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Different frequencies of B lymphocytes committed to produce anti-thyrotropin receptor antibodies in patients at different clinical stages of Graves' disease

Weiguo JIA ; Decai CHEN ; Zhenmei AN ; Songquan WEI

Immunological Journal.2002;(1):1-5. doi:10.3969/j.issn.1000-8861.2002.01.001

Objective To investigate the relationship between the TRAb-producing cell precursors and different clinical stages of Graves' disease.Methods Peripheral lymphocytes were infected with Epstein-Barr virus,a kind of lymphocyte precursor stimulator,and were stimulated to produce TRAb.Results Of the total wells containing Ig-producing B cell precursors,there were 34.6% and 29.1% from 2 untreated patients,30% from 1 relapsing patient and only 1.3% and 3.8% from 2 healthy controls that secreted TRAb,respectively.As for the wells containing B cell precursors from 2 remission patients with undetectable TRAb in circulation,the percentage (10.7%) of TRAb-containing wells from one was higher than that from the other (5.3%).And the latter had similar percentage of TRAb-positive wells with the controls.The isotype of TRAb in supernatants of our EBV-transformed B lymphocytes was predominantly IgM.Conclusion The frequencies of TRAb-specific precursors were different at three distinct clinical stages of the disease.

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Forecasting system on spatial coverage of cumulative phenotypic frequency of HLA class Ⅰ for designing HLA-based vaccines in China

Fuzhong XUE ; Jiezhen WANG ; Ping HU ; Yishou GUO ; Guorong LI

Immunological Journal.2005;(2):136-141. doi:10.3969/j.issn.1000-8861.2005.02.017

Objective To build the forecasting system on spatial coverage of cumulative phenotypic frequency (CPF) of HLA- Ⅰ for designing HLA-based vaccines (epitopes or DNA vaccines) and evaluating their effects in China. Methods The spatial database of HLA- Ⅰgenes of Chinese (across the land of China) was set up, and then the spatial forecasting system of CPF was designed using Kriging technique.Results Using this spatial forecasting system, the vaccine designer could predict the spatial coverage of CPF for any combination of the alleles confined to a single class Ⅰ locus, either HLA-A or HLA-B, as well as for any combination of alleles at these two loci of each given population at any geographical location across the land of China. Conclusions This system is applicable in the following rields: ①To provide for the identification of alleles that represent a desired percentage of populations across the land of China and that can be targeted for vaccine composition. ② To predict the theoretical responder status of vaccines whose HLA restricted epitopes have already been known in given population at any geographical location across the land of China. (③) To identify how many individuals will be non-responders to a HLA-based vaccine across the land of China.

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Humoral and cellular immune responses induced by DNA vaccine based on major outer membrane protein of chlamydia trachomatis

Zhongyu LI ; Yimou WU ; Minjun YU ; Chaoqun CHEN

Immunological Journal.2005;(6):457-459. doi:10.3969/j.issn.1000-8861.2005.06.007

Objective To construct DNA vaccine containing MOMP gene of Chlamydia trachomatis and to observe immune response in mice. Methods Mice of 4 - 6 weeks old were immunized with pcDNA3.1-MOMP or pcDNA3.1 intramuscularly at a dose of 100 μg. Booster immunizations were employed at 2-week interval for two times. Specific antibody in the sera of mice and the level of IFN-γ in murine spleen lymphocyte supernatant were detected by ELISA. The proliferation response of spleen cells was detected by MTT assay. Results Significant specific antibody titers were observed and the highest titer was 1: 1 024 in mice after three times immunization with pcDNA3.1-MOMP. The proli-feration response of spleen cells were significantly higher than that of mice injected with pc DNA3.1. IFN-γ reached(532.0 + 45.4)pg/mL in immunized mice. Conclusion Strong responses of humoral and cellular immunity can be evoked by DNA vaccine of pcDNA3.1-MOMP in mice.

