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Autophagy is an evolutionarily conserved catabolic process that maintains cellular homeostasis under stress conditions such as starvation and pathogen infection. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that plays important roles in inflammation and tumorigenesis. Cytokines such as IL-1β and TNF-α that are induced by MIF have been shown to be involved in the induction of autophagy. However, the actual role of MIF in autophagy remains unclear. Here, we have demonstrated that incubation of human hepatoma cell line HuH-7 cells with recombinant MIF (rMIF) induced reactive oxygen species (ROS) production and autophagy formation, including LC3-II expression, LC3 punctae formation, autophagic flux, and mitochondria membrane potential loss. The autophagy induced by rMIF was inhibited in the presence of MIF inhibitor, ISO-1 as well as ROS scavenger N-acetyl-L-cysteine (NAC). In addition, serum starvation-induced MIF release and autophagy of HuH-7 cells were partly blocked in the presence of NAC. Moreover, diminished MIF expression by shRNA transfection or inhibition of MIF by ISO-1 decreased serum starvation-induced autophagy of HuH-7 cells. Taken together, these data suggest that cell autophagy was induced by MIF under stress conditions such as inflammation and starvation through ROS generation.

Enterovirus 71 (EV71) is one of the most important causes of herpangina and hand, foot, and mouth disease. It can also cause severe complications of the central nervous system (CNS). Brain stem encephalitis with pulmonary edema is the severe complication that can lead to death. EV71 replicates in leukocytes, endothelial cells, and dendritic cells resulting in the production of immune and inflammatory mediators that shape innate and acquired immune responses and the complications of disease. Cytokines, as a part of innate immunity, favor the development of antiviral and Th1 immune responses. Cytokines and chemokines play an important role in the pathogenesis EV71 brain stem encephalitis. Both the CNS and the systemic inflammatory responses to infection play important, but distinctly different, roles in the pathogenesis of EV71 pulmonary edema. Administration of intravenous immunoglobulin and milrinone, a phosphodiesterase inhibitor, has been shown to modulate inflammation, to reduce sympathetic overactivity, and to improve survival in patients with EV71 autonomic nervous system dysregulation and pulmonary edema.

Gold nanoparticles (GNPs), which are generally thought to be bio-inert and non-cytotoxic, have become one of the most ideal nanomaterials for medical applications. Once engulfed by phagocytes, the immunological effects of GNPs are still of concern and require detailed investigation. Therefore, this study explored the immunological significance of GNPs on TLR-mediated innate immunity in murine macrophages. GNP causes specific inhibition of TLR9 (CpG oligodeoxynucleotides; CpG-ODNs) signal in macrophages. The impaired CpG-ODN-induced TNF-α production is GNP concentration- and size-dependent in murine Raw264.7 cells: a GNP of 4 nm in size is more potent than a GNP of 11, 19, 35, or 45 nm in size. Consistent with cytokine inhibition, the CpG-ODN-induced phosphorylation of NF-κB and JNK as well as NF-κB activation are suppressed by GNPs. GNPs accumulate in lysosomes after phagocytosis and also increase TLR9-associated lysosomal cathepsin expression and activities, but this is irrelevant to TLR9 inhibition by GNPs in our studies. In addition, GNPs affected TLR9 translocation in response to CpG-ODNs and to phagosomes. Further exploring how GNPs inhibited TLR9 function, we found that GNPs could bind to high-mobility group box-1 (which is involved in the regulation of TLR9 signaling) inside the lysosomes. The current studies demonstrate that size-dependent inhibition of TLR9 function by GNP may be attributed to its binding to high-mobility group box-1.

Dengue virus infection can lead to life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) in patients. Abnormal activation of the coagulation and fibrinolysis system is one of the hallmarks associated with DHF/DSS patients. However, the mechanisms that cause pathology in DHF/DSS patients are still unclear. Because conversion of plasminogen (Plg) to plasmin (Plm) is the first step in the activation of fibrinolysis, Abs against Plg found in DHF/DSS patients may be important. Therefore, to investigate the specificity, function, and possible origin of these Abs, we generated several Plg cross-reactive mAbs from DENV-immunized mice. An IgG mAb, 6H11, which recognizes an epitope associated with a dengue envelope protein, demonstrated a high level of cross-reactivity with Plg. The 6H11 Ab was further characterized with regard to its effect on Plg activation. Using Plm-specific chromogenic substrate S-2251, we found that mAb 6H11 demonstrated serine protease activity and could convert Plg directly to Plm. The serine protease activity of mAb 6H11 was further confirmed using serine protease chromogenic substrate S-2288. In addition, we found several Plg cross-reactive mAbs that could enhance urokinase-induced Plg activation. Lastly, mAb 6H11 could induce Plm activity and increase the level of D-dimer (a fibrin degradation product) in both human and mouse platelet-poor plasma. Taken together, these data suggest DENV-induced Plg cross-reactive Abs may enhance Plg conversion to Plm, which would be expected to contribute to hyperfibrinolysis in DHF/DSS patients.

The enzyme-linked immunosorbent assay (ELISA) is widely used in medical diagnostics. In order to reduce the diagnosis time and to lower the consumption of sample/reagents in an ELISA assay, a suction-type, automatic, pneumatically-driven microfluidic chip has been designed and fabricated in this study. The microfluidic chip integrates a multi-functional micro-transport/mixing unit, for transporting metering and mixing of samples and reagents in order to automatically perform the entire ELISA protocol. A new surface modification has been adopted which allows for a high processing capacity. The detection sensitivity for the dengue virus is found to be 10(1) PFU/ml, which is much better than a conventional ELISA assay (10(3) PFU/ml). The entire assay time is only 30 min, which is much faster than with 96-well microtiter plates (4 h). The consumed sample and reagent volume is only 12 μl, which is less than a conventional assay (100 μl). The development of this microfluidic chip may be promising for other immunosensing applications.

