ObjectiveTo develop an ELISA method for quantitative determination of enterovirus 71 (EV71) antigen.The method can be applied to detect EV71 antigen contents and analyze the correlation between immunogenicity and immunoprotection.It also can be used for tissue culture infective dose( TCID50 ) assay.MethodsA double antibody sandwich ELISA method was developed for quantitative determination of EV71 antigen on the basis of the high-affinity neutralizing monoclonal antibodies ( K8G2 and Y8H2-HRP).This method was compared with microscopic observation for the detection of EV71 TCID50.The correlation was analyzed between the specific activity of EV71 antigen and the EV71 neutralizing antibody titer in immune serum.ResultsThe linear range of this method was 0.125-4.0 U/ml and the R2 value was 0.9911.The reagent did not react with other antigens except EV71 antigen.The recovery ratio of this method was 0.89-1.16.The coefficient of variation was less than 15％.The heat recovery rate was above 85％ when the reagent was in 37℃ for 9 days.There was a good correlation in TCID50 of EV71 between this method and microscopic observation,r=0.990.The specific activity of EV71 antigen had positive correlation with the neutralization titer of immune serum in 21 EV71 strains,r=0.930.ConclusionThe quantitative ELISA method for EV71 antigen was developed,which could be used to detect EV71 antigen contents and analyze TCID50.The specific activity of EV71 antigen detected by the method could be used to evaluate the immunoprotection of the vaccine potency test.

Objective To investigate the mid-and long-term effects of metal-on-metal hip resurfacing arthroplasty (HRA),and to analyze its related factors.Methods A total of 64 patients (81 hips) who underwent metal-on-metal HRA from June 2005 to January 2013 were recruited in the present study.There were 35 males (44 hips) and 29 females (37 hips) with the mean age of 48.26±10.45 years (20-65 years),including 47 unilateral and 17 bilateral HRAs.The cohort consisted of osteoarthritis secondary to the developmental dysplasia of the hip (23 cases,29 hips),necrosis of the femoral head (19 cases,22 hips),osteoarthritis (8 cases,10 hips),rheumatoid arthritis (5 cases,9 hips),ankylosing spondylitis (6 cases,8 hips),pigmented villonodular synovitis (2 cases,2 hips) and Otto's disease (1 case,1 hip).During the follow-up duration,radiographic features,including acetabular inclination angle,stem-femoral shaft angle,component loosening,osteolysis,femoral neck narrowing and heterotopic ossification,were evaluated by hip X-rays in straight and froglike position.The size and type of pseudotumor were assessed by MRI and ultrasonography.Clinical efficacy was evaluated by Harris hip score and the University of California at Los Angeles (UCLA) hip score.Considering revision surgery as the end point,the component survivorship was calculated.Results The mean follow-up was 7.98±2.21 years,ranging from 5.0 to 12.8 years.The mean postoperative Harris hip score (92.01 ±5.69) was higher than the preoperative score (41.93 ±9.09).The mean postoperative UCLA pain,walking,function,activity scores (9.37±0.86,9.14± 1.01,8.77± 1.09,6.47± 1.27,respectively) were improved when compared with the preoperative UCLA scores (3.57± 1.23,5.99± 1.30,5.00± 1.01,3.84± 1.41,respectively).The postoperative flexion,abduction and adduction,medial and lateral rotation of the hip was larger than the preoperative ones.Complications occurred in 10 hips (12.3％,10/81).Seven patients (8 hips) experienced early and intermediate complications,including one intraoperative femoral nerve injury,one deep femoral artery and saphenous nerve injuries during the same surgery,one unexplained pain of hip,one femoral neck fracture,three hips of heterotopic ossification,and one pseudotumour.There were mid-and long-term complications in two hips,including one narrowing of the neck and one pseudotumour which was occurred at 9 years.There was one patient (2 hips) underwent revision surgery twice at 5 months and 9 years.The former cause of revision was femoral neck fracture and the latter one was pseudotumour.The Kaplan-Meier survivorship was 98.8％ at five years,and 95.0％ at ten years.Conclusion Patients who underwent metal-on-metal HRA could obtain good mid and long-term results.Pseudotumour and unexplained pain of the hip are critical factors which can affect the mid-and long-term results and survivorship of metal-on-metal HRA.

