellular initiation of coagulation and inflammation by increasing TF expression and TNF alpha secretion of monocytes.

Lewis rats given antibodies against denatured acetylcholine receptor become resistant to induction of experimental autoimmune myasthenia gravis.

Experimental autoimmune myasthenia gravis (EAMG) in rats can be produced as the result of immunization with purified acetylcholine receptor (AChR). However, antibodies produced against an irreversibly denatured AChR were not capable of producing detectable AChR-dependent neuromuscular impairment such as that seen following immunization with AChR of intact conformation. This immunopathological difference was observed despite the fact that both immunizations resulted in the production of clonotypically heterogeneous antibodies with similar titers, isotype distribution, and relative binding avidities for conformationally intact AChR. Although they had no apparent disease-causing potential of their own, antibodies produced against denatured AChR could, however, bind AChR at the neuromuscular junction and mediate in vivo AChR-dependent neuromuscular impairment if a second anti-antibody was provided. Finally, immunization against denatured AChR, or administration to naive rats of antibodies obtained by immunization against denatured AChR, resulted in the recipient rats becoming resistant to the usual pathological effects of antibodies produced against intact AChR (either induced by active immunization or following passive antibody transfer). These observations suggest that disease severity in this system may be influenced by relationships between disease-causing and disease-abrogating antibodies.

Implications of urinary basic fibroblast growth factor excretion in patients with urothelial carcinoma.

1. Angiogenesis occurs in response to wounding, and is of vital importance for tumour growth and metastasis. Basic fibroblast growth factor, a well-known angiogenic factor, has been suggested to be a urine marker for urothelial carcinoma. However, the relevance of its detection has not been evaluated in a large number of patients. 2. Immunoassay of basic fibroblast growth factor was performed on urine samples from different aetiologies of urothelial disorder. Expression of basic fibroblast growth factor in the corresponding tumour was correlated with the urine level. 3. The excretion of basic fibroblast growth factor (ng/g creatinine) was significantly elevated in both inflammatory and neoplastic urological diseases compared with normal individuals (P < 0.05), while it was normalized in tumour-free subjects (P < 0.01). Receiver operating characteristic plotting revealed a sensitivity of 40% for tumour diagnosis at the cut-off point of 3.29 ng/g creatinine. The sensitivity of the test in predicting tumour recurrence was only 14%. The basic fibroblast growth factor level in urine showed a positive association with increasing age of cancer patients (P = 0.02) and with tumour grading (P = 0.05). However, no important relationship was observed regarding tumour stage, size, number, shape or degree of local inflammatory reaction (P > 0.01). Pairwise analysis of the basic fibroblast growth factor level in urine and its expression in corresponding tumours did not reveal a conspicuous correlation (r = -0.097, P = 0.43). 4. Our results suggested that estimation of urinary basic fibroblast growth factor cannot be satisfactory as a tumour marker. The measurement may represent one of the tissue responses to injury or the host-tumour interactions. A longitudinal study is required to elucidate the role of basic fibroblast growth factor in order to select the appropriate treatment strategy for urothelial carcinoma.

Lipopolysaccharide binding and antibacterial activities of a synthetic peptide representing amino acids 90-101 of bactericidal/permeability-increasing protein.

The bactericidal/permeability-increasing protein (BPI) of polymorphonuclear leukocytes is a potent antibacterial agent specific for gram-negative bacteria. BPI can bind to lipopolysaccharide (LPS) and neutralize its toxicity. However, little is known about the specific site and mechanisms of the BPI involved in this LPS binding and antibacterial activities. This study compared the amino acid sequences among BPI, cecropin A, magainin 2, and polymyxin B, and identified a common structure among these four bactericidal agents. They share a basic amphipathic alpha helix motif (Baah). A short peptide that represents amino acids 90-101 of BPI was then synthesized to test if it possessed any LPS binding and antibacterial activities. Results from in vitro lymphocyte culture indicated this peptide was able to inhibit LPS-induced lymphocyte proliferation, suggesting that it may interact with LPS. This LPS binding ability of BPI peptide 90-101 was further supported by the results from HPLC assays which showed the mobility of the peptide shifted in the presence of LPS. Furthermore, the antibacterial spectra of this peptide and cecropin peptide 1-11 were very similar to that of polymyxin B, even though the antibacterial activities of these two peptides were less potent than that of polymyxin B. In addition, the antibacterial activities of these two peptides and polymyxin B were inhibited by free LPS or a high concentration of MgCl2. These results thus suggest that a common structure (Baah) and antibacterial mechanism may be involved in these antibacterial agents.