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Screening and identification of recombinant anti-idiotypic antibody against monoclonal antibody MGb1

Yingru ZHENG ; Rongfen LI ; Li LI ; Gang HUANG ; Li ZHANG ; Daqiang HU

Immunological Journal.2006;(1):86-89. doi:10.3969/j.issn.1000-8861.2006.01.025

Objective T9 provide candidate molecules for developing recombinant anti-idiotypic antibody (anti-Id) vaccine of gastric carcinoma by selection of recombinant anti-Id to monoclonal antibody ( McAb) MGb1 directed against the cancer with phage display technique.Methods Balb/c mice were immunized with MGb1 and the mRNA was isolated from the spleens of the immunized mice. The VL and VH cDNAs of the antibody were amplified separately by RT-PCR and assembled into ScFv DNAs with a linker DNA. The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13KO7 helper phage to yield recombinant phage antibody ScFv library. After four rounds of biopanning to the library with MGb1, the MGb1-positive clones were selected from the enriched phages by ELISA. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA. Results The VL and VH cDNAs was about 320 bp and 340 bp, respectively. The ScFv DNA were about 750 bp. After four rounds panning to the phage antibody ScFv library with MGb1, 18 MGb1-positive phage clones displayed anti-Id ScFv were selected from 50 pre-selected phage clones, among which 4 clones displayed β or γ type anti-Id ScFv. Conclusion The phagedisplayed anti-Id ScFvs to McAb MGb1 are successfully selected by recombinant phage antibody technique, which might lay a foundation for screening the anti-Id ScFv possessing the characteristics of inducing anti-gastric carcinoma immunity.

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Expression of T-bet in peripheral blood and its relation with serum IgE in patient with allergic rhinitis

Shenhong QU ; Tianying LI ; Zhiying OU ; Gen XU ; Weiping WEN ; Jianbo SHI ; Zhibin LIN

Immunological Journal.2006;(5):506-510. doi:10.3969/j.issn.1000-8861.2006.05.010

Objective To investigate T-bet mRNA expression on peripheral blood mononuclear cells (PBMCs) and its relations with allergen specific IgE (SIgE), eosinophile cationic protein (ECP) levels, and allergic symptom in patients with allergic rhinitis (AR). Methods The allergen, SIgE, and ECP in serum of patients with AR were detected by Unicap CAP system. Blood samples were taken from 15 healthy controls and 35 house dust mite allergic patients. PBMC was isolated by density gradient centrifugation and one part of them was cultured with mite allergen at a concentration of 50 μg/mL. PBMC was subjected to analysis of T-bet mRNA expression using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Results The ratio of T-bet to β-actin mRNA levels was 0.418 ± 0. 101 in patients of AR and 0.706 ± 0.091 in healthy controls and the difference was significant (P ＜ 0.01). The expression intensity of T-bet mRNA was not related to varying severity of allergic symptom and ECP levels ( r = - 0.227, - 0.033, P ＞ 0.05). However, there was an inverse correlation between expression intensity of T-bet mRNA and SIgE concentration (r = -0.375, P ＜ 0.05). There was a positive correlation between SIgE and allergic symptom scores ( r = 0.426, P ＜ 0.05). After that PBMC was stimulated by mite allergen, the expression intensity of T-bet mRNA, ECP, and SIgE changed very little ( P ＞ 0.05). Conclusion Down-regulated expression of T-bet mRNA in mite-AR patients is not related to serum ECP and symptom scores but one of important links in the mechanism of imbalance of Th1/Th2 in the occurrence of AR. Specific allergen has no effect on T-bet mRNA, ECP, and SIgE of children and adults with AR in vitro. The level of SIgE objectively and directly indicates the severity of allergic symptom, but T-bet did not. T-bet may be one of indirect factors which affect the level of IgE.

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Changes of type Ⅰ fibroblast growth factor receptor gene during development

Chuanlong MIAO ; Ping WANG ; Yongli YU

Immunological Journal.2000;(1):1-3. doi:10.3969/j.issn.1000-8861.2000.01.001

Objective To find the changes of human fibroblast growth factor receptor 1 genone during development.Method Southern blot analysis of genomic DNA isolated from adult and fetal tissues. Result Adult FGFR1 gene structure is different from its embryonic counterpart. Conclusion The disserence might lead to changes of FGFR1 expression as wel as functions of the cells.

