Secretomic Analysis of Host-Pathogen Interactions Reveals That Elongation Factor-Tu Is a Potential Adherence Factor of Helicobacter pylori during Pathogenesis.

The secreted proteins of bacteria are usually accompanied by virulence factors, which can cause inflammation and damage host cells. Identifying the secretomes arising from the interactions of bacteria and host cells could therefore increase understanding of the mechanisms during initial pathogenesis. The present study used a host-pathogen coculture system of Helicobacter pylori and monocytes (THP-1 cells) to investigate the secreted proteins associated with initial H. pylori pathogenesis. The secreted proteins from the conditioned media from H. pylori, THP-1 cells, and the coculture were collected and analyzed using SDS-PAGE and LC-MS/MS. Results indicated the presence of 15 overexpressed bands in the coculture. Thirty-one proteins were identified-11 were derived from THP-1 cells and 20 were derived from H. pylori. A potential adherence factor from H. pylori, elongation factor-Tu (EF-Tu), was selected for investigation of its biological function. Results from confocal microscopic and flow cytometric analyses indicated the contribution of EF-Tu to the binding ability of H. pylori in THP-1. The data demonstrated that fluorescence of EF-Tu on THP-1 cells increased after the addition of the H. pylori-conditioned medium. This study reports a novel secretory adherence factor in H. pylori, EF-Tu, and further elucidates mechanisms of H. pylori adaptation for host-pathogen interaction during pathogenesis.

Enterovirus 71 virion-associated galectin-1 facilitates viral replication and stability.

Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal brain stem encephalitis complicated with pulmonary edema. Several severe EV71 endemics have occurred in Asia-Pacific region, including Taiwan, and have become a serious threat to children's health. EV71 infection is initiated by the attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between viruses and cell surface receptors, soluble factors may also influence the binding of EV71 to host cells. Galectin-1 has been reported to participate in several virus infections, but is not addressed in EV71. In this study, we found that the serum levels of galectin-1 in EV71-infected children were higher than those in non-infected people. In EV71 infected cells, galectin-1 was found to be associated with the EV71 VP1 and VP3 via carbohydrate residues and subsequently released and bound to another cell surface along with the virus. EV71 propagated from galectin-1 knockdown SK-N-SH cells exhibited lower infectivity in cultured cells and less pathogenicity in mice than the virus propagated from parental cells. In addition, this galectin-1-free EV71 virus was sensitive to high temperature and lost its viability after long-term storage, which could be restored following supplement of recombinant galectin-1. Taken together, our findings uncover a new role of galectin-1 in facilitating EV71 virus infection.

Dengue virus infection induces autophagy: an in vivo study.

BACKGROUND: We and others have reported that autophagy is induced by dengue viruses (DVs) in various cell lines, and that it plays a supportive role in DV replication. This study intended to clarify whether DV infection could induce autophagy in vivo. Furthermore, the effect of DV induced autophagy on viral replication and DV-related pathogenesis was investigated. RESULTS AND CONCLUSIONS: The physiopathological parameters were evaluated after DV2 was intracranially injected into 6-day-old ICR suckling mice. Autophagy-related markers were monitored by immunohistochemical/immunofluorescent staining and Western blotting. Double-membrane autophagic vesicles were investigated by transmission-electron-microscopy. DV non-structural-protein-1 (NS1) expression (indicating DV infection) was detected in the cerebrum, medulla and midbrain of the infected mice. In these infected tissues, increased LC3 puncta formation, LC3-II expression, double-membrane autophagosome-like vesicles (autophagosome), amphisome, and decreased p62 accumulation were observed, indicating that DV2 induces the autophagic progression in vivo. Amphisome formation was demonstrated by colocalization of DV2-NS1 protein or LC3 puncta and mannose-6-phosphate receptor (MPR, endosome marker) in DV2-infected brain tissues. We further manipulated DV-induced autophagy by the inducer rapamycin and the inhibitor 3-methyladenine (3MA), which accordingly promoted or suppressed the disease symptoms and virus load in the brain of the infected mice.We demonstrated that DV2 infection of the suckling mice induces autophagy, which plays a promoting role in DV replication and pathogenesis.

