Genetic characteristics of the P1 coding region of Coxsackievirus A16 associated with hand, foot, and mouth disease in China.

Coxsackievirus A16 (CVA16) is one of the major etiological agents of hand, foot, and mouth disease (HFMD) in young children. To investigate the genetic characteristics of the P1 coding region gene of CVA16 associated with HFMD in China, we included the sequences of CVA16 specimens obtained from outbreak investigations and sporadic HFMD cases between 1998 and 2014 in China from GenBank, we genotyped the CVA16 sequences and analyzed P1 coding region sequences that encode structural proteins with bioinformatics software. CVA16 was classified into genotypes A and B1 based on the VP1 gene; the B1b and B1a subgenotypes were the major CVA16 strains and predominated in the coastal areas of China. Four strains were found to show inter- and intra-typic recombination in the P1 region. The amino acid identities of VP1, VP2, VP3, and VP4 proteins in all Chinese CVA16 strains were 88.2-100%, 83.0-100%, 87.6-100%, and 72.4-100%, respectively. A total of 251 amino acid substitution sites were detected in the structural proteins encoded by the P1 coding region gene. The amino acid sequences of the P1 coding region in Chinese CVA16 strains were highly conserved, although some amino acid mutations occurred with high frequency: VP1-T11A (10%), N14S (14%), L23M/V (11%), T98M (16%), V107A (14%), N102D (6.1%), E145V (8.8%), N218D (10%), E241K (22%), T248A/I (6.8%); VP2-I217V (22%), T226A (38%); VP3-N141S/G (5.4%), and N240D (15%). The genetic characteristics of CVA16 in the P1 coding region gene may provide a basis for developing a CVA16 vaccine and preventing and controlling HFMD in China.

Improvement of short-term hypothermic preservation of microencapsulated hepatocytes.

OBJECTIVES: To determine the optimal storage solution containing suitable protective agents for the preservation of microencapsulated hepatocytes at 4 °C as well as the optimum incubation time after hypothermic preservation. RESULTS: L15 was the optimum solution for both maintaining microcapsule integrity and cell viability. Furthermore, 5 %(v/v) PEG (20 or 35 kDa) added to Leibovitz-15 medium was optimal for microencapsulated C3A cells, enhancing cell viability and liver-specific functions, including albumin and urea synthesis as well as CYP1A2 and CYP3A4 activities. The transcription levels of several CYP450-related genes were also dramatically increased in cells incubated in the optimal solution. Pre-incubation for 2 h was the optimal time for restoring favorable levels of CYP1A2 and CYP3A4 activities in microencapsulated C3A cells for short term, 2 day storage. CONCLUSIONS: Leibovitz-15 medium supplemented with 5 % (v/v) PEG is a promising cold solution for microencapsulated hepatocytes at 4 °C, with an incubation of 2 h at 37 °C after hypothermic preservation being the best incubation duration for further cell application.

Co-delivery of azithromycin and ibuprofen by ROS-responsive polymer nanoparticles synergistically attenuates the acute lung injury.

Bacterial infection causes lung inflammation and recruitment of several inflammatory factors that may result in acute lung injury (ALI). During bacterial infection, reactive oxygen species (ROS) and other signaling pathways are activated, which intensify inflammation and increase ALI-related mortality and morbidity. To improve the ALI therapy outcome, it is imperative clinically to manage bacterial infection and excessive inflammation simultaneously. Herein, a synergistic nanoplatform (AZI+IBF@NPs) constituted of ROS-responsive polymers (PFTU), and antibiotic (azithromycin, AZI) and anti-inflammatory drug (ibuprofen, IBF) was developed to enable an antioxidative effect, eliminate bacteria, and modulate the inflammatory milieu in ALI. The ROS-responsive NPs (PFTU NPs) loaded with dual-drugs (AZI and IBF) scavenged excessive ROS efficiently both in vitro and in vivo. The AZI+IBF@NPs eradicated Pseudomonas aeruginosa (PA) bacterial strain successfully. To imitate the entry of bacterial-derived compounds in body, a lipopolysaccharide (LPS) model was adopted. The administration of AZI+IBF@NPs via the tail veins dramatically reduced the number of neutrophils, significantly reduced cell apoptosis and total protein concentration in vivo. Furthermore, nucleotide oligomerization domain-like receptor thermal protein domain associated protein 3 (NLRP3) and Interleukin-1 beta (IL-1ß) expressions were most effectively inhibited by the AZI+IBF@NPs. These findings present a novel nanoplatform for the effective treatment of ALI.

