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The effect of AD injection on immune functions in tumor-bearing mice

Chinese Journal of Immunology.2010;26(3):224-227.

Objective:In order to study the antitumor effect of AD injection and its immunological mechanisms.Methods:Liver cancer H22-bearing mice were treated with AD injection and 7 days later to observe the effect of AD injection on the inhibitory rate of tumor,indexes of thymus and spleen,lymphocytes transformation function,phagocytosis of the peritoneal macrophage,activity of TNF,the level of IL-2 and IFN-α in serum.Results:AD injection could inhibit the growth of tumor in bearing mice significantly and promote the transformation function of lymphocytes and enhance the peritoneal macrophage phagocytosis and the activities of TNF.AD injection could improve the level of IL-2 of spleen cells and IFN-α in serum.Conclusion:These results suggested that AD injection could obviously inhibit the growth of tumor and enhance the immune functions in bearing mice.

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Study on the family-based association Tim-1 promoter polymorphism with child allergic asthma

Qiwen WU ; Chun PU ; Pengcheng CAI ; Lihua HU

Chinese Journal of Immunology.2010;26(4):320-324. doi:10.3969/j.issn.1000-484X.2010.04.008

Objective: To investigate the association between three single nucleotide polymorphisms-2562G＞A,-416C＞G and-232G＞A in Tim-3(T cell immunoglobulin domain and mucin domain protein 3)gene promoter region and child allergic asthma in Chinese Han population by using family-based association study.Methods: Genotypes of 3 SNPs(-2562G＞A,-416C＞G and-232G＞A)in 118 allergic asthma nuclear pedigrees were analyzed by restriction fragment length polymorphism.The genotype data were analyzed by using the family-based transmission disequilibrium test(TDT).Haplotypes and their frequencies were established and analyzed by TRANSMIT software.Results: ①No transmission disequilibrium was found at the-2562G＞A and-232G＞A sites from heterozygous parents onto patients in 118 trios analyzed by TDT(P＞0.05);However,at the-416 C＞G locus,the observed values of G allele from heterozygous parents to offspring were significantly higher than the expected values(P＜0.05)②The haplotype TDT analysis by TRANSMIT showed the observed and the expected value in GCA and GGA haplotype from parents to the affected offsprings had significant difference respectively(P＜0.05).The Global X~2 test results also showed that Tim-1 haplotype were associated with child allergic asthma(X~2 = 17.26, P＜0.01).Conclusion: Tim-1 gene promoter-416C＞G locus are associated with allergic asthma susceptibility in Hubei Chinese Han population and the haplotypes constructed by-416C＞G are also associated with asthma.Tim-1 genetic polymorphism may play an important role in the pathogenesis of asthma.

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Association between HLA-DRB1 allele polymorphism and susceptibility to chronic myelogenous leukemia in Chinese Han

Mingrui HUO ; Haiyan LIU ; Yan YU ; Bin XI ; Xiaojun HUANG ; Dan LI

Chinese Journal of Immunology.2010;26(4):309-311. doi:10.3969/j.issn.1000-484X.2010.04.005

Objective: The aim was to explore the association between the HLA-DRB1 allele polymorphism and susceptibility to chronic myelogenous leukemia(CML)in Chinese Han population.Methods: The polymorphism of HLA-DRB1 alleles in 762 CML patients and 2 264 normal umbihcal cord blood samples were tested by polymerase chain reaction with sequence specific primer(PCR-SSP).Results: The most common allele in DRB1 locus in normal population was DRB1 * 15(17.25%), whereas DRB1 * 10 was the least frequent allele(1.39%).Compared with normal controls, the frequency of HLA-DRB1 * 08 was significantly increased in all CML groups(7.48%vs 5.39%, X~2 =8.963,OR= 1.023, P =0.004)and CML male groups(7.72%vs 5.39%,X~2 = 8.059,OR= 1.025,P = 0.007).But there was no significant difference between the normal controls and CML female patients.Conclusion: It should be concluded that DRB1 * 08 could be considered a susceptible allele for CML male patients.