A new magnetic nanoparticle was synthesized in the form of Gd(3+)-chelated Fe(3)O(4)@SiO(2). The Fe(3)O(4) nanoparticle was octahedron-structured, was highly magnetic (∼94 emu/g), and was the core of an encapsulating mesoporous silica shell. DOTA-NHS molecules were anchored to the interior channels of the porous silica to chelate Gd(3+) ions. Because there were Gd(3+) ions within the silica shell, the transverse relaxivity increased 7-fold from 97 s(-1) mM(-1) of Fe(3)O(4) to 681 s(-1) mM(-1) of Gd(3+)-chelated Fe(3)O(4)@SiO(2) nanoparticles with r(2)/r(1) = 486. The large transversal relaxivity of the Gd(3+)-chelated Fe(3)O(4)@SiO(2) nanoparticles had an effective magnetic resonance imaging effect and clearly imaged lymph nodes. Physiological studies of liver, spleen, kidney, and lung tissue in mice infused with these new nanoparticles showed no damage and no cytotoxicity in Kupffer cells, which indicated that Gd(3+)-chelated Fe(3)O(4)@SiO(2) nanoparticles are biocompatible.

Numerous infectious agents may trigger autoimmunity or even result in autoimmune diseases. Several mechanisms have been proposed for pathogen-triggered autoimmunity including molecular mimicry, cryptic antigens, epitope spreading, bystander activation and polyclonal activation. In the case of dengue virus infection which causes serious public health problems, the mechanisms regarding the pathogenesis of dengue hemorrhagic syndrome are not fully resolved. Our previous studies suggest a mechanism of molecular mimicry in which antibodies directed against dengue virus non-structural protein 1 (NS1) cross-react with human platelets and endothelial cells and cause their damage and dysfunction, which may be related to the clinical features of dengue disease. Several cell surface proteins recognized by patient serum samples and anti-NS1 antibodies have been identified. Based on proteomic studies and sequence analysis, the C-terminal region of dengue virus NS1 shows sequence homology with target proteins. In addition, different regions of dengue virus proteins including core, prM, E and NS1 proteins show sequence homology with different coagulatory molecules. As an example, the amino acid sequence 101-106 of E protein (WGNGCG) shows sequence homology with factors XI, X, IX, VII, II (thrombin), plasminogen and tissue plasminogen activator. Furthermore, single chain variable region against NS1 can interfere with fibrin formation, which leads to prolonged thrombin time. We hypothesize that molecular mimicry between dengue virus proteins and coagulatory molecules may induce cross-reactive autoantibodies that can interfere with coagulation activation. A molecular mimicry pathogenesis for dengue disease which involves cross-reactivity of dengue virus with human endothelial cells, platelets and coagulatory molecules is proposed.

Dengue virus (DENV) infection can cause mild dengue fever or severe dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Serum levels of the macrophage migration inhibitory factor (MIF) have been shown to be correlated with severity and mortality in DENV patients, but the pathogenic roles of MIF in DHF/DSS are still unclear. Increase in vascular permeability is an important hallmark of DHF/DSS. In this study, we found that DENV infection of the human hepatoma cell line (Huh 7) induced MIF production. Conditioned medium collected from DENV-infected Huh 7 cells enhanced the permeability of the human endothelial cell line (HMEC-1) which was reduced in the presence of a MIF inhibitor, ISO-1 or medium from DENV-infected MIF knockdown Huh 7 cells. To further identify whether MIF can alter vascular permeability, we cloned and expressed both human and murine recombinant MIF (rMIF) and tested their effects on vascular permeability both in vitro and in vivo. Indirect immunofluorescent staining showed that the tight junction protein ZO-1 of HMEC-1 was disarrayed in the presence of rMIF and partially recovered when cells were treated with ISO-1 or PI3K/MEK-ERK/JNK signaling pathway inhibitors such as Ly294002, U0126, and SP600215. In addition, ZO-1 disarray induced by MIF was also recovered when CD74 or CXCR2/4 expression of HMEC-1 were inhibited. Last but not least, the vascular permeabilities of the peritoneal cavity and dorsal cutaneous capillary were also increased in mice treated with rMIF. Taken together; these results suggest that MIF induced by DENV infection may contribute to the increase of vascular permeability during DHF/DSS. Therapeutic intervention of MIF by its inhibitor or neutralizing antibodies may prevent DENV-induced lethality., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The innate immune system elicits the first wave of immune responses against pathogen infection. Its operational modes are complex and have yet to be defined. Here, we report the identification of an innate immune regulator termed TAPE (TBK1-associated protein in endolysosomes), previously known as CC2D1A/Freud-1/Aki-1, which modulates the TLR3 and TLR4 pathways. We found that TAPE activated the TBK1, NF-κB, and ERK pathways leading to IFN-β and inflammatory cytokine induction. TAPE was shown to colocalize with endosomal marker Rab5 and lysosomal marker LAMP1 in mammalian cells, suggesting that TAPE resided in endolysosomes. Knockdown of TAPE selectively impaired the TLR3 and endocytic TLR4 pathways to IFN-β induction. Furthermore, TAPE interacted and synergized with Trif to activate IFN-β. TAPE knockdown failed to block Trif-mediated IFN-β induction, whereas Trif knockdown impaired the TLR3 and TAPE cooperation on IFN-β induction, suggesting that TAPE acts upstream of Trif. Together, our data demonstrate a central role for TAPE in linking TLR3 and TLR4 to innate immune defenses at an early step.

The endoplasmic reticulum (ER) is essential for lipid biosynthesis, and stress signals in this organelle are thought to alter lipid metabolism. Elucidating the mechanisms that underlie the dysregulation of lipid metabolism in hepatocytes may lead to novel therapeutic approaches for the treatment of lipid accumulation. We first tested the effects of several inhibitors on lipid dysregulation induced by tunicamycin, an ER stress inducer. Triacsin C, an inhibitor of long-chain acyl-CoA synthetase (ACSL) 1, 3, and 4, was the most potent among these inhibitors. We then analyzed the expression of the ACSL family during ER stress. The expression of ACSL3 was induced by ER stress in HuH-7 cells and in mice livers. ACSL3 shRNA, but not ACSL1 shRNA, inhibited the induction of lipid accumulation. GSK-3β inhibitors attenuated ACSL3 expression and the lipid accumulation induced by ER stress in HuH-7 cells. shRNA that target GSK-3β also inhibited the upregulation of ACSL3 and lipid accumulation in HuH-7 and HepG2 cells. The hepatitis B virus mutant large surface protein, which is known to induce ER stress, increased the lipid content of cells. Similarly, Triacsin C, and GSK-3β inhibitors abrogated the lipid dysregulation caused by the hepatitis B virus mutant large surface protein. Altogether, ACSL3 and GSK-3β represent novel therapeutic targets for lipid dysregulation by ER stress., (Copyright © 2010 Wiley-Liss, Inc.)

