CD44-mediated monocyte transmigration across Cryptococcus neoformans-infected brain microvascular endothelial cells is enhanced by HIV-1 gp41-I90 ectodomain.

BACKGROUND: Cryptococcus neoformans (Cn) is an important opportunistic pathogen in the immunocompromised people, including AIDS patients, which leads to fatal cryptococcal meningitis with high mortality rate. Previous researches have shown that HIV-1 gp41-I90 ectodomain can enhance Cn adhesion to and invasion of brain microvascular endothelial cell (BMEC), which constitutes the blood brain barrier (BBB). However, little is known about the role of HIV-1 gp41-I90 in the monocyte transmigration across Cn-infected BBB. In the present study, we provide evidence that HIV-1 gp41-I90 and Cn synergistically enhance monocytes transmigration across the BBB in vitro and in vivo. The underlying mechanisms for this phenomenon require further study. METHODS: In this study, the enhancing role of HIV-1 gp41-I90 in monocyte transmigration across Cn-infected BBB was demonstrated by performed transmigration assays in vitro and in vivo. RESULTS: Our results showed that the transmigration rate of monocytes are positively associated with Cn and/or HIV-1 gp41-I90, the co-exposure (HIV-1 gp41-I90 + Cn) group showed a higher THP-1 transmigration rate (P < 0.01). Using CD44 knock-down HBMEC or CD44 inhibitor Bikunin in the assay, the facilitation of transmigration rates of monocyte enhanced by HIV-1 gp41-I90 was significantly suppressed. Western blotting analysis and biotin/avidin enzyme-linked immunosorbent assays (BA-ELISAs) showed that Cn and HIV-1 gp41-I90 could increase the expression of CD44 and ICAM-1 on the HBMEC. Moreover, Cn and/or HIV-1 gp41-I90 could also induce CD44 redistribution to the membrane lipid rafts. By establishing the mouse cryptococcal meningitis model, we found that HIV-1 gp41-I90 and Cn could synergistically enhance the monocytes transmigration, increase the BBB permeability and injury in vivo. CONCLUSIONS: Collectively, our findings suggested that HIV-1 gp41-I90 ectodomain can enhance the transmigration of THP-1 through Cn-infected BBB, which may be mediated by CD44. This novel study enlightens the future prospects to elaborate the inflammatory responses induced by HIV-1 gp41-I90 ectodomain and to effectively eliminate the opportunistic infections in AIDS patients.

VP1-binding protein glucose-regulated protein 78 as an important mediator for enterovirus 71 infecting human brain microvascular endothelial cells.

Purpose: Enterovirus 71 (EV71) is one of the main pathogens causing hand, foot and mouth disease, which could even induce severe brain damage in some patients. As the underlying mechanism of the invasion and replication process still remains largely unknown, we investigated the role of candidate proteins expressed during EV71 invasion in human brain microvascular endothelial cells (HBMECs) to delineate the pathophysiological mechanism of EV-71 infection. Materials and Methods: Ninety-one candidate EV71-associated proteins which could bind the major capsid protein (viral protein 1 [VP1]) of EV71 on the HBMEC were identified by applying an analysis of glutathione-S-transferase pull-down coupling with liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). Seventy-eight kDa glucose-regulated protein 78 (GRP78) binding to the VP1 protein was further validated by co-immunoprecipitation, immunofluorescence and western blot analysis. To explore the role of GRP78 in EV71 infection, GRP78 was knocked down and overexpressed in HBMEC and was verified by TCID50 assay. Results: LC-ESI-MS/MS-identified 91 proteins were subjected to gene ontology analysis, and on molecular and biological function analysis revealed GRP78 act as an important binding protein in mediating EV71 infection. In addition, immunofluorescence demonstrated the co-localisation of GRP78 and VP1 in cytoplasm of the infected HBMEC. The TCID50 assay showed that knockdown of GRP78 could attenuate the replication capacity of EV71 in HBMEC, and the overexpression could increase the virus titre in HBEMC at 24 h post-infection suggesting that GRP78 was associated with the replication capacity of EV71 in HBMEC. Conclusion: These findings provided evidence that GRP78 plays an important role during the progression of EV71 infection as a mediator in HBMEC.

iTRAQ-based phosphoproteomic analysis reveals host cell's specific responses to Toxoplasma gondii at the phases of invasion and prior to egress.

