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Objective:To determine the expression of T follicular helper (Tfh) cell-related molecules inducible costimulator (ICOS) and programmed cell death-1 receptor (PD-1) in skin lesions of patients with bullous pemphigoid (BP), and to explore the role of Tfh cells in the pathogenesis of BP.Methods:Twenty-one paraffin-embedded tissue specimens were collected from 21 patients with confirmed BP in Dalian Dermatosis Hospital from 2014 to 2017, including 7 females and 14 males with an average age of 72.57 years. Ten normal skin tissue specimens served as control group. Immunohistochemical SP method was used to determine the expression of ICOS and PD-1 in BP skin lesions and normal skin tissues.Results:In BP lesions, ICOS and PD-1 were mainly expressed in the basal layer, spinous layer, granular layer and stratum corneum, especially in the spinous layer in the epidermis, and also expressed in inflammatory cells in the dermis. Additionally, they were both expressed in the cytoplasm and nuclei, occasionally expressed on the cell membrane. However, ICOS and PD-1 were rarely expressed in the normal skin tissues. The expression rates of ICOS and PD-1 were significantly higher in the BP group (85.71% [18/21], 47.62% [10/21], respectively) than in the normal control group (both were 0; P < 0.001, < 0.05, respectively) .Conclusion:Tfh cell-related molecules ICOS and PD-1 may play important roles in the pathogenesis of BP.
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Glaucoma is a disorder that leads to retinal ganglion cells (RGCs) apoptosis, visual field loss and optic nerve degeneration.The RGCs death is irreversible, which limites their ability for axon regeneration after injury.Bone marrow mesenchymal stem cells (BMSCs) have shown promise as cell-incorporation, cell-supplements and paracrine-mediated therapy for compromised neurons, which have allowed the possibility of the pluripotent BMSCs based regeneration of retinal cells and repair of neurodegenerative diseases.Intravitreal injection, subretinal injection and autologous BMSCs homing transplantation were explored as therapy for various retinal injury conditions.These BMSCs primarily have paracrine trophic effects and can also incorporate into the damaged retina directly, which have regenerative and protective effects on the reduce of RGCs apoptosis and retinal nerve fiber loss, and multiple cell signals and mechanisms are involved.This review provides an update of the current evidence of BMSCs as treatment and potential limitations, and complications for glaucomatous RGCs dysfunction.The researches including induced-differentiation, transplantation methods and the potential neuroprotective mechanism of BMSCs as therapy for glaucomatous retinal degeneration were discussed.
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Objective@#To demonstrate the effects of symbiotic bacteria from lettuce on inactivation of norovirus（NV）.@*Methods@#Symbiotic bacteria were isolated from the lettuces sampled from farmlands and supermarkets. NV mixed with symbiotic bacteria was set as the experimental group,without symbiotic bacteria as the control group. After the inactivation by high temperature,ultraviolet light（UV）and chlorine dioxide,the ratio of NV amount in the experimental group and the control group was calculated to evaluate the effects of symbiotic bacteria. The mechanism of symbiotic bacteria was revealed by detecting their effects on the protection of viral capsid protein from UV and on the adsorption of NV.@*Results@#Eleven symbiotic bacteria were identified from lettuces,all of which were bacilli,mainly Pseudomonas. Ten symbiotic bacteria could improve the heat-resistant ability of NV,with Microbacterium oryzae,Cupriavidus taiwanensis（SC061204）,Pseudomonas furukawaii,Enterobacter tabaci and Pseudomonas resinovorans（SC061211）more significant. Eleven symbiotic bacteria could improve anti-UV ability of NV,with Pseudomonas putida,Microbacterium oryzae and Enterobacter tabaci more significant. Only one strain of Pseudomonas putida could improve anti-chlorine dioxide ability of NV（Class I hazard）. Pseudomonas putida,Microbacterium oryzae and Enterobacter tabaci could significantly reduce the damage of NV capsid protein. Nine symbiotic bacteria could promote NV adsorption into lettuces,with the promotion rates ranged from 1.04% to 46.73%;while Pseudomonas putida and Pseudomonas resinovorans（SC061211） could restrain NV absorption,with the promotion rates of -6.50% and -19.85%.@*Conclusion@#Symbiotic bacteria from lettuce may enhance the anti-inactivation of NV by protecting capsid protein and promoting adsorption of NV. It is recommended to control the presence of symbiotic bacteria in the process of inactivating NV.