Liver cirrhosis and hepatocellular carcinomas are two major causes of morbidity and mortality worldwide, and can synergistically interact to expedite the tumor progression. How fibrosis promotes the hepatoma growth remains completely unexplained. Using an in situ murine hepatoma model together with fibrosis induction by thioacetamide (TAA), the hepatoma growth and the immune factors in the fibrotic liver were analyzed. We found that TAA-fibrosis induction enhanced hepatoma cell growth in the liver and increased the mortality of hepatoma-bearing mice. The tumor-infiltrating CD4(+) or CD8(+) T cells are downregulated by fibrosis induction. The Foxp3(+) regulatory T cells (Treg) cells were induced. We conclude that fibrosis induction causes further immunosuppression, in which Treg cells exert a downregulation effect on the antitumor immunity.

Enterovirus (EV) is an RNA virus that has circulated with different serotypes and genotypes worldwide. Enterovirus 71 (EV71) is a major neurotropic virus that causes severe brain stem encephalitis (BE) in infants and young children. The most vulnerable age for fatal infection is 6 to 11 months. This is associated with the coincident decline in maternal antibodies. The current report describes our finding that EV71 can infect human peripheral blood monocytes. We were able to show that EV71 infection is enhanced in the monocytic cell line THP-1 by the presence of subneutralizing concentrations of anti-EV71 antibodies. We also found that antibody-dependent enhancement (ADE) is mediated in part by Fcγ receptors. These observations support the concept that ADE augments the infectivity of EV71 for human monocytes and contributes to the age-dependent pathogenesis of EV71-induced disease. The ADE phenomenon must be considered during the development of an EV71 vaccine.

Helicobacter pylori is a Gram-negative, spiral-shaped bacterium that infects more than 50% of the human population and can cause gastritis, peptic ulcer, or gastric malignancies. It is generally viewed as an extracellular microorganism. In a gentamicin protection assay on AGS or MKN45 cells, H. pylori could invade the epithelial cells and multiply within double-layer vesicles either on the plasma membrane or in the cytoplasm. A 5-fold increase in the number of bacteria was recultured from the infected cells at 12 h, compared with the number of invading cells at 2.5 h postinfection. The autophagic vesicles induced by H. pylori are the sites of replication and also of the degradation of the replicating bacteria after fusion with lysosomes. Many H. pylori bacteria in coccoid form associated with the plasma membrane can be released into culture. Only cell-penetrating antibiotics can enhance the intracellular killing of the replicating bacteria. The multiplication of H. pylori within cells provides a niche for its resistance to antibacterial therapy and has a significant impact on its biological life cycle.

We have reported both T-cell-dependent and -independent hepatitis in immunocompetent and immunodeficiency mice, respectively, after intravenous injection of Con A in mice. The mode of hepatocyte cell death is different: autophagy for T-cell-independent hepatitis in contrast to apoptosis for T-cell-dependent one. In this study, we further demonstrate that liver blood vessels are the first target in both modes. The infused Con A bond to the hepatic vascular endothelial cells and cause its damage with autophagy. Before the elevation of the serum alanine aminotransferase at 6 h post-injection, the plasma leakage and hemorrhage occur at 1-3 h without inflammation. Con A induces autophagy of endothelial cells and hemorrhage that is enhanced by IFN-gamma. Using the endothelial cell line HMEC-1, a dose- and time-dependent cell death with autophagic LC3-II (microtubule-associated protein light chain 3) conversion was induced by Con A and was enhanced by IFN-gamma. In conclusion, Con A induced autophagy on hepatic endothelial cells; the damage of liver blood vessel occurs before the induction of T-cell-dependent hepatitis via apoptosis or T-cell-independent hepatitis via autophagy.

Aim: Hepatitis B virus (HBV) infection is highly correlated with hepatocellular carcinoma. Previous studies have reported that expression of hepatitis B virus pre-S2 mutant surface antigen is related to hepatoma development. An aberrant carbohydrate metabolism is a hallmark of malignant transformation., Methods: We performed yeast two-hybrid screening with HBV pre-S2-del large surface protein (pre-S2Delta) by using human liver cDNA library, and identified the acid alpha-glucosidase (acid alpha-glucosidase) as the novel cellular interacting protein of pre-S2Delta. The association of pre-S2Delta with the acid alpha-glucosidase was confirmed by confocal immunofluorescence and co-immunoprecipitation assay. Further, the acid alpha-glucosidase activity and glycogen content were analyzed in ML-1 cells expressing pre-S2Delta., Results: The interaction between HBV large surface protein and acid alpha-glucosidase was demonstrated with co-immunoprecipitation in vitro and in vivo, and the binding was mediated through c-terminal region 889-952 amino acid of acid alpha-glucosidase. On the other hand, HBV large surface protein interacted with acid alpha-glucosidase through N-terminal region 1-157 amino acid of HBV large surface protein. Expression of HBV large surface protein enhanced acid alpha-glucosidase activity and resulted in decrease of cellular glycogen., Conclusion: Our result demonstrates that HBV large surface protein interacts with acid alpha-glucosidase which plays an important role in glycogen balance. Together, these data suggest a novel pathway by which HBV large surface protein affects carbohydrate metabolism.