Protein phosphorylation plays a key role in host cell-T. gondii interaction. However, the phosphoproteome data of host cell at various phases of T. gondii infection has not been thoroughly described. In this study, we assessed the host phosphoproteome data with isobaric tags for relative and absolute quantification (iTRAQ) method during the phases of T. gondii invasion (30 min post infection, PI) and prior to egress (28 h PI). Our iTRAQ analysis revealed a total of 665 phosphoproteins, among which the significantly regulated phosphoproteins in different between-group comparisons were further analyzed. Functional analysis of these significantly regulated phosphoproteins suggested that T. gondii modulated host cell processes through phosphorylation including cell cycle regulation, inducing apoptosis, blocking the synthesis of some inflammatory factors, mediating metabolism to support its proliferation at the infection phase prior to egress, and utilizing membrane and energy from host cell, reorganizing cytoskeleton to favor its invasion and PV formation at the phase of invasion. The phosphorylation level of Smad2, CTNNA1, and HSPB1 identified with western blot revealed a consistent trend of change with iTRAQ result. These newly identified and significantly regulated phosphoproteins from our phosphoproteome data may provide new clues to unravel the host cell's complex reaction against T. gondii infection and the interaction between the host cell and T. gondii.

Toxoplasma gondii ROP18 inhibits human glioblastoma cell apoptosis through a mitochondrial pathway by targeting host cell P2X1.

BACKGROUND: Apoptosis plays a critical role in the embryonic development, homeostasis of immune system and host defense against intracellular microbial pathogens. Infection by the obligate intracellular pathogen Toxoplasma gondii can both inhibit and induce host cell apoptosis; however, the parasitic factors involved remain unclear. The T. gondii virulence factor ROP18 (TgROP18) has been reported to regulate host cell apoptosis; nevertheless, results for this regulation have been rarely reported or have provided contradictory findings. Human purinergic receptor 1 (P2X1) is an ATP-gated ion channel that responds to ATP stimulation and functions in cell apoptosis mediation. The precise roles of TgROP18 in T. gondii pathogenesis, and the relationship between TgROP18 and host P2X1 in host cell apoptosis are yet to be revealed. METHODS: Apoptosis rates were determined by flow cytometry (FCM) and TUNEL assay. The interaction between TgROP18 and the host P2X1 was measured by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (co-IP) assay. Calcium influx and mitochondrial membrane depolarization were determined by FCM after JC-1 staining. The translocation of cytochrome C (Cyt C), Bax and Bcl2 proteins, expression of the apoptotic proteins PARP and caspase activation were detected by western blotting. RESULTS: The apoptosis rates of glial or immune cells (human SF268, mouse RAW264.7 and human THP-1 cells) infected by any T. gondii strain (RH-type I, ME49-type II and VEG-type III) were significantly inhibited compared with their uninfected controls. TgROP18 inhibited ATP-induced apoptosis of SF268 with P2X1 expression, but had no effect on RAW264.7 or THP-1 cells without detectable P2X1 expression. It was further identified that TgROP18 interacted with P2X1, and overexpression of ROP18 in COS7 cells significantly inhibited cell apoptosis mediated by P2X1. Moreover, TgROP18 also inhibited P2X1-mediated Ca2+ influx, translocation of cytochrome C from the mitochondria to the cytosol, and ATP-triggered caspase activation. CONCLUSIONS: Toxoplasma gondii infection inhibits ATP-induced host cell apoptosis, regardless of strain virulence and host cell lines. TgROP18 targets the purinergic receptor P2X1 of the SF268 human neural cells and inhibits ATP-induced apoptosis through the mitochondrial pathway, suggesting a sensor role for the host proapoptotic protein P2X1 in this process.

Bacteroides fragilis Strain ZY-312 Defense against Cronobacter sakazakii-Induced Necrotizing Enterocolitis In Vitro and in a Neonatal Rat Model.