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Expression and relationship between HDAg and bcl-2／bax in liver tissue of the patients with hepatitis D

Xiaohong GU ; Qifen LI ; Yuming WANG

Immunological Journal.2001;(2):128-131. doi:10.3969/j.issn.1000-8861.2001.02.016

Objective　To study the role of bcl-2 and bax in the pathogenesis of hepatitis D. Methods　Expression of HDAg， bcl-2 and bax in liver specimens of 79 patients with hepatitis D were studied by immunohistochemistry technique. Meanwhile， the relationship between expression of HDAg and that of bcl-2／bax in liver specimens of patients were studied by double labelling technique and serial sections.Results　bcl-2 was mainly expressed in the cytoplasm of hepatocytes， bax mainly in the cytoplasm of hepatocytes and partly in the nucleus of hepatocytes， and HDAg mainly in the nucleus of hepatocytes. Most of HDAg and bax positive cells were distributed among infiltrating lymphocytes at the periportal region especially at the advancing edges of areas of “piecemeal necrosis”. Most of hepatocytes of bax positive was found to locate near to positive cells of HDAg， and there were positive correlations between degrees of bax expression and HDAg expression（P＜0.05）. Conclusions　The distribution and the expression of bax and HDAg are significantly correlated with the activity of inflammation and the severity of the liver damage， and HDV infection may induce the expression of bax in the hepatocytes，and hepatocyte apoptosis mediated by bcl-2／bax may play an important role in the liver cell injury by HDV infection.

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Expression of IL-18 in the peripheral blood mononuclear cells of chronic hepatitis B patients

Weiqun WEN ; Lian ZHANG ; Zhangyong HU ; Mingxia ZHANG ; Hong XIAO ; Yi QIAN

Immunological Journal.2001;(2):125-127. doi:10.3969/j.issn.1000-8861.2001.02.015

Objective　 To investigated the possible roles of IL-18 in HBV infection, and the expression of IL-18 in the peripheral blood mononuclear cells (PBMC) of chronic hepatitis B. Methods 　In 15 cases of asymptomatic carriers, 30 cases of active and remissive phases of chronic hepatitis and 10 healthy individuals (as normal controls), IL-18 expression in PBMC were quantitatively analyzed with flow cytometric immunological method. The PBMC were separated routinely and stimulated with LPS (SIGMA) and Monensin (SIGMA) for 6 hours. Then the cells were harvested and fixed by PBS/4% paraformaldehyde. The treated cells were stored in liquid nitrogen for detection later. After immunologic stain, the expression of IL-18 in PBMC were examined by flow cytometry. Results　 ① IL-18 was the lowest in asymptomatic carriers. It was fewer in remissive phase than in normal controls (P＜0.01).There was no difference between active phase and normal controls(P=0.25).②Within the group of active phase, IL-18 was significantly different between each grade of inflammatory activity(P＜0.01),and correlated with serum ALT positively(r=0.63,P＜0.01). Conclusion　 IL-18 may relate to disease activity and the inflammation of liver.

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Study of gastroenteric mucosa immune responses in Balb／c mice by oral immunization with helicobacter pylori

Dongshui LU ; Changqing YU ; Quangming ZOU

Immunological Journal.2001;(2):107-109. doi:10.3969/j.issn.1000-8861.2001.02.009

Objective　To study the mucosa immune responses of gastric and intestinal mucosa in Balb／c mice administered orally with Hp sonicate and mucosal adjuvant（LT）．Methods　The changes of antigen specific AFC in gastric and intestinal mucosa were detected by ELISPOT assay． Results　The numbers of sIgA and IgG AFC rise significantly in PP and gastric mucosa， especially the numbers of sIgA-AFC， significant differences were observed between two immunized groups and the control． Conclusions　Locally synthesized specific sIgA antibodies contribute to immunity against gastric helicobacter infection．

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Construction of mammalian cell expression vector of human CD154 gene from active peripheral blood mononuclear cell and analysis of its sequence

Chunyan ZHANG ; Bo NING ; Shunong LI ; Zhifang ZHANG ; Lianqiang FENG

Immunological Journal.2001;(2):88-90. doi:10.3969/j.issn.1000-8861.2001.02.003

Objective　To obtain mammalian cell expression vector of human CD154 gene. Methods　A 820 bp cDNA fragment was amplified by RT-PCR method from total RNA of human peripheral blood mononuclear cell（PBMC） activated with 10 ng／mL PMA and 1 μg／mL PHA for 8 hours. The fragment was cloned into pcDNA3.1（＋） plasmids.The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes BamH Ⅰ and EcoR Ⅰ and sequenced by Sangers-dideory-mediated chain termination. Results　This cDNA fragment included 820 bp entire coding region and a part of the 3 non-coding region. The recombinant mammalian cell expression vector of pcDNA3.1（＋）／hCD154 was constructed， the sequence of the insert was identical to the published sequence encoding human CD154 antigen. Conclusion　The recombinant mammalian cell expression vector of pcDNA3.1（＋）／hCD154 was successfully constructed.

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