Background: Helicobacter pylori, a human pathogen associated with chronic gastritis, peptic ulcer and gastric malignancies, is generally viewed as an extracellular microorganism. Here, we show that H. pylori replicates in murine bone marrow derived-dendritic cells (BMDCs) within autophagosomes., Methodology/principal Findings: A 10-fold increase of CFU is found between 2 h and 6 h p.i. in H. pylori-infected BMDCs. Autophagy is induced around the bacterium and participates at late time points of infection for the clearance of intracellular H. pylori. As a consequence of infection, LC3, LAMP1 and MHC class II molecules are retained within the H. pylori-containing vacuoles and export of MHC class II molecules to cell surface is blocked. However, formalin-fixed H. pylori still maintain this inhibitory activity in BMDC derived from wild type mice, but not in from either TLR4 or TLR2-deficient mice, suggesting the involvement of H. pylori-LPS in this process. TNF-alpha, IL-6 and IL-10 expression was also modulated upon infection showing a TLR2-specific dependent IL-10 secretion. No IL-12 was detected favoring the hypothesis of a down modulation of DC functions during H. pylori infection. Furthermore, antigen-specific T cells proliferation was also impaired upon infection., Conclusions/significance: H. pylori can infect and replicate in BMDCs and thereby affects DC-mediated immune responses. The implication of this new finding is discussed for the biological life cycle of H. pylori in the host.

Taiwan's dengue outbreaks have a unique type of transmission: starting by import from abroad in early summer, spreading out locally, and ending in the winter. This pattern repeats every year. Most of the dengue patients are adults, with dengue fever peaking in the 50-54 year age range, and dengue hemorrhagic fever in the 60-64 year age range. Two patterns of dengue infection were found: DENV-2 in 2002 with 74% of secondary infection in contrast to non-DENV-2 (DENV-1 or DENV-3) in 2004-2007 with approximately 70% of primary infection. Secondary dengue virus infection increases disease morbidity, but not mortality in adults. The active serological surveillance shows two-thirds of the dengue-infected adults are symptomatic post infection. The Taiwanese experience of adult dengue should be valuable for countries or areas where, although dengue is not endemic, the Aedes aegypti vector exists and dengue virus can be introduced by travelers.

Cells infected with dengue virus release a high proportion of immature prM-containing virions. In accordance, substantial levels of prM antibodies are found in sera of infected humans. Furthermore, it has been recently described that the rates of prM antibody responses are significantly higher in patients with secondary infection compared to those with primary infection. This suggests that immature dengue virus may play a role in disease pathogenesis. Interestingly, however, numerous functional studies have revealed that immature particles lack the ability to infect cells. In this report, we show that fully immature dengue particles become highly infectious upon interaction with prM antibodies. We demonstrate that prM antibodies facilitate efficient binding and cell entry of immature particles into Fc-receptor-expressing cells. In addition, enzymatic activity of furin is critical to render the internalized immature virus infectious. Together, these data suggest that during a secondary infection or primary infection of infants born to dengue-immune mothers, immature particles have the potential to be highly infectious and hence may contribute to the development of severe disease.

Hemorrhagic syndrome is a hallmark of severe dengue diseases. We previously suggested a mechanism of molecular mimicry in which antibodies against dengue virus (DV) nonstructural protein 1 (NS1) cross-react with platelets. In the present study, we demonstrate that protein disulfide isomerase (PDI) on the platelet surface is recognized by anti-DV NS1 antibodies. Anti-DV NS1 obtained from hyperimmunized mouse sera inhibited PDI activity and platelet aggregation, and both inhibitory effects were prevented when anti-DV NS1 antibodies were preabsorbed with PDI. Anti-PDI antibodies bound to a peptide consisting of amino acid residues 311-330 (P311-330) of NS1. This peptide was a predicted epitope analyzed by homologous sequence alignments between DV NS1 and PDI. The platelet binding activities of anti-PDI and anti-DV NS1 antibodies were both reduced by P311-330 preabsorption. Similar to the findings using anti-DV NS1, antibodies against P311-330 bound to PDI and platelets, followed by inhibition of PDI activity and platelet aggregation. Furthermore, the cross-reactivity of dengue hemorrhagic fever patient sera with platelets was reduced when patient sera were preabsorbed with PDI or P311-330. Dengue hemorrhagic fever patient sera also inhibited platelet aggregation, while PDI or P311-330 reduced this inhibitory effect. In conclusion, anti-DV NS1 antibodies cross-react with PDI on platelet surface causing inhibition of platelet aggregation, which may provide implications in dengue disease pathogenesis.

The mechanisms underlying dengue hemorrhagic disease are incompletely understood. We previously showed that anti-dengue virus (DV) nonstructural protein 1 (NS1) Abs cross-react with human platelets and inhibit platelet aggregation. Based on sequence homology alignment, the cross-reactive epitopes reside in the C-terminal region of DV NS1. In this study, we compared the effects of Abs against full-length DV NS1 and NS1 lacking the C-terminal aa 271 to 352 (designated DeltaC NS1). Anti-DeltaC NS1 Abs exhibited lower platelet binding activity than that of anti-full-length NS1. Anti-full-length NS1 but not anti-DeltaC NS1 Abs inhibited platelet aggregation, which was shown to involve integrin alpha(IIb)beta(3) inactivation. We found that the bleeding time in full-length NS1-hyperimmunized mice was longer than that in the normal control mice. By contrast, DeltaC NS1-hyperimmunized mice showed a bleeding time similar to that of normal control mice. Passively administered anti-DV NS1, but not anti-DeltaC NS1, Ab level decreased markedly in serum and this decrease was correlated with Ab binding to platelets. A transient platelet loss in the circulation was observed after anti-DV NS1, but not anti-DeltaC NS1, Ab administration. In summary, platelet dysfunction and bleeding tendency are induced by anti-full-length DV NS1 but not by anti-DeltaC NS1 Abs. These findings may be important not only for understanding dengue hemorrhagic disease pathogenesis but also for dengue vaccine development.

Autophagosome formation is induced by Helicobacter pylori infection and these autophagic vesicles are adopted for replication of H. pylori and subsequent eradication of the invading H. pylori in macrophages. Some Taiwanese clinical isolates of H. pylori can replicate in certain macrophage cell lines. After entry, there was a 5-10 fold increment of recultivable H. pylori from the infected permissible cells at 12 h post infection. The dividing H. pylori are observed to reside in double-layered autophagosomes. Therefore, H. pylori can be considered as a kind of intracellular microorganism. The autophagy induction by H. pylori is not only found in macrophages, but also in dendritic cells and gastric epithelial cells. This new finding has several implications for the life cycle of H. pylori in the host. The bacterium's residence inside infected cells will increase its resistance to antimicrobial treatment, avoid neutralization by anti-H. pylori antibodies, impair antigen presentation, and alter the cellular immune response. The replication of H. pylori in autophagic vesicles, and the consequences of this provide an important hint as to why this microorganism causes such a broad spectrum of diseases.