Cronobacter sakazakii is an important pathogen associated with the development of necrotizing enterocolitis (NEC), infant sepsis, and meningitis. Several randomized prospective clinical trials demonstrated that oral probiotics could decrease the incidence of NEC. Previously, we isolated and characterized a novel probiotic, Bacteroides fragilis strain ZY-312. However, it remains unclear how ZY-312 protects the host from the effects of C. sakazakii infection. To understand the underlying mechanisms triggering the probiotic effects, we tested the hypothesis that there was cross talk between probiotics/probiotics-modulated microbiota and the local immune system, governed by the permeability of the intestinal mucosa, using in vitro and in vivo models for the intestinal permeability. The probiotic effects of ZY-312 on intestinal epithelial cells were first examined, and the results revealed that ZY-312 inhibited C. sakazakii invasion, C. sakazakii-induced dual cell death (pyroptosis and apoptosis), and epithelial barrier dysfunction in vitro and in vivo The presence of ZY-312 also resulted in decreased expression of an inflammasome (NOD-like receptor family member pyrin domain-containing protein 3 [NLRP3]), caspase-3, and serine protease caspase-1 in a neonatal rat model. Furthermore, ZY-312 significantly modulated the compositions of the intestinal bacterial communities and decreased the relative abundances of Proteobacteria and Gammaproteobacteria but increased the relative abundances of Bacteroides and Bacillus in neonatal rats. In conclusion, our findings have shown for the first time that the probiotic B. fragilis ZY-312 suppresses C. sakazakii-induced NEC by modulating the proinflammatory response and dual cell death (apoptosis and pyroptosis).IMPORTANCE Cronobacter sakazakii is an opportunistic pathogenic bacterium that can cause necrotizing enterocolitis (NEC). However, the mechanism of pathogenicity of C. sakazakii is largely unknown. Here we have now demonstrated that apoptotic and pyroptotic stimuli are effectors of C. sakazakii-induced NEC. Previously, we isolated a novel probiotic strain candidate from fecal samples from healthy infants and characterized it as Bacteroides fragilis strain ZY-312. Functional characterization reveals that ZY-312 inhibited C. sakazakii invasion, restoring epithelial barrier dysfunction, decreasing the expression of inflammatory cytokines, and reducing dual cell death (pyroptosis and apoptosis). Furthermore, the presence of ZY-132 was sufficient to hinder the adverse reaction seen with C. sakazakii in a C. sakazakii-induced NEC model. Taking the results together, our study demonstrated the utility of ZY-312 as a promising probiotic agent for the prevention of NEC.

Human Chrysomya bezziana myiasis: A systematic review.

BACKGROUND: Myiasis due to Old World screw-worm fly, Chrysomya bezziana, is an important obligate zoonotic disease in the OIE-list of diseases and is found throughout much of Africa, the Indian subcontinent, southeast and east Asia. C. bezziana myiasis causes not only morbidity and death to animals and humans, but also economic losses in the livestock industries. Because of the aggressive and destructive nature of this disease in hosts, we initiated this study to provide a comprehensive understanding of human myiasis caused by C. bezziana. METHODS: We searched the databases in English (PubMed, Embase and African Index Medicus) and Chinese (CNKI, Wanfang, and Duxiu), and international government online reports to 6th February, 2019, to identify studies concerning C. bezziana. Another ten human cases in China and Papua New Guinea that our team had recorded were also included. RESULTS: We retrieved 1,048 reports from which 202 studies were ultimately eligible for inclusion in the present descriptive analyses. Since the first human case due to C. bezziana was reported in 1909, we have summarized 291 cases and found that these cases often occurred in patients with poor hygiene, low socio-economic conditions, old age, and underlying diseases including infections, age-related diseases, and noninfectious chronic diseases. But C. bezziana myiasis appears largely neglected as a serious medical or veterinary condition, with human and animal cases only reported in 16 and 24 countries respectively, despite this fly species being recorded in 44 countries worldwide. CONCLUSION: Our findings indicate that cryptic myiasis cases due to the obligate parasite, C. bezziana, are under-recognized. Through this study on C. bezziana etiology, clinical features, diagnosis, treatment, epidemiology, prevention and control, we call for more vigilance and awareness of the disease from governments, health authorities, clinicians, veterinary workers, nursing homes, and also the general public.

Infection by the nematode Angiostrongylus cantonensis induces differential expression of miRNAs in mouse brain.