Exacerbated muscle dysfunction by procainamide in rats with experimental myasthenia gravis.

The induction of experimental autoimmune myasthenia gravis (EAMG) has long been shown to result in inefficient function of the acetylcholine receptor (AChR) and concomitant impairment of AChR-dependent neuromuscular communication. As an animal model of human myasthenia gravis, AChR-immunized rats demonstrate symptoms of MG very similar to those observed in human patients resulting from the presence of circulating anti-AChR antibodies which interfere with the normal function of the receptor. In addition to antibody antagonists of neuromuscular function, a variety of drugs have been observed to be associated with possible exacerbations of impaired neuromuscular function leading to myasthenic crisis in some MG patients. One drug, the cardiac anti-arrhythmic agent, procainamide, has been reported to cause both pre-synaptic and post-synaptic electrophysiologic effects at the neuromuscular junction. The study described below extends these observations to include the demonstration of perturbed AChR-dependent contractile muscle function in a rat model of MG.

Skewed B cell VH family repertoire in Bcl-2-Ig transgenic mice.

The proto-oncogene Bcl-2 is normally expressed in B lineage cells in a stage specific manner and extends cell survival. Deregulated Bcl-2 expression has been shown to cause a major expansion in surface IgM and IgD positive B cells. In this report, the influence of deregulated expression of Bcl-2 on the VH repertoire of B cells was studied. This was accomplished by stimulating B cells from both adult and fetal Bcl-2-Ig transgenic mice and their normal littermates using the polyclonal activator lipopolysaccharide. Activated cells were then analyzed by in situ hybridization using radiolabeled C mu and VH gene probes. The D-proximal VH families 7183 and Q52 were preferentially expressed in the adult transgenic mice compared to their normal littermates. VH 7183 and Q52 were also over-represented in fetal transgenic mice but not to a greater extent than that observed with normal fetuses. These results demonstrate that the overproduction of Bcl-2, which prolongs cell survival independent of affecting proliferation, substantially alters the VH gene repertoire.

Clonotypic analysis of anti-acetylcholine receptor antibodies from experimental autoimmune myasthenia gravis-sensitive Lewis rats and experimental autoimmune myasthenia gravis-resistant Wistar Furth rats.

A single immunization of Lewis rats with purified acetylcholine receptor (AChR) emulsified in adjuvant typically stimulates the production of oligoclonal AChR-reactive antibodies (as demonstrated by IEF) dominated by the IgG2a subclass, of moderate but clonotypically heterogeneous relative Ag-binding avidity, and capable of inducing symptoms of experimental autoimmune myasthenia gravis. Although similar immunization of Wistar Furth rats produces AChR-reactive antibodies with similar characteristics of clonotypic heterogeneity, avidity, and isotype expression, no detectable signs of AChR-dependent muscle impairment is observed. This contrasts the ability to induce impaired AChR function upon the passive transfer of pre-formed Lewis anti-AChR antibodies into naive Wistar Furth rats, suggesting that disease resistance in this model is not conferred at the level of the AChR itself. Moreover, if more aggressive immunization protocols are used (i.e., multiple injections of AChR), a transient breakthrough of AChR-dependent muscle dysfunction can be induced directly in the Wistar Furth strain indicating that the potential for the production of disease-causing antibodies does exist in the Wistar Furth repertoire. IEF analysis of Wistar Furth anti-AChR antibodies has revealed that hyperimmunization results in modified antibody clonotype expression that might explain changing expression of disease symptoms; however, explanations for the apparent "resistance" of Wistar Furth rats to disease induction are likely to be complex.