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Chinese Journal of Microbiology and Immunology.2017;37(8):640-644. doi:10.3760/cma.j.issn.0254-5101.2017.08.015

Toll-like receptor 5 (TLR5) is one of the pattern recognition receptors and recognizes the flagellin protein of bacteria.It activates innate immune responses and induces production of a series of cytokines.TLR5 functions as a bridge linking innate and adaptive immunities.It is known that TLR5 plays an important role in the occurrence and development of certain infectious diseases.This review summarizes the relationships of TLR5 polymorphisms with the development of infectious diseases and discusses the possible pathogenesis.

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Molecular epidemiological characteristics of norovirus outbreaks in Qingdao, 2014-2016

Dan ZHAO ; Zhilei SU ; Feng ZHANG ; Xiaoyan SHI ; Zhaoguo WANG

Chinese Journal of Microbiology and Immunology.2017;37(8):618-623. doi:10.3760/cma.j.issn.0254-5101.2017.08.011

Objective To analyze the molecular epidemiological characteristics of norovirus (NoV) outbreaks in Qingdao between 2014 and 2016.Methods Stool samples were collected from NoV outbreaks between January 2014 and December 2016 and detected by real-time RT-PCR.NoV open reading frame 1 (ORF1) and ORF2 were partially amplified by RT-PCR.The amplified products were further analyzed by gene sequencing and genotyping.Phylogenetic analysis was conducted by using MEGA 6.0 software package.Results A total of 23 NoV outbreaks, involving 260 cases, were reported during 2014 to 2016.Of all collected stool samples, 128 were positive for NoV including 6 of genogroupⅠ (GⅠ) and 122 of genogroupⅡ (GⅡ).All positive samples were genotyped into 6 genotypes, which were GⅡ.P17-GⅡ.17, GⅡ.P12-GⅡ.3, GⅡ.P7-GⅡ.6、GⅡ.P2-GⅡ.2, GⅠ.Pb-GⅠ.6 and GⅡ.Pg-GⅡ.12.The 23 outbreaks included both single infections and mixed genotype infections, which were 11 of GⅡ.17 single infection, 4 of GⅡ.3 single infection, 3 of GⅡ.17 and GⅡ.3 mixed infection, 2 of GⅡ.17 and GⅡ.6 mixed infection, 1 of GⅠ.6 single infection, 1 of GⅡ.17 and GⅡ.2 mixed infection and 1 of GⅡ.17 and GⅡ.12 mixed infection.Conclusion NoV was an important pathogen responsible for viral diarrhea outbreaks in Qingdao.Several different genotypes were detected.The newly variant GⅡ.P17-GⅡ.17 was the predominant epidemic strain causing norovirus outbreaks in Qingdao during 2014 to 2016.

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Surveillance and drug resistance analysis of Salmonella in Guangdong province in 2015

Dongmei HE ; Bixia KE ; Honghui ZENG ; Tong YANG ; Yuheng LIANG ; Hailing TAN ; Bosheng LI ; Meizhen LIU ; Changwen KE

Chinese Journal of Microbiology and Immunology.2017;37(8):611-617. doi:10.3760/cma.j.issn.0254-5101.2017.08.010