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Objective To establish and preliminarily apply an effective PCR assay for detection of Tupaia(tree shrew)paramyxovirus(TPMV). Methods Using TPMV genomic DNA from NCBI GenBank, bases 8231 to 8720 were synthesized and inserted into a plasmid as a positive standard. One primer pair was designed based on this sequence. In total,60 respiratory swabs and 12 lung tissues from the tree shrews were tested in this PCR assay. Results A PCR method for detection of TPMV was successfully established,with high specificity and sensitivity of 11.5 × 10 -5μg/mL. PCR result testing 60 respiratory swabs and 12 lung tissues were negative. Conclusions PCR for detecting TPMV has good specificity and high sensitivity and can be used for conventional tree shrew paramyxovirus detection.
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Objective:To evaluate the effects of different growth conditions on E.faecalis growth in the microfluid chip and the penetration of E.faecalis into microtubes.Methods:Four units on the chip were randomly selected as control,BHI,nutrient-Tdeprived (PBS) and pH 10 groups.The growth of E.faecalis was monitored by microscope for a period of 72 h after the suspension of E.faecalis had been added into the chip.Results:The microscopic analysis showed a distinct variation in the growth rate and morphological feature under different experimental conditions.he depth of bacterial penetration was significantly greater in BHI group.Conclusion:This study demonstrated that environmental changes can significantly influence the growth and penetration of E.faecalis into the micro tubes.
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Objective To understand the characteristics of minipigs infected withJapanese encephalitis virus(JEV).Methods After the brain tissues were treated, the pig brain tissue treatment solution was inoculated with BHK21 cells.Then, virus culture,indirect immunofluorescence assay, neutralization test, electron microscopic observation, and reverse transcription-polymerase chain reaction (RT-PCR) amplification of the new isolate E segment and PrM segment nucleotide sequence were performed and the genotype was identified.Results BHK21 cells were inoculated into 25 pigbrain tissues.Among them, three tissue-treated fluid couldinduce shrinkage and aggregation of BHK21 cells, and immunofluorescence staining showed strong green fluorescence response.The results of neutralization test showed that the neutralization titer of these three new isolates was 1:64, and the size of the virus particles was about 40nm under the electron microscope.The homology of both RT-PCR product sequencing results and E-segment of vaccine strain were 95%.Three new isolates were type GIII JEV.Conclusion The results ofthisstudydemonstrate that there is G III type Japanese encephalitis virus infection in the minipig farm.
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Objective To researching the effect of gestational diabetes mellitus (GDM) prevention and control management by WeChat consultation on the Internet platform. Methods There were 586 pregnant women who performed prenatal care regularly and accepted glucose tolerance test for GDM at 24 to 28 weeks in our hospital during 2015 and 2016 as research objects. Determination of the observation group:in 2016, the 308 people who match the condition of the study during annual prenatal examination in line, in addition to routine guidance outside the clinic and joined the division to establish a dedicated WeChat group for a comprehensive health management consulting. Determination of the control group:in 2015, the 278 people who match the condition of the study during annual prenatal examination in line were treated as control groups. The two groups of observation indexes included the fasting blood glucose and two-hour postprandial blood glucose control in GDM pregnant women, the weight growth rate of the whole pregnancy, cesarean section rate, birth rate of newborns, neonatal referral rate and so on. Results The rate of fasting blood glucose and two-hour postprandial blood glucose control in the observation group was significantly better than that in the control group (t=2.01-3.11, all P<0.05). According to the pre-pregnancy physical index, the weight growth of the observation group was significantly better than the control group (t=2.12-3.76, all P<0.05).The cesarean section rate, birth rate and neonatal referral rate were 21.10％(65/308), 4.87％(15/308), 7.14％(22/308)in the observation group , and 28.78％(80/278), 10.07％(28/278), 12.23％(34/278)in the control group, there were significant differences (U = 2.10-2.42, all P<0.05). Conclusions Gestational diabetes prevention and control management in the Internet platform for WeChat consultation application effect is remarkable, which can effectively improve the maternal and child outcomes. In the protection of maternal and child health is of great significance.
