Enterovirus 71 (EV71) is the major etiological agent of hand, foot, and mouth disease (HFMD). Early studies showed that EV71-infected patients with severe complications exhibited elevated plasma levels of IL-1β, indicating that EV71 may activate inflammasomes. Our current study demonstrates that the NLRP3 inflammasome plays a protective role against EV71 infection of mice in vivo. EV71 replication in myeloid cells results in the activation of the NLRP3 inflammasome and secretion of IL-1β. Conversely, EV71 counteracts inflammasome activation through cleavage of NLRP3 by viral proteases 2A and 3C, which cleave NLRP3 protein at the G493-L494 or Q225-G226 junction, respectively. Moreover, EV71 3C interacts with NLRP3 and inhibits IL-1β secretion when expressed in mammalian cells. These results thus reveal a set of reciprocal regulations between enterovirus 71 and the NLRP3 inflammasome.

Enterovirus 71 (EV71) has emerged as a significant pathogen causing large outbreaks in China for the past 3 years. Developing an EV71 vaccine is urgently needed to stop the spread of the disease; however, the adaptive immune response of humans to EV71 infection remains unclear. We examined the neutralizing antibody titers in HFMD patients and compared them to those of asymptomatic healthy children and young adults. We found that 80% of HFMD patients became positive for neutralizing antibodies against EV71 (GMT = 24.3) one day after the onset of illness. The antibody titers in the patients peaked two days (GMT = 79.5) after the illness appeared and were comparable to the level of adults (GMT = 45.2). Noticeably, the antibody response was not correlated with disease severity, suggesting that cellular immune response, besides neutralizing antibodies, could play critical role in controlling the outcome of EV71 infection in humans.

Chlamydia trachomatis is an obligate intracellular bacterium that is a globally important human pathogen. The chlamydial plasmid is an attenuating virulence factor, but the molecular basis for attenuation is not understood. Chlamydiae replicate within a membrane-bound vacuole termed an inclusion, where they undergo a biphasic developmental growth cycle and differentiate from noninfectious into infectious organisms. Late in the developmental cycle, the fragile chlamydia-laden inclusion retains its integrity by surrounding itself with scaffolds of host cytoskeletal proteins. The ability of chlamydiae to developmentally free themselves from this cytoskeleton network is a fundamental virulence trait of the pathogen. Here, we show that plasmidless chlamydiae are incapable of disrupting their cytoskeletal entrapment and remain intracellular as stable mature inclusions that support high numbers of infectious organisms. By using deletion mutants of the eight plasmid-carried genes (Δpgp1 to Δpgp8), we show that Pgp4, a transcriptional regulator of multiple chromosomal genes, is required for exit. Exit of chlamydiae is dependent on protein synthesis and is inhibited by the compound C1, an inhibitor of the type III secretion system (T3S). Exit of plasmid-free and Δpgp4 organisms, which failed to lyse infected cells, was rescued by latrunculin B, an inhibitor of actin polymerization. Our findings describe a genetic mechanism of chlamydial exit from host cells that is dependent on an unknown pgp4-regulated chromosomal T3S effector gene.

This study was conducted to determine the efficacy of 4 Chinese herbal ingredients (CHI) as immune stimulators for an active vaccine in chickens using both in vitro and in vivo assays. The CHI used were Astragalus polysaccharide (APS), Isatis root polysaccharide (IRPS), Propolis polysaccharide, and Epimedium flavone at various concentrations. Two hundred 14-d-old male White Roman chickens were randomly divided into 10 groups. Chickens in groups 1 to 9 were inoculated with the New-castle disease virus (NDV) strain IV vaccine by intranasal and intraocular administration. Chickens in groups 1 to 8 were also administered subcutaneously on the dorsal region of the neck with 0.5 mL of the corresponding CHI at 2 doses: 29 and 58 mg/kg of BW for APS and IRPS and 7.25 and 14.5 mg/kg of BW for the others, once daily for 3 successive days. In group 9 (CHI-free control) and group 10 (both vaccine- and CHI-free control), chickens were injected with 0.5 mL of physiological saline. New-castle disease virus-specific serum hemagglutination inhibition antibody (Ab) production in immunized chickens was quantified using established methods. The results indicate that a majority of the CHI used at appropriate concentrations were effective in enhancing in vitro proliferation of chick embryo fibroblasts in response to the NDV infection. In vivo administration of CHI to vaccinated chickens (7.25 to 58 mg/kg of BW, depending on type) increased serum anti-NDV hemagglutination inhibition Ab titer concentrations, compared with the administration the NDV alone. For all CHI, a beneficial effect on the Ab production was observed on d 21 after the initiation of the vaccination. On the basis of the in vivo doses used, Propolis polysaccharide and Epimedium flavone were more potent than APS and IRPS in promoting the humoral immune response in the young birds (P < 0.05). Collectively, these findings suggest that appropriate doses of CHI can be used as novel, effective immune stimulators for chickens.