Objective To analyze the serotype distribution and antibiotic resistance characteristics of Salmonella strains isolated in Guangdong province for better understanding the condition of Salmonella infection in patients with diarrhea.Methods Fecal samples collected from patients with diarrhea in Guangdong province were used to isolate Salmonella strains.Biochemical analysis was performed to identify these isolated strains.Serotyping and antimicrobial susceptibility testing were carried out for further analysis of the isolated Salmonella strains.Results The rate of Salmonella infection was 7.64％in 2015, and the male to female patient ratio was 1.52∶1.A total of 2 377 patients of all age groups were positive for Salmonella infection and the patients aged 0-6 years accounted for 81.74％.The isolation rate of Salmonella strains in the summer and autumn was higher than that in the winter and spring (10.73％ vs 4.24％;X2=463.77, P<0.01).The Salmonella isolation rates in different areas were as follows: 16.82％ in Zhuhai, 15.85％ in Heyuan, 11.81％ in Yangjiang, 10.68％ in Jiangmen, 8.49％ in Zhongshan, 8.07％ in Maoming, 8.05％ in Jieyang, 7.35％ in Shaoguan, 6.97％ in Foshan, 6.03％ in Dongguan, 5.48％ in Guangzhou and 0.00％ in Zhanjiang.And the differences between different regions were statistically significant (X2=367.67, P<0.01).The 2 377 isolated Salmonella strains were classified into 108 serotypes except for oneSalmonella strain that could not be classified.The top four predominant serotypes were 4,5,12:i:-, Salmonella enteritidis,Salmonella stanley and Salmonella typhimurium.Most Salmonella strains were sensitive to imipenem, azithromycin, ceftazidime, cefotaxime and trimethoprim/sulfamethoxazole, but multidrug resistance was common among those strains.Conclusion Salmonella serotypes of 4,5,12:i:-and Salmonella enteritidis are the predominant pathogens causing human Salmonella infections in Guangdong province.Ceftazidime and cefotaximeare are preferred in the treatment of Salmonella infections.Surveillance for drug resistance in Salmonella should be strengthened as multidrug resistant strains have become a serious problem in Guangdong province.

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Capability of internal transcribed spacer (ITS) region sequencing analysis to identify clinical isolates of filamentous fungi

Ying LI ; Lina GUO ; Yingchun XU

Chinese Journal of Microbiology and Immunology.2017;37(8):607-610. doi:10.3760/cma.j.issn.0254-5101.2017.08.009

Objective To evaluate the capability of internal transcribed spacer (ITS) region sequencing analysis to identify clinical isolates of filamentous fungi.Methods A total of 267 filamentous fungi isolates collected from clinical specimens were analyzed by ITS region sequencing analysis.Alignment of acquired sequences with known sequences in GenBank and MycoBank was conducted to identify the species of those isolates.Results ITS sequences of the 267 isolates were amplified successfully.Among these isolates, 53.9％ (144/267) were identified to species level and 44.2％ (118/267) to genus level.Only five isolates were failed to be identified at genus level as they shared >95％ homology in ITS sequence with multiple genera.Conclusion ITS region sequencing analysis is preferred for identification of clinical isolates of filamentous fungi at genus level for its high universality and great capability.When species-level identification is required, some informative DNA markers besides ITS region should be included accordingly.

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Predominant antigenic epitopes on Hap adhesin of nontypeable Haemophilus influenzae and their immunogenicity

Zhongxiu WU ; Rongshan WANG ; Yanying HUANG ; Hongxing JIN ; Jie YAN

Chinese Journal of Microbiology and Immunology.2017;37(8):594-600. doi:10.3760/cma.j.issn.0254-5101.2017.08.007

Objective To investigate the distribution and sequence conservation of Hap adhensin encoding gene (hap) in clinical isolates of nontypeable Haemophilus influenzae (NTHi), to screen out and identify the predominant T-and B-cell (T-B) combined antigenic epitopes on Hap protein and to analyze their immunogenicity.Methods Sequence conservation of hap genes in NTHi strains and T-B combined antigenic epitopes were predicted using bioinformatic softwares.PCR was used to amplify the 156 bp segment at 5′-end and the 855 bp segment at 3′-end of hap gene (hap-5′-156 and hap-3′-855) and the amplified products were sequenced.Phage display systems of seven T-B combined antigenic epitopes located on the 55 aa segment at N-terminal and the 285 aa segment at C-terminal of Hap protein (Hap-N52 and Hap-C285) were constructed.Western blot assay and ELISA were performed to detect the antigenicity and immunoreactivity of different T-B combined epitopes displayed by recombinant phage PⅢ protein (rPⅢ).Results Hap protein encoded by the hap gene in NTHi was located on membrane surface.Sequences of the 156 bp segment at 5′-end and the 855 bp segment at 3′-end of hap genes extracted from different NTHi strains were relatively conservative, but many mutations were found in sequences at the middle regions of these hap genes.All of the 56 NTHi strains carried hap-5′-156 and hap-3′-855 segments and shared 92.3％-100％ identities in nucleotide and amino acid sequences of these segements.Hap-N5-24 in the Hap-N52 segment as well as Hap-C4-27, Hap-C28-47, Hap-C114-129, Hap-C150-173, Hap-C200-227 and Hap-C241-267 in the Hap-C285 segment was predicted as the T-B combined antigenic epitope with a higher score and less mutations.Results of Western blot assay and ELISA confirmed that the rPⅢ-displayed Hap-C4-27 and Hap-C150-173 epitopes presented clear hybridization bands with NTHi antisera, and 96.9％ (63/65) and 92.3％ (60/65) of serum samples from children with NTHi infection were positive for antibodies against Hap-C4-27 and Hap-C150-173 epitopes, respectively.Conclusion The gene of hap is widely distributed in clinical isolates of NTHi.Moreover, sequences of the 156 pb segment at 5′-end and the 855 bp segment at 3′-end of hap gene are conservative.Hap-C4-27 and Hap-C150-173 are the predominant T-B combined antigenic epitopes on Hap protein, suggesting that they can be used as epitope candidates for developing multiple antigenic peptide vaccines against NTHi.