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Objective To verify the detection ability of experimental animal quality detection laboratories in China for Staphylococcus aureus.Methods The testing samples for Staphylococcus aureus detection were prepared by bacterial culture, homogeneity test and stability test, according to the study plan approved by CNAS.Then the samples and operation instruction were sent to the participant laboratories.The detection reports from these laboratories should be submitted before the deadline expires, and the collected data were summarized and analyzed.Results There were 28 laboratories which joined to this test plan.Among them 22 laboratories ( 78.57%) achieved satisfactory test results, and six laboratories (21.43%) had unsatisfactory test results.27 Laboratories used the national standard detection assay, while only one labo-ratory used PCR assay.Conclusions Most of experimental animal quality testing laboratories in China have sufficient pro-ficiency in detection of Staphylococcus aureus.The obtained information are very helpful for the laboratory ability verification testing in future.
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Objective Through the proficiency validation of testing of mammalian orthoreovirus 3 (Reo3)antibody in laboratory mice, to investigate the capacity of experimental animal quality control laboratories, so as to improve the de-tection capacity.Methods According to the program approved by CNAS, serum samples after calibration were tested for stability and homogeneity, and numbered randomly.The random samples were issued to the participant laboratories with the Standard Operation Procedure( SOP) .The participants must submit the test reports and original records in time.The feed-back results were judged by the concordance rate with the anticipated results.Results 27 laboratories from 17 provinces were enrolled in this evaluation project, all of them submitted detection results on time.Results All the 27 laboratories were marked as pass or excellent, with a pass rate of 100%.ELISA was used in 26 laboratories, and immunofluorescence assay was used in one laboratory.Conclusions The ability for detection of Reo3 antibody in laboratory mice in animal test laboratories is high.The implementation of proficiency testing can reflect the inspection level of quality control laborato-ries.
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Objective To analyze and evaluate the population genetic quality of 3 subbreeds of China Agricultural University miniature pigs in Beijing.Method According to the local standard DB11/T828.3 -2011, 25 pairs of microsatellite primers were used in 3 subbreeds of China Agricultural University miniature pigs, and software Popgen32 was used to process the data.Results 24 microsatellite loci shared 130, 122 and 138 alleles in the China Agricultural University miniature pigs I, II, III, respectively. The average heterozygosity was 0.6759, 0.5967 and 0.6779, respectively, while the average polymorphism information content ( PIC) was 0.6344, 0.5540 and 0.6403, respectively. The genetic distance between China Agricultural University miniature pig II and III was 0.4251, while the genetic distance between China Agricultural University miniature pig I and II was 0.2084.Conclusions In the 3 subbreeds, China Agricultural University miniature pigs II and III have genetic stability and genetic diversity, and both of which satisfy with the genetic characteristics of closed colony laboratory animal.