ROS-responsive polymer nanoparticles with enhanced loading of dexamethasone effectively modulate the lung injury microenvironment.

The acute lung injury (ALI) is an inflammatory disorder associated with cytokine storm, which activates various reactive oxygen species (ROS) signaling pathways and causes severe complications in patients as currently seen in coronavirus disease 2019 (COVID-19). There is an urgent need for medication of the inflammatory lung environment and effective delivery of drugs to lung to reduce the burden of high doses of medications and attenuate inflammatory cells and pathways. Herein, we prepared dexamethasone-loaded ROS-responsive polymer nanoparticles (PFTU@DEX NPs) by a modified emulsion approach, which achieved high loading content of DEX (11.61 %). DEX was released faster from the PFTU@DEX NPs in a ROS environment, which could scavenge excessive ROS efficiently both in vitro and in vivo. The PFTU NPs and PFTU@DEX NPs showed no hemolysis and cytotoxicity. Free DEX, PFTU NPs and PFTU@DEX NPs shifted M1 macrophages to M2 macrophages in RAW264.7 cells, and showed anti-inflammatory modulation to A549 cells in vitro. The PFTU@DEX NPs treatment significantly reduced the increased total protein concentration in BALF of ALI mice. The delivery of PFTU@DEX NPs decreased the proportion of neutrophils significantly, mitigated the cell apoptosis remarkably compared to the other groups, reduced M1 macrophages and increased M2 macrophages in vivo. Moreover, the PFTU@DEX NPs had the strongest ability to suppress the expression of NLRP3, Caspase1, and IL-1ß. Therefore, the PFTU@DEX NPs could efficiently suppress inflammatory cells, ROS signaling pathways, and cell apoptosis to ameliorate LPS-induced ALI. STATEMENT OF SIGNIFICANCE: The acute lung injury (ALI) is an inflammatory disorder associated with cytokine storm, which activates various reactive oxygen species (ROS) signaling pathways and causes severe complications in patients. There is an urgent need for medication of the inflammatory lung environment and effective delivery of drugs to modulate the inflammatory disorder and suppress the expression of ROS and inflammatory cytokines. The inhaled PFTU@DEX NPs prepared through a modified nanoemulsification method suppressed the activation of NLRP3, induced the polarization of macrophage phenotype from M1 to M2, and thereby reduced the neutrophil infiltration, inhibited the release of proteins and inflammatory mediators, and thus decreased the acute lung injury in vivo.

A study on mineralization behavior of amino-terminated hyperbranched polybenzimidazole membranes.

Amino-bearing polymers, coated with apatite or similar minerals, have attracted significant attention for their potential in medical applications. In this study, an amino-terminated hyperbranched polybenzimidazole (HBPBI) membrane was used as a substrate for apatite growth. The membrane was soaked in solutions of CaCl2, Na2HPO4 and SBF to yield an apatite coating. The structure and morphology of the layers were characterized by FTIR-ATR, XRD and FESEM. The results indicate that the high densities of amino, imide and imidazole groups on the amino-terminated HBPBI membrane provide active sites for the growth of apatite.

In vitro large-scale cultivation and evaluation of microencapsulated immortalized human hepatocytes (HepLL) in roller bottles.