A microfluidic immunomagnetic bead-based system for the rapid detection of influenza infections: from purified virus particles to clinical specimens.

Seasonal and novel influenza infections have the potential to cause worldwide pandemics. In order to properly treat infected patients and to limit its spread, a rapid, accurate and automatic influenza diagnostic tool needs to be developed. This study therefore presents a new integrated microfluidic system for the rapid detection of influenza infections. It integrated a suction-type, pneumatic-driven microfluidic control module, a magnetic bead-based fluorescent immunoassay (FIA) and an end-point optical detection module. This new system can successfully distinguish between influenza A and B using a single chip test within 15 min automatically, which is faster than existing devices. By utilizing the micromixers to thoroughly wash out the sputum-like mucus, this microfluidic system could be used for the diagnosis of clinical specimens and reduced the required sample volume to 40 µL. Furthermore, the results of diagnostic assays from 86 patient specimens have demonstrated that this system has 84.8 % sensitivity and 75.0 % specificity. This developed system may provide a powerful platform for the fast screening of influenza infections.

Autoimmunity in dengue pathogenesis.

Dengue is one of the most important vector-borne viral diseases. With climate change and the convenience of travel, dengue is spreading beyond its usual tropical and subtropical boundaries. Infection with dengue virus (DENV) causes diseases ranging widely in severity, from self-limited dengue fever to life-threatening dengue hemorrhagic fever and dengue shock syndrome. Vascular leakage, thrombocytopenia, and hemorrhage are the major clinical manifestations associated with severe DENV infection, yet the mechanisms remain unclear. Besides the direct effects of the virus, immunopathogenesis is also involved in the development of dengue disease. Antibody-dependent enhancement increases the efficiency of virus infection and may suppress type I interferon-mediated antiviral responses. Aberrant activation of T cells and overproduction of soluble factors cause an increase in vascular permeability. DENV-induced autoantibodies against endothelial cells, platelets, and coagulatory molecules lead to their abnormal activation or dysfunction. Molecular mimicry between DENV proteins and host proteins may explain the cross-reactivity of DENV-induced autoantibodies. Although no licensed dengue vaccine is yet available, several vaccine candidates are under development. For the development of a safe and effective dengue vaccine, the immunopathogenic complications of dengue disease need to be considered.

Factors contributing to the disturbance of coagulation and fibrinolysis in dengue virus infection.

Hemorrhage is one of the hallmarks of dengue hemorrhagic fever. However, the mechanisms that cause hemorrhage are unclear. In this review we focus on the possible factors that may be involved in the disturbance of coagulation and fibrinolysis during dengue virus (DENV) infection. Factors such as autoantibodies and cytokines induced by DENV infection as well as hemostatic molecules expressed on DENV-infected cells, and DENV viral proteins may all contribute to the defect of hemostasis during DENV infection. It is the combination of these viral and host factors that may tilt the balance of coagulation and fibrinolysis toward bleeding in dengue patients.

Targeting NFKB by autophagy to polarize hepatoma-associated macrophage differentiation.

Tumor-associated macrophages (TAMs) have been linked to promoting tumor progression by stimulating angiogenesis, cell growth and inflammation. NFKB activity in TAMs may mediate inflammation-associated tumor formation. However, most isolated TAMs from established tumors express a M2 phenotype with less NFKB activation and show a strong immunosuppressive phenomenon. How tumors affect the dynamic of NFKB activity in TAMs, and hence maintain their pro-tumor M2 phenotype is still poorly understood. We recently found that hepatoma-derived toll-like receptor 2 (TLR2)-related ligands are capable of stimulating M2 macrophage differentiation via controlling NFKB RELA/p65 protein homeostasis by selective autophagy. TLR2 signal induces NFKB RELA cytosolic ubiquitination and leads to its degradation by SQSTM1/p62-mediated autophagy. Inhibition of autophagy will rescue NFKB activity and shape the phenotype of hepatoma-polarized M2 macrophages. This suggests that autophagy might play a role in manipulating TAM functions and tumor-associated immune responses. Our study also demonstrates that autophagy can directly control a transcriptional factor in addition to its regulatory molecules. This finding uncovers a new role of autophagy in controlling cellular functions.