BACKGROUND: The parasitic nematode Angiostrongylus cantonensis is the primary pathogen causing eosinophilic meningitis and meningoencephalitis in nonpermissive hosts. The larval parasites are eliminated by the host's immune responses in the central nervous system (CNS) through infiltration of eosinophils and lymphocytes. This study aimed to determine primary alterations of microRNA (miRNA) during A. cantonensis infection in mice. METHODS: miRNA array was used to analyze the expression of miRNA in uninfected and A. cantonensis-infected mouse brains at 21 days postinfection (dpi). Target genes were predicted by miRDB software, and protein-protein interaction network was analyzed using STRING v9.1. Expression levels of selected miRNAs and cytokine production were verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Twenty-five mature miRNAs showed differential expression in infected mouse brains, of which 24 were upregulated and one was downregulated compared to the uninfected control. These 25 miRNAs were divided into five clusters, and the first upregulated cluster was selected for further bioinformatics analysis. Target gene prediction and gene ontology (GO) enrichment analysis revealed that the miRNAs were mainly related to the immune response. Furthermore, six target genes of mmu-miR-146a-5p were predicted to interact with tumor necrosis factor alpha (TNF-α). The in vitro study suggested that transfected mmu-miR-146a-5p inhibitor upregulated TNF-α and its target gene Traf6 in microglia following stimulation with A. cantonensis larval antigen. CONCLUSION: This study suggested a critical role of miRNAs in the host defense during A. cantonensis infection, providing new insights into the molecular mechanisms underlying the interaction between mmu-miR-146a-5p and TNF-α in angiostrongyliasis in nonpermissive hosts.

C-Met Inhibitors are Potential Novel Therapeutic Agents Against Listeria monocytogenes Infection Through Blocking the Bacteria Entry into Nonphagocytic Cells.

High lethality of infections caused by Listeria monocytogenes still remains a major clinical problem in spite of their susceptibility to a wide spectrum of antibiotics. The refractoriness towards treatment is primarily due to its amazing capacity to invade non-phagocytic cells and replicate there in, imparting the dual protection from immune response and antimicrobials. Therefore, generating new anti-infective drugs against intracellular infections has emerged as an urgent issue in the therapeutics of listeriosis. Researches have demonstrated that, internalization of Listeria monocytogenes into nonphagocytic cells is mediated by the interactions between the two bacterial invasion proteins, InlA and InlB, and their cellular surface receptors, E-cadherin and c-Met. As InlB promotes entry into various cell types, such as hepatocytes, epithelial cells and endothelial cells, targeting of InlB-c-Met mediated invasion is important for specifically blocking their intracellular infection. Furthermore, our preliminary in vitro studies have shown that a GA (Geldanamycin, GA) analogue, 17-AAG (tanespimycin) which is widely used in cancer therapy have important therapeutic potential by significantly enhancing the capacity of ampicillin to kill intracellular L. monocytogenes, and to protect the infected HBMECs from the cytocidal effects of this bacterium. We report here, the feasibility of tanespimycin as a potential anti-intracellular infective drug and its clinical relevance in a broader prospective, including the significant advancements in therapeutic approaches, drug effectiveness and toxicity. Exploring the therapeutic effects of c-Met inhibitors such as tanespimycin on L. monocytogenes intracellular infection may provide an alternative novel strategy for the development of antimicrobial agents for treatment of infectious diseases.

Lactobacillus rhamnosus GG supernatant enhance neonatal resistance to systemic Escherichia coli K1 infection by accelerating development of intestinal defense.

The objective of this study was to determine whether Lactobacillus rhamnosus GG culture supernatant (LCS) has a preventive effect against gut-derived systemic neonatal Escherichia coli (E. coli) K1 infection. The preventive effects were evaluated in human colonic carcinoma cell line Caco-2 and neonatal rat models. Our in vitro results showed that LCS could block adhesion, invasion and translocation of E. coli K1 to Caco-2 monolayer via up-regulating mucin production and maintaining intestinal integrity. In vivo experiments revealed that pre-treatment with LCS significantly decrease susceptibility of neonatal rats to oral E. coli K1 infection as reflected by reduced bacterial intestinal colonization, translocation, dissemination and systemic infections. Further, we found that LCS treated neonatal rats have higher intestinal expressions of Ki67, MUC2, ZO-1, IgA, mucin and lower barrier permeability than those in untreated rats. These results indicated that LCS could enhance neonatal resistance to systemic E. coli K1 infection via promoting maturation of neonatal intestinal defense. In conclusions, our findings suggested that LCS has a prophylactic effect against systemic E. coli K1 infection in neonates. Future studies aimed at identifying the specific active ingredients in LCS will be helpful in developing effective pharmacological strategies for preventing neonatal E. coli K1 infection.