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Objective To acquire the prevalence and molecular identification data on Syphacia muris and provide reference for the revision of national standard. Methods 923 batches of 5199 SPF animals ( including one batch of 5 monkeys, 3 batches of 25 mini?pigs, 28 batches of 55 rabbits, 13 batches of 248 hamsters, 37 batches of 198 guinea pigs, 93 batches of 459 rats, 742 batches of 4179 mice, 5 batches of 25 chickens and one batch of 5 ducks) and 145 batches of 1389 clean animals ( including one batch of 3 rabbits, 4 batches of 31 hamsters, 16 batches of 157 guinea pigs, 32 batches of 268 rats and 92 batches of 930 mice ) came from 50 different manufactures in China. Direct microscopy real?time dynamic video recording techniques in combination with morphological identification method were applied to screen the Syphacia muris infestation. A multiple polymerase chain reaction ( multiple?PCR ) testing of the isolate based on amplification of the conserved portions of the Syphacia muris internal transcribed spacer (ITS), 28S ribosomal RNA (28S rRNA), NADH dehydrogenase subunits 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes, and the molecular sequencing of the multiple?PCR amplicons was used to confirm the Syphacia muris infection. Results Syphacia muris eggs, larvae and adults were detected by using direct microscopy real?time dynamic video recording technique. Syphacia muris were detected based on the morphology and size of ovum, larvae, and female and male adult worms. Multiple?PCR and sequencing were performed to identify ITS, 28S rRNA, nad1 and cox1 genes of DNA extracted from the single egg, larva and adult parasite Syphacia muris. This approach allowed the specific identification with no amplicon being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the amplified sequences. Molecular characterization by multiple?PCR amplification and sequencing of the ITS, 28S rRNA, nad1 and cox1 genes demonstrated the presence of Syphacia muris. Multiple?PCR followed by sequencing confirmed 285 of 5199 SPF and 135 of 1389 clean animal samples classified as positive by using direct microscopy real?time dynamic video recording technique in the study as containing Syphacia muris?specific DNA. Comparison of the partial sequences of the ITS, 28S rRNA, nad1 and cox1 genes revealed 100% similarity amongst Syphacia muris from different animals. The prevalence of Syphacia muris infection in SPF and clean animals were 5?5% (285/5199) and 9?7% (135/1389), respectively. Conclusions Direct microscopy real?time dynamic video recording technique, multiple?PCR and sequencing can be used to rapidly detect and accurately identify Syphacia muris. The zoonotic nature of Syphacia muris can be regard as a public health alter, hence the good quality control of animal has an important role in protecting human health and safeguarding people safety. This is the first molecular identification and infection investigation of Syphacia muris in SPF and clean animals in China.
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OBJECTIVE:To establish a method for the content determination of amygdalin in Lianhua qingwen capsule. METH-ODS:HPLC was performed on the column of Phenomenex Kinetex XB-C18 with mobile phase of acetonitrile-0.2%Phosphoric acid so-lution(6∶94,V/V)at a flow rate of 1.0 ml/min,the detection wavelength was 207 nm,column temperature was 30℃,and the injec-tion volume was 10μl. RESULTS:The linear range of amygdalin was 43.16-215.80 μg/ml(r=0.999 7);the limit of detection was 0.431 6μg/ml,the limit of quantitation was 1.294 8μg/ml;RSDs of precision,stability and reproducibility tests no more than 0.69%;recovery was 95.16%-100.49%(RSD=1.67%,n=9). CONCLUSIONS:The method is simple and rapid with high accuracy and well reproducibility,and can be used for the content determination of amygdalin in Lianhua qingwen capsule.
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Objective Through the detection of rabbit hemorrhagic disease virus( RHDV) antibody, to investigate the capacity of experimental animal quality control laboratories, so as to improve their detection proficiency.Methods According to the program approved by CNAS, the screened samples were numbered randomly and tested for their stability and homogeneity.The random samples were issued to the participant laboratories with the Standard Operation Procedure ( SOP) .The participant laboratories must submit the test reports and original records in time.The feedback results were judged by the rate of concordance with the anticipated results.Results Twenty laboratories from 14 provinces were en-rolled in the evaluation, and all of them submitted detection results on time.ELISA methods were used in 14 laboratories, and hemagglutination inhibition ( HAI) assay was used in 6 laboratories.The results of 17 laboratories were marked as pass or excellent, with a rate of pass of 85%.Conclusions The ability for detection of RHDV antibody in animal test labora-tories in China is high.The implementation of capacity testing can reflect the level of quality control laboratories.
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Intein is a part of polypeptide in the premature protein with the capability of self-splicing, which is widely applied in protein purification, protein conjuction, cyclopeptide preparation, protein labeling and biosensor. In this review, we summarized the development of intein used in protein purification, discussed intein-mediated chromatographic and non-chromatographic purification systems, and summarized the researches in manipulating intein cleavage reaction. This work is to provide clues for improvement of intein-mediated protein purification.