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IFN-γ inhibits acute allergic airway inflammation in mice

Qiuhong GAN ; Lili JIANG ; Qianyang LI ; Liu YANG ; Zhaojing ZHANG ; Fuguang LI

Chinese Journal of Microbiology and Immunology.2017;37(8):573-579. doi:10.3760/cma.j.issn.0254-5101.2017.08.003

Objective To investigate the inhibitory effect of IFN-γ on acute allergic airway inflammation induced by IL-33 in mice.Methods Twenty-four female C57BL/6 mice (6-8 weeks) were randomly divided into four groups: IL-33 model group, IFN-γ treatment group, IL-33+IFN-γ treatment group and PBS control group.A mouse model of acute allergic airway inflammation was induced by IL-33.Samples of bronchial alveolar lavage fluid (BALF) and lung tissues were collected.Group 2 innate lymphoid cells (ILC2s) and eosinophils were analyzed by flow cytometry.Levels of IL-5 and IL-13 in the supernatants of lung homogenate and BALF were measured by ELISA.Expression of IL-5, IL-13 and ST2 at mRNA level was detected by real-time PCR.Pathological changes in lung tissues were observed following hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) staining.Results Compared with the PBS control group, no infiltration with inflammatory cells, goblet cell hyperplasia or mucus secretion was observed in the IFN-γ group;the numbers of ILC2s and eosinophils were not affected by IFN-γ;the levels of IL-5 and IL-13 in the supernatants of BALF and lung homogenate, and the expression of IL-5, IL-13 and ST2 at mRNA level in lung tissues were not significantly changed by IFN-γ (P>0.05).Compared with the PBS control group, massive infiltration with inflammatory cells, excessive mucus secretion, increased numbers of ILC2s and eosinophils, up-regulated levels of IL-5 and IL-13 in the supernatants of BALF and lung homogenate, and enhanced expression of IL-5, IL-13 and ST2 at mRNA level in lung tissues were detected in the IL-33 model group (P<0.05).Compared with the IL-33 model group, the combined treatment with IL-33 and IFN-γ significantly alleviated inflammatory cell infiltration, inhibited mucus secretion, reduced the numbers of ILC2s and eosinophils, down-regulated the levels of IL-5 and IL-13 in the supernatants of BALF and lung homogenate, and suppressed the expression of IL-5, IL-13 and ST2 at mRNA in lung tissues (P<0.05).Conclusion IFN-γ can inhibit the proliferation of eosinophils and ILC2s induced by IL-33, and reduce the secretion of IL-5 and IL-13, which indicates that IFN-γ has an inhibitory effect on acute allergic airway inflammation induced by IL-33 in mice.