Probiotic Mixture Golden Bifido Prevents Neonatal Escherichia coli K1 Translocation via Enhancing Intestinal Defense.

Escherichia coli (E. coli) K1 sepsis and meningitis is a severe infection characterized by high mortality in neonates. Successful colonization and translocation across the intestinal mucosa have been regarded as the critical steps for E. coli K1 sepsis and meningitis. We recently reported that the probiotic mixture, Golden Bifido (containing live Lactobacillus bulgaricus, Bifidobacterium, and Streptococcus thermophilus, LBS) has a preventive role against neonatal E. coli K1 bacteremia and meningitis. However, the interaction between the neonatal gut barrier, probiotics and E. coli K1 is still not elucidated. The present study aims to investigate how LBS exerts its protective effects on neonatal gut barrier during E. coli K1 infection. The beneficial effects of LBS were explored in vitro and in vivo using human colon carcinoma cell lines HT-29 and rat model of neonatal E. coli K1 infection, respectively. Our results showed that stimulation with E. coli K1 was able to cause intestinal barrier dysfunction, which were reflected by E. coli K1-induced intestinal damage and apoptosis of intestinal epithelial cells, reduction of mucin, immunoglobulin A (IgA) and tight junction proteins expression, as well as increase in intestinal permeability, all these changes facilitate E. coli K1 intestinal translocation. However, these changes were alleviated when HT-29 cells were treated with LBS before E. coli K1 infection. Furthermore, we found that LBS-treated neonatal rats (without E. coli K1 infection) have showed higher production of mucin, ZO-1, IgA, Ki67 in intestinal mucosa as well as lower intestinal permeability than that of non-treated rats, indicating that LBS could accelerate the development of neonatal intestinal defense. Taken together, our results suggest that enhancement of the neonatal intestinal defense to fight against E. coli K1 translocation could be the potential mechanism to elucidate how LBS confers a protective effect against neonatal E. coli K1 bacteremia and meningitis. This indirect mechanism makes LBS exert preventive effect on most of gut-derived pathogenic infections rather than only E. coli.

Developmental piRNA profiles of the invasive vector mosquito Aedes albopictus.

BACKGROUND: In eukaryotic organisms, Piwi-interacting RNAs (piRNAs) control the activities of mobile genetic elements and ensure genome maintenance. Recent evidence indicates that piRNAs are involved in multiple biological pathways, including transcriptional regulation of protein-coding genes, sex determination and even interactions between host and pathogens. Aedes albopictus is a major invasive species that transmits a number of viral diseases in humans. Ae. albopictus has the largest genome and the highest abundance of repetitive sequences when compared with members that belong to Culicidae with a published genome. Analysis of piRNA profiles will provide a developmental and evolutionary perspective on piRNAs in Ae. albopictus. METHODS: piRNAs were identified and characterized during the development of Ae. albopictus, and piRNA expression patterns in adult males and females as well as sugar-fed females and blood-fed females were compared. RESULTS: Our results reveal that, despite the large genome size of Ae. albopictus, the piRNA pool of Ae. albopictus (1.2 × 107) is smaller than those of Aedes aegypti (1.7 × 107) and Drosophila melanogaster (1.6 × 107). In Ae. albopictus, piRNAs displayed the highest abundance at the embryo stage and the lowest abundance at the pupal stage. Approximately 50 % of the piRNAs mapped to intergenic regions with no known functions. Approximately 30 % of the piRNAs mapped to repetitive elements, and 77.69 % of these repeat-derived piRNAs mapped to Class I TEs; 45.42 % of the observed piRNA reads originated from piRNA clusters, and most of the top 10 highest expressed piRNA clusters and 100 highest expressed piRNAs from each stage displayed biased expression patterns across the developmental stages. All anti-sense-derived piRNAs displayed a preference for uridine at the 5' end; however, the sense-derived piRNAs showed adenine bias at the tenth nucleotide position and a typical ping-pong signature, suggesting that the biogenesis of piRNAs was conserved throughout development. Our results also show that 962 piRNAs displayed sex-biased expression, and 522 piRNAs showed higher expression in the blood-fed females than in the sugar-fed females. CONCLUSIONS: Our results suggest that piRNAs, aside from silencing transposable elements in Ae. albopictus, may have a role in other biological pathways.