Objectives: Dengue virus (DV) infections can cause severe life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). However, the mechanism to cause hemorrhage in DV infections remains poorly understood. Thrombomodulin (TM), expressed on the surface of endothelial cells and monocytes, is very important in regulation of coagulation and inflammation. Therefore, the effect of DV on the TM expression was studied in vitro using both endothelial cells and monocytes., Methods and Results: The expression of TM in human endothelial cell line, HMEC-1, monocytic cell line THP-1 and peripheral blood mononuclear cells derived from human blood was increased after DV infection, UV-inactivated DV or recombinant DV envelop protein domain III stimulation as demonstrated by flow cytometry and immunofluorescent staining. Western blot analysis further confirmed only DV but not enterovirus 71 infection of HMEC-1 cells increased TM protein expression. In addition, RT-PCR analysis showed the increase of TM mRNA as well as other protein C activation-related molecules in DV stimulated HMEC-1 in a dose-dependent manner., Conclusion: These results suggest that DV stimulation of human endothelial cells and monocytes can increase the expression of TM, which may contribute to the anticoagulant properties of cells during DV infection.

Helicobacter pylori has developed several mechanisms to evade the intracellular killing after phagocytosis. In this study, we reported that some Taiwanese clinical isolated H. pylori can multiply in human monocytic cells, such as THP-1 or U937 cells, but not in murine macrophage Raw264.7 cells. After internalization, there was a 5- to 10-fold increment of re-cultivable H. pylori from the infected THP-1 cells at 12 hrs post infection. The dividing H. pylori was found in a double-layer vesicle, which is characteristic of autophagosome. The formation of autophagosomes is associated with the multiplication of H. pylori in THP-1 cells. Its modulation with rapamycin or 3-MA affects the level of H. pylori replication. Furthermore, the VacA or CagA mutants of H. pylori have lower levels of multiplication in macrophages. We conclude that H. pylori infection induces autophagosome formation, and these autophagic vesicles were adapted for the multiplication of H. pylori in the host.

The preparation of nanoparticles is essential in the application of many nanotechnologies and various preparation methods have been explored in the previous decades. Among them, iron oxide nanoparticles have been widely investigated in applications ranging from bio-imaging to bio-sensing due to their unique magnetic properties. Recently, microfluidic systems have been utilized for synthesis of nanoparticles, which have the advantages of automation, well-controlled reactions, and a high particle uniformity. In this study, a new microfluidic system capable of mixing, transporting and reacting was developed for the synthesis of iron oxide nanoparticles. It allowed for a rapid and efficient approach to accelerate and automate the synthesis of the iron oxide nanoparticles as compared with traditional methods. The microfluidic system uses micro-electro-mechanical-system technologies to integrate a new double-loop micromixer, two micropumps, and a microvalve on a single chip. When compared with large-scale synthesis systems with commonly-observed particle aggregation issues, successful synthesis of dispersed and uniform iron oxide nanoparticles has been observed within a shorter period of time (15 min). It was found that the size distribution of these iron oxide nanoparticles is superior to that of the large-scale systems without requiring any extra additives or heating. The size distribution had a variation of 16%. This is much lower than a comparable large-scale system (34%). The development of this microfluidic system is promising for the synthesis of nanoparticles for many future biomedical applications.

Liver cancer is the predominant cause of cancer mortality in males of Southern China and Taiwan. The current therapy is not satisfactory, and more effective treatments are needed. In the search for new therapies for liver tumor, we found that Concanavalin A (Con A), a lectin from Jack bean seeds, can have a potent anti-hepatoma effect. Con A after binding to the mannose moiety on the cell membrane glycoprotein is internalized preferentially to the mitochondria. An autophagy is triggered which leads to cell death. Con A as a T cell mitogen subsequently activates the immune response in the liver and results in the eradication of the tumor in a murine in situ hepatoma model. The liver tumor nodule formation is inhibited by the CD8+ T cells, and a tumor antigen-specific immune memory is established during the hepatic inflammation. The dual properties (autophagic cytotoxicity and immunomodulation) via the specific carbohydrate binding let Con A exert a potent anti-hepatoma therapeutic effect. The novel mechanism of the Con A anti-hepatoma effect is discussed. The prototype of Con with an anti-hepatoma activity gives support to the search for other natural lectins as anti-cancer compounds.

Background: Brainstem encephalitis (BE) is a serious neurological complication of enterovirus 71 (EV71) infection. The present study was designed to determine the characteristics of the chemokine response in the blood and cerebrospinal fluid (CSF) of patients with EV71-associated BE., Methods: Thirty-one patients with BE were studied. They consisted of 12 with uncomplicated BE, 9 with autonomic nervous system (ANS) dysregulation, and 10 with pulmonary edema (PE); 13 healthy control subjects were also studied. Plasma and CSF concentrations of various chemokines were determined by a particle-based flow cytometry immunoassay., Results: Plasma levels of interferon (IFN)-gamma-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, monokine induced by IFN-gamma (MIG), and interleukin (IL)-8 were significantly higher in patients with PE than in those with uncomplicated BE. CSF levels of MIG were significantly higher in patients with PE than in those with uncomplicated BE and ANS dysregulation. The ratios of mean CSF to plasma levels for MCP-1 and IL-8 were highest in patients with uncomplicated BE and tended to fall with increasing severity of the disease., Conclusions: Overexpression of the chemokine cascade in the central nervous system compartment appears to play an important role in the elicitation of the immune response to EV71. The chemokine CSF to plasma ratios suggest that IL-8, IP-10, MCP-1, and possibly MIG-but not RANTES-are synthesized in the brain in response to encephalitis.