Macrophages are widely distributed immune cells that contribute to tissue homeostasis. Human THP-1 cells have been widely used in various macrophage-associated studies, especially those involving pro-inflammatory M1 and anti-inflammatory M2 phenotypes. However, the molecular characterization of four M2 subtypes (M2a, M2b, M2c, and M2d) derived from THP-1 has not been fully investigated. In this study, we systematically analyzed the protein expression profiles of human THP-1-derived macrophages (M0, M1, M2a, M2b, M2c, and M2d) using quantitative proteomics approaches. The commonly and specially regulated proteins of the four M2 subtypes and their potential biological functions were further investigated. The results showed that M2a and M2b, and M2c and M2d have very similar protein expression profiles. These data could serve as an important resource for studies of macrophages using THP-1 cells, and provide a reference to distinguish different M2 subtypes in macrophage-associated diseases for subsequent clinical research.
Humans ; Macrophages/metabolism* ; Phenotype ; Proteomics ; THP-1 Cells

Direct secretory expression of active microbial transglutaminase (MTG) using heterologous hosts is a promising strategy, although its production level still needs to be improved for industrial production. Pichia pastoris is one of the most efficient expression systems developed in recent years. In this study, secretory expression of active MTG was successfully achieved by co-expressing the pro sequence and mature MTG genes in P. pastoris. Furthermore, we optimized the copy number of pro/MTG expression cassettes and the fermentation conditions. MTG production level reached 7.3 U/mL in 1-liter fermentor through high density fermentation, providing the feasiblity for industrial scale preparation of MTG.
Fermentation ; Genetic Vectors ; genetics ; Pichia ; enzymology ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Streptomyces ; enzymology ; Transglutaminases ; biosynthesis ; genetics

The structure of N-glycans on specific proteins can regulate innate and adaptive immunity via sensing environmental signals. Meanwhile, the structural diversity of N-glycans poses analytical challenges that limit the exploration of specific glycosylation functions. In this work, we used THP-1-derived macrophages as examples to show the vast potential of a N-glycan structural interpretation tool StrucGP in N-glycoproteomic analysis. The intact glycopeptides of macrophages were enriched and analyzed using mass spectrometry (MS)-based glycoproteomic approaches, followed by the large-scale mapping of site-specific glycan structures via StrucGP. Results revealed that bisected GlcNAc, core fucosylated, and sialylated glycans (e.g., HexNAc₄Hex₅Fuc₁Neu5Ac₁, N₄H₅F₁S₁) were increased in M1 and M2 macrophages, especially in the latter. The findings indicated that these structures may be closely related to macrophage polarization. In addition, a high level of glycosylated PD-L1 was observed in M1 macrophages, and the LacNAc moiety was detected at Asn-192 and Asn-200 of PD-L1, and Asn-200 contained Lewis epitopes. The precision structural interpretation of site-specific glycans and subsequent intervention of target glycoproteins and related glycosyltransferases are of great value for the development of new diagnostic and therapeutic approaches for different diseases.
Humans ; B7-H1 Antigen ; Glycosylation ; Polysaccharides/metabolism*

Biodegradable polyamines have long been studied as potential recombinant viral gene vectors. Spermine (SPE) is an endogenous tetra-amine with excellent biocompatibility yet poor gene condensation capacity. We have previously synthesized a polyspermine based on SPE and poly(ethylene glycol) (PEG) diacrylate (SPE-alt-PEG) for enhanced transfection performance, but the synthesized SPE-alt-PEG still lacked specificity towards cancer cells. In this study, folic acid (FA) was incorporated into SPE-alt-PEG to fabricate a targeted gene delivery vector (FA-SPE-PEG) via an acylation reaction. FA-SPE-PEG exhibited mild cytotoxicity in both cancer cells and normal cells. FA-SPE-PEG possessed higher transfection efficiency than PEI 25 K and Lipofectamine(®) 2000 in two tested cancer cell lines at functional weight ratios, and its superiority over untargeted SPE-alt-PEG was prominent in cells with overexpressed folate receptors (FRs). Moreover, in vivo delivery of green fluorescent protein (GFP) with FA-SPE-PEG resulted in highest fluorescent signal intensity of all investigated groups. FA-SPE-PEG showed remarkably enhanced specificity towards cancer cells both in vivo and in vitro due to the interaction between FA and FRs. Taken together, FA-SPE-PEG was demonstrated to be a prospective targeted gene delivery vector with high transfection capacity and excellent biocompatibility.