Comparative evaluation of the efficiency of the BG-Sentinel trap, CDC light trap and Mosquito-oviposition trap for the surveillance of vector mosquitoes.

BACKGROUND: The surveillance of vector mosquitoes is important for the control of mosquito-borne diseases. To identify a suitable surveillance tool for the adult dengue vector Aedes albopictus, the efficacy of the BG-Sentinel trap, CDC light trap and Mosquito-oviposition trap (MOT) on the capture of vector mosquitoes were comparatively evaluated in this study. METHODS: The capture efficiencies of the BG-Sentinel trap, CDC light trap and Mosquito-oviposition trap for common vector mosquitoes were tested in a laboratory setting, through the release-recapture method, and at two field sites of Guangzhou, China from June 2013 to May 2014. The captured mosquitoes were counted, species identified and compared among the three traps on the basis of species. RESULTS: In the release-recapture experiments in a laboratory setting, the BG-Sentinel trap caught significantly more Aedes albopictus and Culex quinquefasciatus than the CDC light trap and Mosquito-ovitrap, except for Anopheles sinensis. The BG-Sentinel trap had a higher efficacy in capturing female rather than male Ae. albopictus and Cx. quinquefasciatus, but the capture in CDC light traps displayed no significant differences. In the field trial, BG-Sentinel traps collected more Aedes albopictus than CDC light traps and MOTs collected in both urban and suburban areas. The BG-Sentinel trap was more sensitive for monitoring the population density of Aedes albopictus than the CDC light trap and MOT during the peak months of the year 2013. However, on an average, CDC light traps captured significantly more Cx. quinquefasciatus than BG-Sentinel traps. The population dynamics of Cx. quinquefasciatus displayed a significant seasonal variation, with the lowest numbers in the middle of the year. CONCLUSIONS: This study indicates that the BG-Sentinel trap is more effective than the commonly used CDC light trap and MOT in sampling adult Aedes albopictus and Culex quinquefasciatus. We recommend its use in the surveillance of dengue vector mosquitoes in China.

The expression profile of Aedes albopictus miRNAs is altered by dengue virus serotype-2 infection.

BACKGROUND: Aedes albopictus is an important vector of Dengue virus (DENV) and it has quickly invaded the tropical and temperate environments worldwide. A few studies have shown that, microRNAs (miRNAs) regulate mosquito defense against pathogens. However, there is no systematic analysis of the impact of DENV infection on miRNA expression in Ae. albopictus. We conducted this study to investigate the miRNA expression of Ae. albopictus upon DENV-2 infection using Illumina RNA sequencing. RESULTS: A total of 103 known and 5 novel candidate miRNAs were identified in DENV-2 infected and non-infected adult female Ae. albopictus. Comparative analysis indicated that 52 miRNAs were significantly down-regulated and 18 were up-regulated significantly after infection. Furthermore, RT-qPCR validated the expression patterns of eleven of these differentially expressed miRNAs. Targets prediction and functional analysis of these regulated miRNAs suggested that miR-34-5p and miR-87 might be involved in the anti-pathogen and immune responses. CONCLUSION: This is the first systematic study on the impact of DENV infection on miRNA expression in Ae. albopictus. Complex changes in miRNA expression suggest a potential role of miRNAs in antiviral responses by regulating immune-related genes. This investigation provides information concerning DENV-induced miRNAs and offers clues for identifying potential candidates for vector based antiviral strategies.

Role of uropathogenic Escherichia coli outer membrane protein T in pathogenesis of urinary tract infection.