Clinical manifestations of severe dengue diseases include thrombocytopenia, vascular leakage, and liver damage. Evidence shows that hepatic injury is involved in the pathogenesis of dengue infection; however, the mechanisms are not fully resolved. Our previous in vitro studies suggested a mechanism of molecular mimicry in which antibodies directed against dengue virus (DV) nonstructural protein 1 (NS1) cross-reacted with endothelial cells and caused inflammatory activation and apoptosis. In this study, the pathogenic effects of anti-DV NS1 antibodies were further examined in a murine model. We found, in liver sections, that anti-DV NS1 antibodies bound to naive mouse vessel endothelium and the binding activity was inhibited by preabsorption of antibodies with DV NS1. Active immunization with DV NS1 resulted in antibody deposition to liver vessel endothelium, and also apoptotic cell death of liver endothelium. Liver tissue damage was observed in DV NS1-immunized mice by histological examination. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased in mice either actively immunized with DV NS1 protein or passively immunized with antibodies obtained from DV NS1-immunized mice. Furthermore, histological examination revealed mononuclear phagocyte infiltration and cell apoptosis in mice passively immunized with antibodies obtained from mice immunized with DV NS1. Increased AST and ALT levels were observed in mice passively immunized with purified immunoglobulin G (IgG) from dengue patients compared with normal control human IgG-immunized mice. The increased AST and ALT levels were inhibited when dengue patient serum IgG was preabsorbed with DV NS1. In conclusion, active immunization with DV NS1 protein causes immune-mediated liver injury in mice. Passive immunization provides additional evidence that anti-DV NS1 antibodies may play a role in liver damage, which is a pathologic manifestation in dengue virus disease.

Dengue haemorrhagic fever and dengue shock syndrome, the most severe responses to dengue virus (DV) infection, are characterized by plasma leakage (due to increased vascular permeability) and low platelet counts. CLEC5A (C-type lectin domain family 5, member A; also known as myeloid DAP12-associating lectin (MDL-1)) contains a C-type lectin-like fold similar to the natural-killer T-cell C-type lectin domains and associates with a 12-kDa DNAX-activating protein (DAP12) on myeloid cells. Here we show that CLEC5A interacts with the dengue virion directly and thereby brings about DAP12 phosphorylation. The CLEC5A-DV interaction does not result in viral entry but stimulates the release of proinflammatory cytokines. Blockade of CLEC5A-DV interaction suppresses the secretion of proinflammatory cytokines without affecting the release of interferon-alpha, supporting the notion that CLEC5A acts as a signalling receptor for proinflammatory cytokine release. Moreover, anti-CLEC5A monoclonal antibodies inhibit DV-induced plasma leakage, as well as subcutaneous and vital-organ haemorrhaging, and reduce the mortality of DV infection by about 50% in STAT1-deficient mice. Our observation that blockade of CLEC5A-mediated signalling attenuates the production of proinflammatory cytokines by macrophages infected with DV (either alone or complexed with an enhancing antibody) offers a promising strategy for alleviating tissue damage and increasing the survival of patients suffering from dengue haemorrhagic fever and dengue shock syndrome, and possibly even other virus-induced inflammatory diseases.

Unlabelled: Concanavalin A (ConA), a lectin with mannose specificity that can induce acute hepatic inflammation, was tested for its therapeutic effect against hepatoma. ConA is cytotoxic or inhibitory to hepatoma cells, which is mediated by the autophagic pathway through mitochondria. Once it was bound to cell membrane glycoproteins, the ConA was internalized and preferentially localized onto the mitochondria. The mitochondria membrane permeability changed, and an autophagic pathway including LC3-II generation, double-layer vesicle, BNIP3 induction, and acidic vesicular organelle formation was induced. Either 3-MA or siRNA for BNIP3 and LC3, but neither beclin-1 nor ATG 5, partially inhibited the ConA-induced cell death. In addition to the autophagy induction, ConA is known to be a T cell mitogen. Using an in situ hepatoma model, ConA can exert an anti-hepatoma therapeutic effect, inhibiting tumor nodule formation in the liver and prolonging survival., Conclusion: ConA can be considered as an anti-hepatoma agent therapeutically because of its autophagic induction and immunomodulating activity. This dual function of ConA provides a novel mechanism for the biological effect of lectin.

Background: Several epidemics of enterovirus 71 (EV71) infections occurred in Taiwan since 1998., Objectives: We performed the study to determine the changes in cytokine profiles associated with administration of intravenous immunoglobulin (IVIG) in patients with EV71-associated brainstem encephalitis complicated by autonomic nervous system (ANS) dysfunction and pulmonary edema., Study Design: Plasma cytokine concentrations (IL-1beta, IL-6, IL-8, IFN-gamma, TNF-alpha, IL-2, IL-4, IL-5, IL-10, and IL-13) were monitored on admission and within 12-24h after administration of IVIG in a cohort of children (n=22) with virologically confirmed EV71 infection, from March 2000 through April 2004., Result: Plasma levels of IFN-gamma, IL-6, IL-8, IL-10, and IL-13 levels significantly decreased in patients with pulmonary edema after administration of IVIG, P<0.05. Plasma levels of IL-6 and IL-8 were significantly decreased in patients with ANS dysregulation after administration of IVIG, P<0.05. Administration of IVIG was not associated with significant changes in plasma concentration of IL-1beta, IL-2, IL-4, IL-5 IL-10, IL-13 and TNF-alpha in patients with ANS dysregulation., Conclusions: These findings suggest that IVIG might be considered to have a therapeutic role in EV71-associated brainstem encephalitis. A clinical trial is needed to support this hypothesis.

T-cell depletion is found in enterovirus 71 (EV71)-infected patients with pulmonary edema. However, the mechanism that causes T-cell depletion is unclear. To address this question, the effects of EV71 infection on the cell viability of human Jurkat T cells were studied. Viable viruses were recovered from both the culture supernatant and the cell lysate of Jurkat cells after EV71 infection. Results from reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay confirmed further the presence of EV71 negative-strand RNA and antigen, respectively, in EV71-infected Jurkat cells. The viability of the Jurkat cells decreased after 48 hr of EV71 infection. Both terminal transferase end labeling (TUNEL) and DNA fragmentation assays demonstrated that the apoptosis of EV71-infected Jurkat cells had increased. In addition, the expression of Fas ligand (FasL) in EV71-infected Jurkat cells increased at both mRNA and surface expression levels. Taken together, these results confirmed that EV71 infected T cells and induced FasL expression, which may contribute to T-cell apoptosis during EV71 infection.