Background and purposeSystemic upregulation of inflammatory cytokines is characteristic of critical severe hand, foot, and mouth disease (HFMD) with pulmonary edema. Thus, immunomodulatory medicines such as steroids, including methylprednisolone, have been proposed to treat patients with severe HFMD in China, because it is postulated that inflammatory cytokines play a role in the development of severe complications. This study is to further investigate the inflammatory response in the relatively mild HFMD patients, and whether steroid treatment has a beneficial effect on the suppression of inflammation in HFMD patients.MethodWe measured the levels of 50 kinds of chemokines, cytokines, growth factors and soluble receptors in serum samples from control patients without HFMD and the HFMD patients with or without prior treatment of intravenous methylprednisolone.ResultsOur present study found that even relatively mild HFMD patients without central nervous system (CNS) complications had elevated serum levels of inflammatory cytokines, including interleukin (IL)-3, IL-6, IL-12p40, and tumor necrosis factor (TNF)-α, which suggested systemic inflammation. In contrast, these patients also have decreased levels of other serum biomarkers, including IL-1Ra, IL-8, IL-16, soluble ICAM-1, CXCL-1, and CCL27. The dysregulation of cytokine and chemokine expression may be involved in CNS complications and unbalanced circulating leukocytes in HFMD patients. Surprisingly, patients treated with methylprednisolone had no difference in the expression levels of HFMD-associated biomarkers instead had slightly increased levels of IL-17A, which was not associated with the occurrence of HFMD.ConclusionWhether steroid treatment has any beneficial effect on the prognosis of HFMD patients requires to be further investigated.

Enterovirus 71 (EV71) is the most virulent pathogen among enteroviruses that cause hand, foot and mouth disease in children but rarely in adults. The mechanisms that determine the age-dependent susceptibility remain largely unclear. Here, we found that the paucity of invariant natural killer T (iNKT) cells together with immaturity of the immune system was related to the susceptibility of neonatal mice to EV71 infection. iNKT cells were crucial antiviral effector cells to protect young mice from EV71 infection before their adaptive immune systems were fully mature. EV71 infection led to activation of iNKT cells depending on signaling through TLR3 but not other TLRs. Surprisingly, iNKT cell activation during EV71 infection required TLR3 signaling in macrophages, but not in dendritic cells (DCs). Mechanistically, interleukin (IL)-12 and endogenous CD1d-restricted antigens were both required for full activation of iNKT cells. Furthermore, CD1d-deficiency led to dramatically increased viral loads in central nervous system and more severe disease in EV71-infected mice. Altogether, our results suggest that iNKT cells may be involved in controlling EV71 infection in children when their adaptive immune systems are not fully developed, and also imply that iNKT cells might be an intervention target for treating EV71-infected patients.