OmpT is one of the members of the outer membrane protein family that has been identified as a virulence factor in most of the uropathogenic Escherichia coli (UPEC). However, the exact role of OmpT in the urinary tract infections (UTIs) remains unclear. To determine the role of OmpT in the pathogenesis of UPEC, an isogenic deletion mutant of ompT (COTD) was constructed by the λ Red recombination. Human bladder epithelial cell line 5637(HBEC 5637) was used to evaluate the ability of bacterial adhesion/invasion. A murine model of UTI was established to study the formation of intracellular bacterial communities (IBCs) in the process of UTIs. The cytokines were also examined during the pathogenesis. The results showed that the COTD strain was deficient in bacterial adhesion and invasion as well as in IBC formation compare to the parent strain. ELISA quantification analysis of cytokines showed that the levels of TNF-α, IL-6 and IL-8 in the serum, bladder and kidney tissues of the mice infected with COTD were lower than that of the CFT073 group. In summary, these results suggest that OmpT plays a multifaceted role in pathogenesis of UTI, including increased bacterial adhesiveness/invasiveness, formation of IBCs and upregulated proinflammatory cytokines.

Mucin2 is Required for Probiotic Agents-Mediated Blocking Effects on Meningitic E. coli-Induced Pathogenicities.

Mucin2 (MUC2), an important regulatory factor in the immune system, plays an important role in the host defense system against bacterial translocation. Probiotics known to regulate MUC2 gene expression have been widely studied, but the interactions among probiotic, pathogens, and mucin gene are still not fully understood. The aim of this study was to investigate the role of MUC2 in blocking effects of probiotics on meningitic E. coli-induced pathogenicities. In this study, live combined probiotic tablets containing living Bifidobacterium, Lactobacillus bulgaricus, and Streptococcus thermophilus were used. MUC2 expression was knocked down in Caco-2 cells by RNA interference. 5-Aza-2'-deoxycytidine (5-Aza-CdR), which enhances mucin-promoted probiotic effects through inducing production of Sadenosyl- L-methionine (SAMe), was used to up-regulate MUC2 expression in Caco-2 cells. The adhesion to and invasion of meningitic E. coli were detected by competition assays. Our studies showed that probiotic agents could block E. coli-caused intestinal colonization, bacteremia, and meningitis in a neonatal sepsis and meningitis rat model. MUC2 gene expression in the neonatal rats given probiotic agents was obviously higher than that of the infected and uninfected control groups without probiotic treatment. The prohibitive effects of probiotic agents on MUC2-knockdown Caco-2 cells infected with E44 were significantly reduced compared with nontransfected Caco-2 cells. Moreover, the results also showed that 5- Aza-CdR, a drug enhancing the production of SAMe that is a protective agent of probiotics, was able to significantly suppress adhesion and invasion of E44 to Caco-2 cells by upregulation of MUC2 expression. Taken together, our data suggest that probiotic agents can efficiently block meningitic E. coli-induced pathogenicities in a manner dependent on MUC2.

miR-281, an abundant midgut-specific miRNA of the vector mosquito Aedes albopictus enhances dengue virus replication.

BACKGROUND: Emerging evidence indicates that microRNAs (miRNAs) are involved in host-virus interaction. We previously reported that some miRNAs were differentially expressed in sugar-fed and blood-fed females of Aedes albopictus (Ae. albopictus). Here, we analysis the role in the host-virus system of an abundant midgut-specific miRNA in the mosquito Ae. albopictus. METHODS: The expression profiles of miR-281 in different body parts of Ae. albopictus and following dengue virus infection were determined using RT-qPCR and Northern blot. miR-281 mimics, antagomiRs and corresponding negative controls were designed and their overexpression and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting female mosquitoes. Dengue virus serotype-2 (DENV-2) viral genomic RNA abundance was determined by RT-qPCR. The levels of DENV-2 E protein were detected using Western blot. Virus titers were tested using TCID50. RNAhybrid was used to predict targets of miR-281 in the DENV-2 genome. The EGFP plasmid-based reporter system was used to investigate the interaction between miR-281 and the predicted binding site in the C6/36 cell line. RESULTS: miR-281 is specifically expressed in the female midgut where dengue virus first invades. After DENV-2 infection, this miRNA is up-regulated in response to viral infection. Functional intervention analyses in vitro with specifically designed miR-281 mimics and corresponding antagomiRs indicated that miR-281 enhances DENV-2 viral replication. Further depletion of miR-281 in female mosquitoes by injection of its specific antagomiRs led to a significant reduction in DENV-2 abundance. The interaction between miR-281 and its predicted target sequence, the DENV-2 genomic 5'-untranslated region (UTR), is confirmed in the context of a plasmid-based reporter system. CONCLUSION: These findings confirm that miR-281, an abundant midgut-specific miRNA, facilitates DENV-2 replication.

