Development and evaluation of a reverse transcription-loop-mediated isothermal amplification assay for rapid detection of enterovirus 71.

Human enterovirus 71 (EV71) is the major etiological agent of hand, foot, and mouth disease (HFMD), which is a common infectious disease in young children and infants. EV71 can cause various clinical manifestations and has been associated with severe neurological complications; it has resulted in fatalities during recent outbreaks in Asian-Pacific regions since 1997. The early and rapid detection is critical for prevention and control of EV71 infection, since no vaccine or antiviral drugs are currently available. In this study, a simple and sensitive reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid detection of EV71. The detection limit of the RT-LAMP assay was approximately 0.01 PFU per reaction mixture, and no cross-reactive amplification with other enteroviruses was observed. The assay was evaluated further with 40 clinical specimens and exhibited 92.9% sensitivity and 100% specificity. This RT-LAMP assay may become a useful alternative in clinical diagnosis of EV71, especially in resource-limited hospitals or rural clinics of China and other countries in the Asian-Pacific region.

Construction of Microunits by Adipose-Derived Mesenchymal Stem Cells Laden with Porous Microcryogels for Repairing an Acute Achilles Tendon Rupture in a Rat Model.

OBJECTIVE: Tissue engineering approaches seem to be an attractive therapy for tendon rupture. Novel injectable porous gelatin microcryogels (GMs) can promote cell attachment and proliferation, thus facilitating the repair potential for target tissue regeneration. The research objectives of this study were to assess the efficacy of tissue-like microunits constructed by multiple GMs laden with adipose-derived mesenchymal stem cells (ASCs) in accelerated tendon regeneration in a rat model. METHODS: Through a series of experiments, such as isolation and identification of ASCs, scanning electron microscopy, mercury intrusion porosimetry (MIP), laser scanning confocal microscopy and the CCK-8 test, the biocompatibility of GMs was evaluated. In an in vivo study, 64 rat right transected Achilles tendons were randomly divided into four groups: the ASCs+GMs group (microunits aggregated by multiple ASC-laden GMs injected into the gap), the ASCs group (ASCs injected into the gap), the GMs group (GMs injected into the gap) and the blank defect group (non-treated). At 2 and 4 weeks postoperatively, the healing tissue was harvested to evaluate the gross observation and scoring, biomechanical testing, histological staining and quantitative scoring. Gait analysis was performed over time. The 64 rats were randomly assigned into 4 groups: (1) micro-unit group (ASCs+GMs) containing ASC (105)-loaded 120 GMs in 60 µL DMEM; (2) cell control group (ASCs) containing 106 ASCs in 60 µL DMEM; (3) GM control group (GMs) containing 120 blank GMs in 60 µL DMEM; (4) blank defect group (Defect) containing 60 µL DMEM, which were injected into the defect sites. All animals were sacrificed at 2 and 4 weeks postsurgery (Table 1). RESULTS: In an in vitro study, GMs (from 126 µm to 348 µm) showed good porosities and a three-dimensional void structure with a good interpore connectivity of the micropores and exhibited excellent biocompatibility with ASCs. As the culture time elapsed, the extracellular matrix (ECM) secreted by ASCs encased the GMs, bound multiple microspheres together, and then formed active tendon tissue-engineering microunits. In animal experiments, the ASCs+GMs group and the ASCs group showed stimulatory effects on Achilles tendon healing. Moreover, the ASCs+GMs group was the best at improving the macroscopic appearance, histological morphology, Achilles functional index (AFI), and biomechanical properties of repair tissue without causing adverse immune reactions. CONCLUSION: Porous GMs were conducive to promoting cell proliferation and facilitating ECM secretion. The ASCs-GMs matrices showed an obvious therapeutic efficiency for Achilles tendon rupture in rats.

Phenotypic and genomic characterization of human coxsackievirus A16 strains with distinct virulence in mice.