Sulfated proteoglycans have been shown to be involved in the binding of sporozoites of malaria parasites to hepatocytes. In this study, we have evaluated the effect of sulfated glycosaminoglycans on the invasion of erythrocytes by Plasmodium falciparum merozoites and cytoadherence of parasitized erythrocytes (PRBC) to endothelial cells. Invasion of erythrocytes by HB3EC-6 (an HB3 line selected for high binding to endothelial cells) was inhibited by dextran sulfate 500K, dextran sulfate 5K, sulfatides, fucoidan, and heparin but not by chondroitin sulfate A. With the exception of sulfatides, the invasion-inhibitory effect was not mediated by killing of parasites. Cytoadherence of HB3EC-6 to human microvascular endothelial cells (HMEC-1) and HB3C32-6 (an HB3 line selected for high binding to C32 melanoma cells) to C32 melanoma cells was also inhibited by these sulfated glycoconjugates. The highly sulfated dextran sulfate 500K had the highest inhibitory effect on both invasion and cytoadherence. Both unsulfated dextran 500K and hyaluronic acid had no significant effect on invasion or cytoadherence, whereas the positively charged protamine sulfate promoted cytoadherence. Because preincubation of PRBC with sulfated glycosaminoglycans and treatment of target cells with heparinase had no significant inhibition on cytoadherence, it is unlikely that sulfated glycoconjugates are used directly by endothelial cells as cytoadhesion receptors. In an in vivo experiment, we found that the administration of dextran sulfate 500K to CBA/Ca mice infected with Plasmodium berghei ANKA reduced parasitemia and delayed the death associated with anemia. These observations suggest that sulfated polyanions inhibit the invasion of erythrocytes by merozoites and cytoadherence of PRBC to endothelial cells by increasing the negative repulsive charge and sterically interfering with the ligand-receptor interaction after binding to target cells.

Abstract. Because microvascular damage is a common feature of cerebral malaria, we have examined the role eicosanoid metabolites (prostaglandins and leukotrienes) in experimental cerebral malaria. Eighty ICR mice were infected with Plasmodium berghei ANKA, with 40 uninfected mice as controls. Half of the infected mice were treated on days 4 and 5 with aspirin, a prostaglandin synthesis inhibitor. Infected mice started to die of cerebral malaria on day 6, and by day 17, all infected mice died. In contrast, all infected mice treated with aspirin died by day 12. Infected mice had increased phospholipase A2 mRNA expression in the spleen and cyclooxygenase 1 (COX1) and COX2 expression in the brain. At the peak of cerebral malaria, infected mice had higher serum leukotriene B4 levels than control mice, and aspirin-treated infected mice had higher serum leukotriene B4 levels than untreated infected mice. These results suggest that prostaglandins are protective whereas leukotrienes are detrimental in cerebral malaria.Microvascular damage is a common feature of both primate and murine cerebral malaria. Humans who die of cerebral malaria caused by Plasmodium falciparum infection have petechial and ring hemorrhage and edema in the brain. [1][2][3][4][5][6] Similar findings are also seen in cerebral malaria of rhesus monkeys caused by P. coatneyi 7 and of mice caused by P. berghei ANKA. 8 The mechanisms involved in the induction of microvascular damage during cerebral malaria are not well studied. Because cerebral malaria is associated with the over-production of T helper-1 and proinflammatory cytokines (tumor necrosis factor-␣ [tnF-␣], interferon-␥ [IFN-␥], and interleukin-1 [IL-1]), 9-11 it is suggested that these cytokines cause cerebral malaria by up-regulation of intercellular adhesion molecule-1 (a cytoadhesion molecule involved in the adhesion of parasitized erythrocytes and mononuclear cells to endothelial cells) expression and nitric oxide production, which lead to mechanic blockage and disrupted neurotransmission. 11-13 However, whether endothelial blockage and activation themselves cause brain hemorrhage is not clear. The role of platelets in cerebral malaria has attracted some attention, although detailed pathways for the plateletinduced pathology have not yet been studied. 14 An alternative effect of the proinflammatory cytokines is the modulation on the production of eicosanoids (prostaglandins and leukotrienes), which are important in the maintenance of the structure and function of blood vessels. 15 Increased phospholipase A2 activities have been reported in adults infected with P. falciparum. 16 Children with cerebral malaria also have higher phospholipase A2 expression than those with uncomplicated malaria. 17 Because the expression of other enzymes such as cyclooxygenase 1 and 2 (COX1 and COX2) (for prostaglandin synthesis) and lipoxygenase (for leukotriene synthesis) in malaria infection is not known, it is not clear which eicosanoid pathway is involved in the pathogenesis. Malaria infection is known to ...