Strongyloidiasis--an insight into its global prevalence and management.

BACKGROUND: Strongyloides stercoralis, an intestinal parasitic nematode, infects more than 100 million people worldwide. Strongyloides are unique in their ability to exist as a free-living and autoinfective cycle. Strongyloidiasis can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection. The most common risk factors for these complications are immunosuppression caused by corticosteroids and infection with human T-lymphotropic virus or human immunodeficiency virus. Even though the diagnosis of strongyloidiasis is improved by advanced instrumentation techniques in isolated and complicated cases of hyperinfection or dissemination, efficient guidelines for screening the population in epidemiological surveys are lacking. METHODOLOGY AND RESULTS: In this review, we have discussed various conventional methods for the diagnosis and management of this disease, with an emphasis on recently developed molecular and serological methods that could be implemented to establish guidelines for precise diagnosis of infection in patients and screening in epidemiological surveys. A comprehensive analysis of various cases reported worldwide from different endemic and nonendemic foci of the disease for the last 40 years was evaluated in an effort to delineate the global prevalence of this disease. We also updated the current knowledge of the various clinical spectrum of this parasitic disease, with an emphasis on newer molecular diagnostic methods, treatment, and management of cases in immunosuppressed patients. CONCLUSION: Strongyloidiasis is considered a neglected tropical disease and is probably an underdiagnosed parasitic disease due to its low parasitic load and uncertain clinical symptoms. Increased infectivity rates in many developed countries and nonendemic regions nearing those in the most prevalent endemic regions of this parasite and the increasing transmission potential to immigrants, travelers, and immunosuppressed populations are indications for initiating an integrated approach towards prompt diagnosis and control of this parasitic disease.

Urbanization increases Aedes albopictus larval habitats and accelerates mosquito development and survivorship.

INTRODUCTION: Aedes albopictus is a very invasive and aggressive insect vector that causes outbreaks of dengue fever, chikungunya disease, and yellow fever in many countries. Vector ecology and disease epidemiology are strongly affected by environmental changes. Urbanization is a worldwide trend and is one of the most ecologically modifying phenomena. The purpose of this study is to determine how environmental changes due to urbanization affect the ecology of Aedes albopictus. METHODS: Aquatic habitats and Aedes albopictus larval population surveys were conducted from May to November 2013 in three areas representing rural, suburban, and urban settings in Guangzhou, China. Ae. albopictus adults were collected monthly using BG-Sentinel traps. Ae. albopictus larva and adult life-table experiments were conducted with 20 replicates in each of the three study areas. RESULTS: The urban area had the highest and the rural area had the lowest number of aquatic habitats that tested positive for Ae. albopictus larvae. Densities in the larval stages varied among the areas, but the urban area had almost two-fold higher densities in pupae and three-fold higher in adult populations compared with the suburban and rural areas. Larvae developed faster and the adult emergence rate was higher in the urban area than in suburban and rural areas. The survival time of adult mosquitoes was also longer in the urban area than it was in suburban and rural areas. Study regions, surface area, water depth, water clearance, surface type, and canopy coverage were important factors associated with the presence of Ae. albopictus larvae. CONCLUSIONS: Urbanization substantially increased the density, larval development rate, and adult survival time of Ae. albopictus, which in turn potentially increased the vector capacity, and therefore, disease transmissibility. Mosquito ecology and its correlation with dengue virus transmission should be compared in different environmental settings.

Functional characterization of three MicroRNAs of the Asian tiger mosquito, Aedes albopictus.

BACKGROUND: Temporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, which are temporally up-regulated in the various developmental stages of Ae. albopictus mosquitoes. METHODS: miRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, hatching rate and hatching time in embryos, eclosion rate in larvae, longevity and fecundity in the adult mosquitoes. RESULTS: The knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, both by in vitro expression in C6/36 mosquito cell lines after transfection as well as in in vivo expression in developmental stages of mosquitoes by microinjection. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in Ae. albopictus development, detected by a decrease in the hatching rate of embryos and eclosion rate in larvae and a marked reduction in longevity and fecundity in adults. CONCLUSION: This study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection is a novel study to delineate the importance of the miRNA expression in regulating mosquito development. The knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development and maintenance of the vectorial population density and pathogen transmissibility of this mosquito vector.