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Chromatography, Affinity , Inteins , Protein Splicing , ProteinsABSTRACT
Objective To develop a duplex PCR assay for detection of rat parvovirus H-1 and KRV and its application.Methods To design specific primers on the basis of H-1 ( NC_001358 ) and KRV ( U790330 ) genome sequences published in NCBI and establish a duplex PCR assay using H-1 and KRV DNA as templates.To verify the sensitivity and specificity of the method after optimizing PCR.The rats were infected by oral inoculation.The rats were divided into three groups:H-1 infection, KRV infection and mixed infection groups.To collect feces at the 4th, 6th, 8th and 10th days postinfection.Rats were euthanized on the 10th day and samples from heart, liver, spleen, lung, kidney and cecal contents were collected from each rat, then all the samples were screened with the duplex PCR.Results The 183 bp and 302 bp bands were amplified using H-1 and KRV as templates.The sensitivity test showed that the PCR method can detect as low as 3.8 pg/mL H-1 and 0.73 pg/mL KRV.There were no bands amplified when mouse minus virus, canine parvovirus and feline parvovirus were used as templates, showing that the specificity of the duplex PCR assay is very good. The nucleic acids of H-1 or KRV were detected in all rat feces on the 2th day postinfection and there was no obvious clinical symptoms in all the infected rats.The positive rates of H-1 were as follows:50%(4/8) heart tissues, 50%(4/8) liver tissues, 62.5%(5/8) spleen tissues, 50%(4/8) lung tissues, 37.5%(3/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of single infection group was higher than that of mixed infection group.The positive rates of KRV were as follows:0 (0/8) heart tissues, 25% (2/8) liver tissues, 87.5% (7/8) spleen tissues, 12.5% (1/8) lung tissues, 25%(2/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of mixed infection group was higher than that of single infection group.Conclusions The duplex PCR assay for H-1 and KRV established in this study can effectively detect H-1 or KRV infection in rat feces and other tissues, and can be used as an effective supplement to the national standard of lab animals.
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Objective To develop RT-PCR for detection of TMEV and apply the method .Methods To design specific primers on the basis of GD VII ( GI:62039) genome sequences published in NCBI and establish RT-PCR.To verify the sensitivity and specificity of method after optimizing PCR .We infected 9 BALB/c mice intracerebrally and collected brain, heart, liver, spleen, lung, kidnet, cecal contents and serum samples the 6th day postinfection.The samples were tested by the TMEV RT-PCR.100 mouse cecal contents samples were also detected to apply the established method . Results The 371bp single band was amplified using GDVII as template .Sensitivity test showed that the RT-PCR method can detect as low as 0.69 pg/μL GDVII cDNA.There were no objective band amplified when encephalomyocarditis virus , lymphocytic choriomeningitis virus , Japanese B encephalitis virus , murine norovirus and normal mouse brain tissue were used as case-control .All infected mice showed symptom of different degrees such as depression and hind limb paralysis the 3th day postinoculation and two of infected mice died the 5th day postinoculation.Tissues such as heart, liver, spleen, lung, kidney, brain, cecal contents and serum were collected and tested for TMEV .All the brain samples were detected positive for GDVII and other tissues were all negative;The 100 cecal contents samples were tested and all were negative . Conclusions RT-PCR for TMEV GDVII strain can detect virus infection in mouse tissues efficiently and can be used as a powerful supplement for the national standard of lab animal .
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Objective To establish multiplex PCR assay for detection of chicken embryo lethal orphan virus (CELO)and egg drop syndrome virus (EDS).Methods According to GenBank gene sequence , two pairs of specific primers designed were amplified CELO long fiber protein and EDS hexon protein gene sequence .The specificity and sensitivity of multiplex PCR were tested .We also use the multiplex PCR to detect exogenous CELO and EDS in influenza virus.Results Two target bands have been successfully amplified and verified by sequencing .The specificity of the method is better , and the sensitivity is 10 -4μg/mL.The results of detecting exogenous CELO and EDS in 12 influenza virus were negative .Conclusion The multiplex PCR assay for detection of CELO and EDS was established successfully , which have good specificity and high sensitivity , and have high value and application prospect for detecting exogenous CELO and EDS in influenza virus .