The serotype-independent but concentration-dependent enhancing antibodies among Thai dengue patients.

Antibody-dependent enhancement of infection (ADE) is central to explaining the development of severe disease at the end of post-dengue virus infection. Non-neutralizing anti-dengue antibodies bound to the dengue virion enhances the virus entrance into the target cells via the Fc receptor. The titer of enhancing antibodies in dengue patients is not determined during dengue virus infection. Sensitive flow cytometry detecting dengue virus-infected K562 cells was used to quantitate enhancing activity among Thai DF and DHF patients against four serotypes and the patient's dengue isolate. The titer was defined as the reciprocal of the final dilution that loses enhancing activity. The serum of Thai patients confirmed to have dengue infection were found to have high titers of enhancing antibodies and increased gradually through the convalescent phase of infection. The enhancing antibody titers were not different among the four serotypes or from the infecting isolate. The anti-dengue antibodies from dengue patients can enhance dengue virus infections in a concentration-dependent, serotype-independent manner.

Cytokine immunopathogenesis of enterovirus 71 brain stem encephalitis.

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.

The regulatory T cells in anti-influenza antibody response post influenza vaccination.

The efficacy and effectiveness of influenza vaccines depend primarily on the vaccine recipient and the virus similarity to the endemic virus. Regulatory T cells (Tregs) and cytokines are known to restrict immune responses against viral infections. We conducted this study to explore the role of Tregs, cytokines, and antibody production after influenza vaccination. The whole blood was collected from healthy subjects (n = 36) before and two weeks after influenza vaccine immunization for two or three consecutive years. The cell surface markers, intracellular staining of Foxp3(+) Tregs, and Th1/Th2 cytokines were determined. The antibody titer was detected using the hemagglutination inhibition test. The CD3(+), CD127(+), CD4(+)CD25(+) and CD4(+)Foxp3(+) cells were increased significantly post vaccination. The plasma level of the transforming growth factor (TGF-ß), but not interleukin (IL)-2, IL-4, IL-5, IL-10, IFN-γ, TNF-α, was also found to increase significantly after vaccination. We further correlated the cytokine fold-increases with the anti-influenza antibody titer for individual post vaccination. It was found that the IL-10 level after vaccination correlated with the fold-increases of anti-H1N1, anti-H3N2, anti-B/Yamagata, and anti-B/Victoria antibodies. But, a negative relationship occurs between the TGF-ß level and fold-increases of anti-H1N1, anti-H3N2, anti-B/Yamagata, and anti-B/Victoria antibodies post vaccination. Treg cells and TGF-ß seem to participate in the downregulation of the anti-influenza antibody response post influenza vaccination. Alteration of Treg activity might enhance influenza vaccine antibody responses and efficacy.

TBK1-associated protein in endolysosomes (TAPE)/CC2D1A is a key regulator linking RIG-I-like receptors to antiviral immunity.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are key RNA viral sensors for triggering antiviral immunity. The underlying mechanisms for RLRs to trigger antiviral immunity have yet to be explored. Here we report the identification of TAPE (TBK1-associated protein in endolysosomes) as a novel regulator of the RLR pathways. TAPE functionally and physically interacts with RIG-I, MDA5, and IPS-1 to activate the IFN-ß promoter. TAPE knockdown impairs IFN-ß activation induced by RLRs but not IPS-1. TAPE-deficient cells are defective in cytokine production upon RLR ligand stimulation. During RNA virus infection, TAPE knockdown or deficiency diminishes cytokine production and antiviral responses. Our data demonstrate a critical role for TAPE in linking RLRs to antiviral immunity.

Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation.

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-1ß 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.

Endothelial cell surface expression of protein disulfide isomerase activates ß1 and ß3 integrins and facilitates dengue virus infection.

Infection with dengue virus (DENV) causes diseases ranging from mild dengue fever to severe hemorrhage or shock syndrome. DENV infection of endothelial cells may cause cell apoptosis or vascular leakage and result in clinical illness of hemorrhage. However, the endothelial cell molecules involved in DENV infection and the mechanisms governing virus-cell interactions are still uncertain. Since protein disulfide isomerase (PDI) reducing function at the cell surface was shown to be required for entry of certain viruses and bacteria, we explored the role of PDI expressed on endothelial cell surface in DENV infection. Using siRNA to knock down PDI, DENV infection was reduced which could be reversed by treating cells with a reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). DENV-induced PDI surface expression was mediated through the Lys-Asp-Glu-Leu (KDEL) receptor-Src family kinase signal pathway. Furthermore, cell surface PDI colocalized with ß1 and ß3 integrins after DENV infection, and the activation of integrins was blocked by PDI inhibition. Finally, blockade of PDI inhibited DENV entry into endothelial cells. Our findings suggest a novel mechanism whereby surface PDI which causes integrin activation is involved in DENV entry, and DENV infection further increases PDI surface expression at later time points. These findings may have implications for anti-DENV drug design.

Loss of outer membrane protein C in Escherichia coli contributes to both antibiotic resistance and escaping antibody-dependent bactericidal activity.

Outer membrane proteins (OMPs) serve as the permeability channels for nutrients, toxins, and antibiotics. In Escherichia coli, OmpA has been shown to be involved in bacterial virulence, and OmpC is related to multidrug resistance. However, it is unclear whether OmpC also has a role in the virulence of E. coli. The aims of this study were to characterize the role of OmpC in antimicrobial resistance and bacterial virulence in E. coli. The ompC deletion mutant showed significantly decreased susceptibility to carbapenems and cefepime. To investigate the survival of E. coli exposed to the innate immune system, a human blood bactericidal assay showed that the ompC mutant increased survival in blood and serum but not in complement-inactivated serum. These effects were also demonstrated in the natural selection of OmpC mutants. Also, C1q interacted with E. coli through a complex of antibodies bound to OmpC as a major target. Bacterial survival was increased in the wild-type strain in a dose-dependent manner by adding free recombinant OmpC protein or anti-C1q antibody to human serum. These results demonstrated that the interaction of OmpC-specific antibody and C1q was the key step in initiating the antibody-dependent classical pathway for the clearance of OmpC-expressing E. coli. Anti-OmpC antibody was detected in human sera, indicating that OmpC is an immunogen. These data indicate that the loss of OmpC in E. coli is resistant to not only antibiotics, but also the serum bactericidal effect, which is mediated from the C1q and anti-OmpC antibody-dependent classical pathway.

Size-dependent attenuation of TLR9 signaling by gold nanoparticles in macrophages.

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 nonstructural protein NS1 binds to prothrombin/thrombin and inhibits prothrombin activation.