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Immunopathological mechanism of TNBS-induced inflammatory bowel disease in mice

Lei YANG ; Qinghong MENG ; Li ZUO

Chinese Journal of Microbiology and Immunology.2017;37(8):567-572. doi:10.3760/cma.j.issn.0254-5101.2017.08.002

Objective To study the possible pathogenesis of TNBS (2,4,6-trinitrobenzenesulfonic acid)-induced inflammatory bowel disease (IBD) in a mouse model by analyzing histological changes in colon and the expression of cytokines and transcription factor RORγt related to T cell subsets in mesenteric lymph nodes.Methods Female BALB/c mice aged 6-8 weeks were randomly grouped into two groups: IBD model and normal control groups.The mouse model of IBD was established by treating mice with 200 μl of 5％ TNBS/50％ ethanol solution (1∶1) through intestinal instillation, while the mice in the normal control group were instilled with PBS.Pathological changes in colon samples of mice were observed.Real-time PCR was performed to detect the dynamic expression of Th1 cytokines (IL-2, IFN-γ and IL-12p40), Th2 cytokine (IL-4), Treg-related cytokine (IL-10), Th17 cell-related cytokines (IL-17, IL-21 and IL-23) and transcription factor RORγt in mesenteric lymph nodes.Results The mice in the model group begun to show abnormal vital signs such as diarrhea, loss of weight and reduced activity, and mild hyperemia of intestinal mucosa and edema from the third day after modeling.Slight lesions were observed in histological slices of colon tissues stained with hematoxylin and eosin (HE).The expression of IL-21, IL-23 and IL-17 at mRNA level were significantly increased, while the expression of other cytokines showed no significant change.On the sixth day after modeling, many pathological symptoms and intestinal mucosal lesions were aggravated, and marked infiltration of inflammatory cells was observed in histological slices of colon tissues, which indicated that the IBD model was successfully induced by TNBS.Compared with the control group, the IBD model group showed significantly enhanced expression of IL-2, IL-12p40 and IL-10 in mesenteric lymph nodes at mRNA level on the sixth day after modeling.Although the expression of IL-21, IL-23, IL-17 and RORγt at mRNA level on the sixth day were down-regulated to different extent as compared with those on the third day, they were still significantly higher than those of the control group.Conclusion Th17 cell-related cytokines play an important role in the early stage of TNBS-induced IBD.With the progression of the disease, both Th1 and Th17 cells are involved in the immunopathological injury of colon tissues.

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Sequence analysis, prokaryotic expression and purification of Salmonella typhimurium Ssek3 protein

Fuyu DU ; Chengshui LIAO ; Yadong YANG ; Xiaoli WANG ; Chuan YU ; Yinju LI ; Xiangchao CHENG ; Chunjie ZHANG

Chinese Journal of Microbiology and Immunology.2017;37(11):849-855. doi:10.3760/cma.j.issn.0254-5101.2017.11.008

Objective To study the sequence structure of Salmonella typhimurium Ssek3 gene and to express it at protein level in a prokaryotic expression system .Methods Sequence of Ssek3 gene was ob-tained from Salmonella typhimurium SL1344 strain.Bioinformatics methods were used for systematic analy-sis .A prokaryotic expression system for expressing Sse3k gene was constructed and the expressed protein was purified by Ni-NTA affinity chromatography .Results Sequence analysis showed that the Ssek3 gene of Sal-monella typhimurium was 1008 bp in length, encoding a protein of 335 amino acids and 72 amino acid resi-dues.The molecular weight, molecular formula and isoelectric point of Ssek3 protein was 37.89×103, C1700 H2629 N463 O497 S12 and 6.7, which indicated that it was a stable and hydrophilic protein .Ssek3 protein was a membrane protein without signal peptide or transmembrane region , containing five N-glycosylation sites , three O-glycosylation sites , 33 phosphorylation sites , 22 linear B-cell epitopes , 11 T-cell epitopes and 21 di-sulfide bonds.The secondary structure of Ssek3 protein contained 114 α-helices (Hh) (34.03％), 72 ex-tended chain (Ee) (21.49％), 30β-sheets (Tt) (8.96％) and 119 random coils (Cc) (35.52％).Re-sults of SDS-PAGE showed that the fusion protein Ssek 3 expressed in the prokaryotic expression system was a secretory protein with a molecular weight of about 40×103 .Conclusion The Ssek3 gene of Salmonella typh-imurium is successfully cloned , sequenced and expressed in this study , which will lay a foundation for fur-ther studying the role of Ssek3 protein in host cells during Salmonella typhimurium infection.