Volume replacement was studied prospectively in 208 infants with dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The mean volume of intravenous fluid used was 110.4 mL/kg administered over a mean period of 25.8 hours. The mean volumes of intravenous fluid replacement in infants with DSS was significantly higher than in those with non-shock DHF (129.8 mL/kg versus 102.1 mL/kg; P = 0.001). Patients with DSS had significantly higher proportional requirements for dextran and blood transfusions than non-shock infants. Recurrent shock, prolonged shock, and acute respiratory failure were recorded in 8, 6, and 13 patients, respectively. Four patients with DSS died of severe complications. Intravenous fluid replacement with special care to avoid fluid overload requires careful attention to established indications for use of colloidal solutions and blood transfusions. To improve case fatality rates, special efforts need to be directed to infants with DHF/DSS accompanied by severe complications.

Substantial evidence suggests that inflammatory cytokines, immune cells, and angiogenesis are important for endometriosis. In this study, we investigated the role of the sequential events in the development of endometriosis in a mouse model. Uterine tissue was transplanted into the peritoneum of ovariectomized mice and then supplemented with estrogen or vehicle. On different days after transplantation, cell proliferation, angiogenesis, and infiltrated immune cells in ectopic tissue were examined using immunochemical staining. Many disintegrated blood vessels but no bromodeoxyuridine-positive cells in ectopic tissue were observed in the estrogen-treated group on posttransplantation d 1 and 2. On d 4-7, bromodeoxyuridine-positive cells were detected in the blood vessels of ectopic tissue, indicating that angiogenesis was initiated in this stage. Angiogenesis also occurred in ectopic tissue in the vehicle-treated group. Profound infiltration of neutrophils in ectopic tissue occurred on d 1-4, when the number of neutrophils and levels of macrophage inflammatory protein (MIP)-1alpha and MIP-2 chemokines in peritoneal fluids also reached their peak. Peritoneal macrophage numbers did not change, but secretions of TNFalpha, IL-6, MIP-1alpha, and MIP-2 from macrophages isolated on d 2 were higher than on d 0. In vitro studies showed that peritoneal neutrophils and macrophages secreted vascular endothelial growth factor, which was up-regulated by TNFalpha and IL-6. Our results suggest that neutrophils and macrophages may promote angiogenesis in the early stage of endometriosis and that chemokines and cytokines amplify the angiogenic signal for the growth of endometriotic tissue.

Aim: Toll-like receptor 4 (TLR4) has been shown to be important for bacterial infection, especially to lipopolysaccharide signaling. Its possible role in HBV infection is studied in the present study., Materials and Methods: pHBV3.6 plasmid, containing full-length HBV genome was used in the murine model of acute HBV expression by hydrodynamics in vivo transfection. TLR4 normal or mutant mouse strain was compared to investigate the possible role of TLR4 in acute HBV expression., Results: After pHBV3.6 injection, the infiltrating leukocytes expressed TLR4 were observed nearby the HBsAg-expressing hepatocytes. The HBV antigenemia as well as the replication and transcription were higher in TLR4-mutant C3H/HeJ mice than in normal C3H/HeN mice. The HBV-specific immune responses were impaired in the liver or spleen of the C3H/HeJ mice. Their inducible nitric oxide synthase (iNOS) expression on the hepatic infiltrating cells was also impaired. When adoptively transferring splenocytes from C3H/HeN mice to C3H/HeJ mice, the HBV replication was inhibited to the level as that of C3H/HeN., Conclusion: These results suggest that TLR4 plays an anti-HBV role in vivo through the induction of iNOS expression and HBV-specific immune responses after HBV expression.

The association between sex, nutritional status, and the severity of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), and immune status was investigated in 245 Vietnamese infants with predominantly primary infections with dengue virus. Male and female infants were at equal risk of developing DHF/DSS. However, infants of low height and weight for age were under-represented among DHF/DSS cases compared with 533 healthy baby clinic infant controls. Acute illness phase blood levels of selected cytokines (interferon-gamma and tumor necrosis factor-alpha) and serum levels of antibodies to dengue virus were elevated in the same range in male and female infants with DHF/DSS, as well as in infants with and without malnutrition.

Hand, foot, and mouth disease and herpangina are the major clinical manifestations of enterovirus 71 (EV71) infections. Brain-stem encephalitis and pulmonary edema are severe complications that can lead to death. This study was designed to evaluate the potential therapeutic effect of milrinone, a phosphodiesterase (PDE) inhibitor, in the treatment of patients with EV71-induced pulmonary edema. We conducted a historically controlled trial of 24 children with severe EV71-induced pulmonary edema from April 1998-June 2003 in southern Taiwan. Patients were divided into groups treated before and after the introduction of milrinone therapy. Etiological diagnosis was established by viral cultures and confirmed by specific immunofluorescence and neutralization tests. All 24 patients were below 5 years of age. The mortality was lower in the milrinone-treated vs. nontreated group (36.4% vs. 92.3%, P=0.005). Sympathetic tachycardia was decreased in patients treated with milrinone compared to controls (144 +/- 17/min vs. 206 +/- 26/min, P=0.004). A marked decrease in IL-13 (77 +/- 9 pg/ml vs. 162 +/- 88 pg/ml, P=0.001) was observed in milrinone-treated patients compared to controls. There was a significant reduction in white blood cell (10,838 +/- 4,537/mm3 vs. 19,475 +/- 7,798/mm3, P=0.009) and platelet (257 +/- 45 x 10(3)/mm3 vs. 400 +/- 87 x 10(3)/mm3, P=0.001) counts in milrinone-treated patients compared to controls. These results were associated with improvement in sympathetic regulation and decrease in IL-13 production. Milrinone therapy may provide a useful therapeutic approach for this highly lethal disorder.