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Objective:To analyze the distribution of UGT1A1 gene polymorphisms in Chinese Han patients with extensive-disease small cell lung cancer(ED-SCLC),and evaluate the correlation between UGT1A1 gene polymorphisms and toxicity and efficacy of irino-tecan(CPT-11) based regimen in the patients with ED-SCLC. Methods: The analysis of UGT1A1?28 and UGT1A1?6 gene poly-morphisms was performed in 67 patients with ED-SCLC admitted in our hospital from June 2011 to January 2013. The 67 cases with ED-SCLC treated with irinotecan(CPT-11) based regimen were enrolled to observe the adverse events and efficacy during the chemo-therapy, including objective responserate rate ( ORR) , progression free survival ( PFS) and overall survival ( OS) . The incidence of different genotypes was compared. Results:The distribution of UGT1A1 genotypes in the 67 patients was follows:UGT1A1?28 wild-type (WT) genotype TA6/6 (56, 83. 6%), heterozygous genotype TA6/7 (11, 16. 4%);UGT1A1?6 wild-type (WT) genotype G/G (45,67. 2%), heterozygous genotype G/A (22,32. 8%). No significant difference of PFS and OS was observed between the differ-ent genotypes. The incidence of grade 3 and 4 delayed diarrhea and neutropenia in the patients carrying UGT1A1?6 G/A was higher than that in those with WT genotype (36. 4% vs. 6. 6%, P<0. 05;27. 2% vs. 4. 4%, P<0. 05, respectively). The incidence of grade 3 and 4 thrombocytopenia in the patients carrying UGT1A1?28 TA6/7 was higher than that in those with WT genotype (27. 2%vs. 1. 8%, P<0. 05). The patients simultaneously carrying UGT1A1?28 TA6/7 and UGT1A1?6 G/A were prone to suffering 3 and 4 delayed diarrhea and neutropenia. Conclusion: UGT1A1 polymorphisms may predict the adverse events of CPT-11 in ED-SCLC, while can not predict the efficacy of CPT-11.
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Most pathogens initiate their infections at the human mucosal surface. Therefore, mucosal vaccination, especially through oral or intranasal administration routes, is highly desired for infectious diseases. Meanwhile, protein-based antigens provide a safer alternative to the whole pathogen or DNA based ones in vaccine development. However, the unique biopharmaceutical hurdles that intranasally or orally delivered protein vaccines need to overcome before they reach the sites of targeting, the relatively low immunogenicity, as well as the low stability of the protein antigens, require thoughtful and fine-tuned mucosal vaccine formulations, including the selection of immunostimulants, the identification of the suitable vaccine delivery system, and the determination of the exact composition and manufacturing conditions. This review aims to provide an up-to-date survey of the protein antigen-based vaccine formulation development, including the usage of immunostimulants and the optimization of vaccine delivery systems for intranasal and oral administrations.
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Humans , Adjuvants, Immunologic , Pharmacology , Administration, Intranasal , Administration, Oral , Antigens , Drug Delivery Systems , Proteins , VaccinationABSTRACT
Objective To establish and apply an effective PCR assay for detection of the Tupaia ( tree shrew) adenovirus ( TAV) .Methods According to NCBI Genbank, TAV genome DNA from 19418 to 19917 were synthetized and inserted into a plasmid as positive standards.One pair of primers was designed based on this sequence.Sixty blood samples and fifty-six stool samples from tree shrew were detected with this PCR assay.Results A PCR method for detection of TAV was successfully established, with a high specificity and the sensitivity was 13.5 ×10 -7μg/mL.The PCR results of testing sixty tree shrew blood DNA samples were negative.24 positive cases were tested among 56 stool DNA samples.Sequencing of the samples confirmed a 42.9%infection rate of TAV in tree shrew stool samples, well consistent with the PCR results.Conclusions The PCR method for detecting TAV established in this study has good specificity and high sensitivity, therefore, can be used in conventional detection of tree shrew adenovirus.