OBJECTIVES: Dengue virus (DENV) infection may result in severe dengue hemorrhage fever (DHF). However the mechanisms to cause hemorrhage during DENV infection are not fully understood. The sera level of secreted DENV nonstructural protein 1 (NS1) is correlated with the development of DHF. However, whether secreted NS1 can interfere with coagulation and contribute to the hemorrhage in DHF is unknown. Since thrombin plays a very important role in the activation of coagulation, we investigated whether NS1 can bind to thrombin and affect its formation or activity. METHODS AND RESULTS: We first demonstrated that NS1 could bind to thrombin and formed NS1/thrombin complex in dengue patients' sera by enzyme-linked immunosorbent assay (ELISA). The ability of NS1 binding to prothrombin or thrombin was further confirmed using recombinant NS1 (rNS1) by ELISA, co-immunoprecipitation, and rNS1-affinity column purification. Even though the binding of rNS1 to thrombin showed no effect on thrombin activity, rNS1 could inhibit prothrombin activation and prolong activated partial thromboplastin time (APTT) of human platelet poor plasma. CONCLUSION: These results suggest secreted DENV NS1 may bind to prothrombin and inhibit it activation, which in turn, may contribute to the APTT prolongation and hemorrhage in DHF patients.

Concanavalin A/IFN-gamma triggers autophagy-related necrotic hepatocyte death through IRGM1-mediated lysosomal membrane disruption.

Interferon-gamma (IFN-γ), a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A) can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP) is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1) translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/-) mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.

A single nucleotide in stem loop II of 5'-untranslated region contributes to virulence of enterovirus 71 in mice.

BACKGROUND: Enterovirus 71 (EV71) has emerged as a neuroinvasive virus responsible for several large outbreaks in the Asia-Pacific region while virulence determinant remains unexplored. PRINCIPAL FINDINGS: In this report, we investigated increased virulence of unadapted EV71 clinical isolate 237 as compared with isolate 4643 in mice. A fragment 12 nucleotides in length in stem loop (SL) II of 237 5'-untranslated region (UTR) visibly reduced survival time and rate in mice was identified by constructing a series of infectious clones harboring chimeric 5'-UTR. In cells transfected with bicistronic plasmids, and replicon RNAs, the 12-nt fragment of isolate 237 enhanced translational activities and accelerated replication of subgenomic EV71. Finally, single nucleotide change from cytosine to uridine at base 158 in this short fragment of 5'-UTR was proven to reduce viral translation and EV71 virulence in mice. Results collectively indicated a pivotal role of novel virulence determinant C158 on virus translation in vitro and EV71 virulence in vivo. CONCLUSIONS: These results presented the first reported virulence determinant in EV71 5'-UTR and first position discovered from unadapted isolates.

Dengue virus-induced autoantibodies bind to plasminogen and enhance its activation.

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.

Exogenous interleukin-6, interleukin-13, and interferon-γ provoke pulmonary abnormality with mild edema in enterovirus 71-infected mice.

BACKGROUND: Neonatal mice developed neurological disease and pulmonary dysfunction after an infection with a mouse-adapted human Enterovirus 71 (EV71) strain MP4. However, the hallmark of severe human EV71 infection, pulmonary edema (PE), was not evident. METHODS: To test whether EV71-induced PE required a proinflammatory cytokine response, exogenous pro-inflammatory cytokines were administered to EV71-infected mice during the late stage of infection. RESULTS: After intracranial infection of EV71/MP4, 7-day-old mice developed hind-limb paralysis, pulmonary dysfunction, and emphysema. A transient increase was observed in serum IL-6, IL-10, IL-13, and IFN-γ, but not noradrenaline. At day 3 post infection, treatment with IL-6, IL-13, and IFN-γ provoked mild PE and severe emphysema that were accompanied by pulmonary dysfunction in EV71-infected, but not herpes simplex virus-1 (HSV-1)-infected control mice. Adult mice did not develop PE after an intracerebral microinjection of EV71 into the nucleus tractus solitarii (NTS). While viral antigen accumulated in the ventral medulla and the NTS of intracerebrally injected mice, neuronal loss was observed in the ventral medulla only. CONCLUSIONS: Exogenous IL-6, IL-13, and IFN-γ treatment could induce mild PE and exacerbate pulmonary abnormality of EV71-infected mice. However, other factors such as over-activation of the sympathetic nervous system may also be required for the development of classic PE symptoms.