BACKGROUND/AIMS: Microencapsulated hepatocytes have been proposed as promising bioactive agents for packed-bed or fluidized-bed bioartificial liver assist devices (BLaDs) and for hepatocyte transplantation because of the potential advantages they offer of high mass transport rate and an optimal microenvironment for hepatocyte culture. We developed a large-scale and high-production alginate-chitosan (AC) microcapsule roller bottle culture system for the encapsulation of hepLL immortalized human hepatocytes. In this study, the efficacy of upscaling encapsulated hepLL cells production with roller bottle cultivation was evaluated in vitro. METHODS: Microencapsulated hepLL cells were grown at high yield in large-scale roller bottles, with free cells cultured in roller bottle spinners serving as controls. The mechanical stability and the permeability of the AC microcapsules were investigated, and the growth, metabolism and functions of the encapsulated hepLL cells were evaluated as compared to free cells. RESULTS: The microcapsules withstood well the shear stress induced by high agitation rates. The microcapsules were permeable to albumin, but prevented the release of immunoglobulins. Culture in roller bottles of immortalized human hepatocytes immobilized in the AC microcapsules improved cell growth, albumin synthesis, ammonia elimination and lidocaine clearance as compared with free cells cultured in roller bottles. CONCLUSIONS: Encapsulated hepLL cells may be cultured on a large scale in roller bottles. This makes them possible candidates for use in cell-based liver assist therapies.

Growth and metabolism of human hepatocytes on biomodified collagen poly(lactic-co-glycolic acid) three-dimensional scaffold.

Hepatic tissue engineering offers a promising approach toward alleviating the need for donor liver, yet many challenges must be overcome including choice of scaffold, cell source, and immunologic barriers. Poly(lactic-co-glycolic acid) (PLGA) polymers are innovative biodegradable materials that have been shown to be useful as scaffolds for seeding and culturing various types of cells. In this study, a porous sponge scaffold of modified PLGA polymer with collagen was investigated for its ability to improve the growth and metabolism of human hepatocytes. We evaluated the biocompatibility of collagen-modified PLGA (C-PLGA) scaffolds with hepatocytes isolated from human liver. Cell adhesion and function (cell density, culture lifespan, albumin synthesis, urea synthesis, and ammonia elimination and diazepam clearance) were assessed during different culture periods. The number of hepatocytes cultured in C-PLGA scaffolds was higher compared with those cultured in PLGA scaffolds without collagen modification, and the lifespan of hepatocytes cultured in C-PLGA scaffolds was longer than that of cells cultured in PLGA scaffolds. Albumin and urea synthesis and ammonia elimination from attached hepatocytes were greater in C-PLGA than in PLGA scaffolds, with the exception of diazepam clearance. Collagen-modified PLGA scaffold is a promising biomaterial for hepatic tissue engineering.

3D PLGA scaffolds improve differentiation and function of bone marrow mesenchymal stem cell-derived hepatocytes.

UNLABELLED: Liver tissue engineering with hepatic stem cells provides a promising alternative to liver transplantation in patients with acute and chronic hepatic failure. In this study, a three-dimensional (3D) bioscaffold was introduced for differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocytes. For hepatocyte differentiation, third passage BMSCs isolated from normal adult F344 rats were seeded into collagen-coated poly(lactic-co-glycolic acid) (C-PLGA) 3D scaffolds with hepatocyte differentiation medium for 3 weeks. Hepatogenesis in scaffolds was characterized by reverse transcript PCR, western blot, confocal laser scanning microscopy (CLSM), periodic acid-Schiff staining, histochemistry, and biochemical assays with hepatic-specific genes and markers. A monolayer culture system was used as a control differentiation group. The results showed that isolated cells possessed the basic features of BMSCs. Differentiated hepatocyte-like cells in C-PLGA scaffolds expressed hepatocyte-specific markers [eg, albumin (ALB), alpha-fetoprotein, cytokeratin 18, hepatocyte nuclear factor 4alpha, and cytochrome P450] at mRNA and protein levels. Most markers were expressed in C-PLGA group 1 week earlier than in the control group. Results of biocompatibility indicated that the differentiated hepatocyte-like cells grew more stably in C-PLGA scaffolds than that in controls during a 3-week differentiation period. The significantly higher metabolic functions in hepatocyte-like cells in the C-PLGA scaffold group further demonstrated the important role of the scaffold. CONCLUSION: As the phenomenon of transdifferentiation is uncommon, our successful transdifferentiation rates of BMSCs to mature hepatocytes prove the superiority of the C-PLGA scaffold in providing a suitable environment for such a differentiation. This material can possibly be used as a bioscaffold for liver tissue engineering in future clinical therapeutic applications.