Fourteen cytokines or chemokines were analyzed on 88 RT-PCR-confirmed severe acute respiratory syndrome (SARS) patients. IFN-gamma, IL-18, TGF-beta, IL-6, IP-10, MCP-1, MIG, and IL-8, but not of TNF-alpha, IL-2, IL-4, IL-10, IL-13, or TNFRI, were highly elevated in the acute phase sera of Taiwan SARS patients. IFN-gamma was significantly higher in the Ab(+) group than in the Ab(-) group. IFN-gamma, IL-18, MCP-1, MIG, and IP-10 were already elevated at early days post fever onset. Furthermore, levels of IL-18, IP-10, MIG, and MCP-1 were significantly higher in the death group than in the survival group. For the survival group, IFN-gamma and MCP-1 were inversely associated with circulating lymphocytes count and monocytes count, but positively associated with circulating neutrophils count. It is concluded that an interferon-gamma-related cytokine storm was induced post SARS coronavirus infection, and this cytokine storm might be involved in the immunopathological damage in SARS patients.

Expression of mutant proteins or viral infection may interfere with proper protein folding activity in the endoplasmic reticulum (ER). Several pathways that maintain cellular homeostasis were activated in response to these ER disturbances. Here we investigated which of these ER stress-activated pathways induce COX-2 and potentially oncogenesis. Tunicamycin and brefeldin A, two ER stress inducers, increased the expression of COX-2 in ML-1 or MCF-7 cells. Nuclear translocation of NF-kappaB and activation of pp38 MAPK were observed during ER stress. IkappaBalpha kinase inhibitor Bay 11-7082 or IkappaBalpha kinase dominant negative mutant significantly inhibited the induction of COX-2. pp38 MAPK inhibitor SB203580 or eIF2alpha phosphorylation inhibitor 2-aminopurine attenuated the nuclear NF-kappaB DNA binding activity and COX-2 induction. Expression of mutant hepatitis B virus (HBV) large surface proteins, inducers of ER stress, enhanced the expression of COX-2 in ML-1 and HuH-7 cells. Transgenic mice showed higher expression of COX-2 protein in liver and kidney tissue expressing mutant HBV large surface protein in vivo. Similarly, increased expression of COX-2 mRNA was observed in human hepatocellular carcinoma tissue expressing mutant HBV large surface proteins. In ML-1 cells expressing mutant HBV large surface protein, anchorage-independent growth was enhanced, and the enhancement was abolished by the addition of specific COX-2 inhibitors. Thus, ER stress due either to expression of viral surface proteins or drugs can stimulate the expression of COX-2 through the NF-kappaB and pp38 kinase pathways. Our results provide important insights into cellular carcinogenesis associated with latent endoplasmic reticulum stress.

A prospective study of clinical and cytokine profiles of 107 infants with dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS) was conducted. Fever, petechiae on the skin, and hepatomegaly were the most common clinical findings associated with DHF/DSS in infants. DSS occurred in 20.5% of the patients. Hemoconcentration and thrombocytopenia were observed in 91.5% and 92.5% of the patients, respectively. Serologic testing revealed that almost all of the patients (95.3%) had primary dengue virus infections. These data demonstrate that clinical and laboratory findings of DHF/DSS in infants are compatible with the World Health Organization's clinical diagnostic criteria for pediatric DHF. The present study is the first to report evidence of production of cytokines in infants with DHF/DSS and to describe the difference between the cytokine profile of infants with primary dengue virus infections and children with secondary infections. Overproduction of both proinflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) and anti-inflammatory cytokines (interleukin-10 and -6) may play a role in the pathogenesis of DHF/DSS in infants.

Background/aims: For subjects with isolated antibody to hepatitis B core antigen, vaccination can help discriminate various diagnostic possibilities. The aim of this study was to evaluate the clinical significance of isolated antibody to hepatitis B core antigen., Methodology: A total of 1403 hospital personnel were screened for hepatitis B surface antigen, antibody to hepatitis B surface antigen and antibody to hepatitis B core antigen by radioimmunoassay. Thirty subjects were confirmed to have isolated antibody to hepatitis B core antigen, and 278 subjects lacked all hepatitis B virus markers. Twenty-five of the 30 subjects (group I) and 136 of the 278 subjects (group II) were vaccinated by hepatitis B vaccine at months 0, 1, 6; followed by checking antibody to hepatitis B surface antigen in geometric mean titres at months 1, 2, and 7., Results: The geometric mean titres of antibody to hepatitis B surface antigen were higher in group I than in group II at month 1 (9.1 +/- 8.6 vs. 3.2 +/- 6.0, p < 0.05), and were lower in group I than group II at month 7 (267.2 +/- 17.2 vs. 2315.3 +/- 5.1, p < 0.05). Furthermore, primary response was higher in group II than group I (73.3% vs. 35.7%, p < 0.05), but anamnestic response and non-response were higher in group I than group II (50% vs 26.7%, p = 0.116 with a trend; 14.3% vs. 0%, p < 0.01, respectively)., Conclusions: For subjects with isolated antibody to hepatitis B core antigen, a strategy concerning sequential vaccination followed by checking antibody to hepatitis B surface antigen might be adopted to avoid two extra vaccine dosages and ineffective vaccination.

Taiwan experienced several epidemics of enterovirus 71 (EV71) infections, which were associated with brainstem encephalitis (BE) and pulmonary edema (PE). To elucidate the role of immune mechanisms in the pathogenesis of BE caused by EV71 and its fatal complication, PE, we analyzed the laboratory findings, cytokine, and immunophenotypes of 73 EV71-infected patients with BE. Patients were stratified by disease: PE (n=14), autonomic nervous system (ANS) dysregulation (n=25), and isolated BE (n=34). The mortality rate for PE was 64.3%. Leukocytosis and thrombocytosis were significantly more frequent among patients with PE. A significant elevation of plasma interleukin (IL)-10, IL-13, and interferon (IFN)-gamma levels observed in patients with PE. Patients with PE also had lower circulating CD4(+) T cells, CD8(+) T cells, and natural killer (NK) cells. An extensive peripheral and central nervous system inflammatory response with abnormal IL-10, IL-13, and IFN-gamma cytokine production and lymphocyte depletion appears to be responsible for the pathogenesis of EV71-associated PE.