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Digital gene expression profiling of dendritic cells treated with Seabuckthorn favones

Bingchun LIU ; Ying QIU ; Ruidong ZHAO ; Xia HAN ; Feiyu YUN ; Erxia ZHOU ; Sheng YUN

Chinese Journal of Microbiology and Immunology.2017;37(11):840-848. doi:10.3760/cma.j.issn.0254-5101.2017.11.007

Objective To study the changes in morphology , phenotypes and gene expression pro-files of dendritic cells (DCs) following treatment with Seabuckthorn flavones (SF).Methods DCs were treated with 200μg/ml of SF and then cultured for 7 days.Changes in the morphology of DCs were observed under light microscope .Flow cytometry was used to detect DC surface molecules .Total RNA was extracted to construct the library for digital gene expression profiling ( DGE ) .Differentially expressed genes were screened out and further analyzed by gene ontology ( GO) enrichment analysis and Kyoto encyclopedia of genes and genomes ( KEGG ) pathway enrichment analysis .Results Compared with control group , SF treatment significantly enhanced the expression of HLA-DR, CD80, CD83 and CD86 on DCs.A total of 355 differentially expressed genes were screened out by DGE , including 176 up-regulated genes and 179 down-regulated genes .GO enrichment was mainly involved in the regulation and development of the immune sys -tem and other biological processes .KEGG pathway analysis showed that the significantly enriched pathways were closely related to inflammation , the immune system, cancer and other diseases .Conclusion SF can promote the expression of DC co-stimulatory molecules and pro-mature molecules, and regulate the expres-sion of immunity-related genes such as CD11a, SLAMF6, LMCD1, TSC22D3 and IKZF3.

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Flow cytometry analysis of circulating and splenic follicular helper T ( Tfh) cells in a mouse model of allergic rhinitis

Zhiqi LIU ; Kun YANG ; Lisi LIU

Chinese Journal of Microbiology and Immunology.2017;37(11):834-839. doi:10.3760/cma.j.issn.0254-5101.2017.11.006

Objective To investigate changes in the percentages of circulating and splenic follicular helper T ( Tfh) cells and the significance of Tfh cells in a mouse model of allergic rhinitis .Meth-ods BALB/c mice were sensitized by injection of OVA and aluminum hydroxide to establish the model of allergic rhinitis .Flow cytometry was performed to measure the percentages of circulating and splenic CD 4+CXCR5+after staining Tfh cells with anti-mouse CD4-FITC and anti-mouse CXCR5-PE.Concentrations of OVE-specific IgE ( OVA-sIgE ) in serum samples were measured by enzyme-linked immunosorbent assay ( ELISA ) .Student′s t-test and Pearson ′s correlation analysis were used for statistical analysis .Results Enhanced infiltration of eosinophils in submucosa was observed in the experimental group .After sensitizing the mice with OVA and aluminum hydroxide , the concentration of OVA-sIgE increased from 43 .47 pg/ml [(43.47±2.58) pg/ml] to 50.44 pg/ml [(50.44±1.40) pg/ml] (P=0.029); the percentage of circu-lating CD4+CXCR5+was up-regulated from 6.25％ [(6.25±1.19)％] to 13.94％ [(13.94±2.77)％] (P=0.026); the percentage of splenic CD4+CXCR5+was down-regulated from 18.04％ [(18.04 ± 4.97)％] to 7.26％[(7.26±0.96)％] (P=0.019).The concentration of OVA-specific IgE was posi-tively correlated with the percentage of circulating Tfh cells (r=0.954, P=0.002), but negatively correla-ted with the percentage of splenic Tfh cells (r=-0.801, P=0.028).Moreover, a negative correlation was found between circulating and splenic Tfh cells (r=-0.787, P=0.032).Conclusion Tfh cells might be involved in the immune responses against allergic rhinitis in mice .

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