Human coxsackievirus A16 (CA16) infection results in hand, foot, and mouth disease (HFMD) along with other severe neurological diseases in children and poses an important public health threat in Asian countries. During an HFMD epidemic in 2009 in Guangdong, China, two CA16 strains (GD09/119 and GD09/24) were isolated and characterized. Although both strains were similar in plaque morphology and growth properties in vitro, the two isolates exhibited distinct pathogenicity in neonatal mice upon intraperitoneal or intracranial injection. Complete genome sequences of both CA16 strains were determined, and the possible virulence determinants were analyzed and predicted. Phylogenetic analysis revealed that these CA16 isolates from Guangdong belonged to the B1b genotype and were closely related to other recent CA16 strains isolated in mainland China. Similarity and bootscanning analyses of these CA16 strains detected homologous recombination with the EV71 prototype strain BrCr in the non-structural gene regions and the 3'-untranslated regions. Together, the phenotypic and genomic characterizations of the two clinical CA16 isolates circulating in China were compared in detail, and the potential amino acid residues responsible for CA16 virulence in mice were predicted. These findings will help explain the evolutionary relationship of the CA16 strains circulating in China, warranting future studies investigating enterovirus virulence.

Elevated antigen-specific Th2 type response is associated with the poor prognosis of hand, foot and mouth disease.

The immunopathogenesis of severe hand, foot and mouth disease (HFMD) remains elusive. This study revealed that enterovirus 71 (EV71) epitope-specific CD4+ T cell responses of HFMD patients were skewed toward a Th2 cytokine profile. Patients that demonstrated higher levels of IL-4 expression in their CD4 T cells following antigen stimulation in vitro tended to have a more prolonged period of high fevers and a longer duration of illness. Thus, an increase of EV71 epitope-specific Th2 type response may portend the poor prognosis for some HFMD patients.

Global transcriptomic analysis of human neuroblastoma cells in response to enterovirus type 71 infection.

Human enterovirus type 71 (EV71) is the major pathogen of hand-foot-and-mouth disease (HFMD) and has been associated with severe neurological disease and even death in infants and young children. The pathogenesis of EV71 infection in the human central nervous system remains unclear. In this study, human whole genome microarray was employed to perform transcriptome profiling in SH-SY5Y human neuroblastoma cells infected with EV71. The results indicated that EV71 infection lead to altered expression of 161 human mRNAs, including 74 up-regulated genes and 87 down-regulated genes. Bioinformatics analysis indicated the possible roles of the differentially regulated mRNAs in selected pathways, including cell cycle/proliferation, apoptosis, and cytokine/chemokine responses. Finally, the microarray results were validated using real-time RT-PCR with high identity. Overall, our results provided fundamental information regarding the host response to EV71 infection in human neuroblastoma cells, and this finding will help explain the pathogenesis of EV71 infection and virus-host interaction.

Tribological behavior study on Ti-Nb-Sn/hydroxyapatite composites in simulated body fluid solution.

In this study, Ti-35Nb-2.5Sn/xhydroxyapatite (HA) composites were sintered by pulse current activated sintering (PCAS) from powders milled for different time. These sintered composites were expected to be potential biomaterials. Ca(3)(PO(4))(2) phase which could increase hardness of sintered composites was found in the Ti-35Nb-2.5Sn/15HA composite sintered from 12 h milled powders. The sintered composites had low elastic modulus (18∼26 GPa) and high compression strength. Due to the importance of friction and wear in biomaterials application, the tribological behavior of sintered composites was studied in simulated body fluid (SBF) solution. Results revealed that milling time and HA content of powders could affect wear properties of sintered composites. The major wear mechanism was abrasive wear in the wear test. The wear rate and friction coefficient decreased when milling time and HA content of powders increased. The lowest friction coefficient (0.1223) was obtained in the Ti-35Nb-2.5Sn/15HA composite sintered from 12 h milled powders, and this composite had superior wear resistance.

Ti-Nb-Sn-hydroxyapatite composites synthesized by mechanical alloying and high frequency induction heated sintering.

A ß-type Ti-based composite, Ti-35Nb-2.5Sn-15-hydroxyapatite (HA), has been synthesized by mechanical alloying and powder metallurgy. The effects of milling time on microstructure, mechanical properties and biocompatibility of the sintered composites were investigated by scanning electronic microscopy (SEM), X-ray diffraction (XRD), microhardness tests, compression tests and cells culture. The results revealed when milling time increased, the homogeneity and relative density of the sintered composite increased, but the finished sintering temperature decreased. The compression Young's modulus of sintered composite from 12 h milled powders was about 22 GPa and its compression strength was 877 MPa. The cell culture results indicated cell viability for these sintered composites was very good. These results revealed the Ti-35Nb-2.5Sn-15HA composite could be useful for medical implants.