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Regulatory role of BTLA signal in the initiation and early phase of T cell activation

Yueying WANG ; Shijie ZHANG ; Yi SHAO ; Yuping JIANG ; Zongjiang GU

Chinese Journal of Immunology.2010;26(4):304-308. doi:10.3969/j.issn.1000-484X.2010.04.004

Objective:To observe the expression of BTLA on T cells during activation and further analyze its inhibitory effects on T cell activation in different phases.Methods: T cells from PBMC were enriched by negative selection using magnetic beads.Expression of BTLA,CTLA-4 and PD-1 on freshly isolated human T cells and kinetics expression of BTLA,CILA-4 and PD-1 on CD3 mAb stimulated T cells were examined by flow cytometry.T cells were stimulated by anti-CD3 mAb combined with anti-CD28 mAb in the presence of anti-BTLA mAb 8H9,then T cell proliferation was tested by MTT assay in the different culture time.Immature DCs were generated from monocytes cultured in the medium containing GM-CSF and IL-4, and further driven to maturation by anti-CD40 mAb.Expression of HVEM on DCs was measured by flow cytometry.T cells were co-cultured with DCs in the presence of soluble 8H9 or anti-HVEM antibody to block HVEM-BTLA interaction,T cell proliferation was measured by MTT assay.Results:Freshly isolated T cells exhibited high levels of BTLA expression, but not CTLA-4 and PD-1.After T cell activation, BTLA expression decreased on first 2 days, with rapidly increasing to high levels.Unlike BTLA, expression of CTLA-4 and PD-1 was gradually increased during T cell activation.8H9 significantly inhibited the proliferation of T cell stimulated by CD3 mAb and CD28 mAb.8H9 could still exhibit inhibitory effect on T cell proliferation after 24 h or 48 h of preactivation by CD3 mAb plus CD28 mAb stimulation.HVEM was highly expressed on immature DCs, and down-regulated on mature DCs.Blockade of BTLA by soluble 8H9 or anti-HVEM antibody enhanced DC-mediated T cell proliferation within 48 h.Conclusion: BTLA signal enhances the threshold of T cell activation and plays importantly negative regulatory role in the initiation and early phase of T cell activation.

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Expression of the estrogen receptor α and β in mouse thymus

Guoning GUO ; Guoyan ZHU ; Yongjun SHANG ; Liang WEN ; Shiwei XU

Chinese Journal of Immunology.2010;26(4):291-293. doi:10.3969/j.issn.1000-484X.2010.04.001

Objective: To detect estrogen receptor α and β(ER-α, ER-β)protein expression in different age of mouse thymus.Methods:Protein expression of ER-α and ER-β in thymus was analyzed via immunohistochemistry.Moreover,the relationship between ER-α and cytokeratin 18(epithelial cell marker)was further tested through fluorescence double-staining.Results: Immunohistochemical analysis revealed that both ER-α and β protein was found in nuclei of some thymocytes of 3 month mice.However,expression of ER-β was absence while ER-α was still positive in aged mice, such as 12 months and 16 months old.Double staining further confirmed that lots of ER-α/β positive cells were Foxp3 negative cells.Conclusion: Expression of ER-β is absent while ER-α is still positive in thymus of aged mice, which indicates ER-α is the critical estrogen receptor that involves in thymic involution.Moreover, ER-α/β do not participate in Treg development within thymus.

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Preliminary study on the induction and differentiation of γδ T cells-Ⅰ

Chaoying HU ; Liu QIAN ; Weizhi CHENG ; Qiuyu HUANG ; Ping WANG ; Qiwen YU ; Jiying ZHANG ; Xuehua CHEN ; Dongqing ZHANG

Chinese Journal of Immunology.2010;26(4):368-371. doi:10.3969/j.issn.1000-484X.2010.04.019

Objective: To investigate the variation of γδ T cells from healthy human peripheral blood(PB)and neonatus cord blood (CB)in proliferation and subtypes with isopentenyl pyrophosphate(IPP), and to acquire enough γδ T cells possessing distinct characteristics for experimental study.Methods: Mononuclear-cells from peripheral blood and cord blood induced by IPP were stained separately with monoclonal antibodies,which were fluorescein-labeled,and then used for assaying the expressing condition of surfaco molecules,as well as to evaluate the variation of γδ T cells on the percentage, subtypes and pbenotypes by FCM.Results:γδ T cells only account for a small proportion in both PB and CB.However,there was a significant difference in the heterogeneity between peripheral blood and cord blood γδ T cells.Vγ9Vδ2 T cells were dominant in peripheral blood γδ T cells.Most of Vγ9Vδ2 T cells in fresh isolated PBMC were central memory-type(CD27~+ CD45RA~-)and effector memory-type(CD27~-CD45RA~-)with IPP, PB γδ T cells proliferated strongly;The effector memory-typo(CD27~-CD45RA~-)turned into the main subtype in all Vγ9Vδ2 T cells,and HLA-DR and B7 molecules were highly expressed on the populations.But the cord blood γδ T cells showed rather complex subgroup heterogeneity,and Vγ9Vδ2 T cells were almost na(i)ve-type(CD27~+ CD45RA~+); Though γδ T cells were expanded(the percent of γδ T cells was increased),and Vγ9Vδ2 T cells were differentiated towards central memory-type and effector memory-type on day 14 with IPP,most of γδ T celLs still remained in the phase of na(i)ve-type(CD27~+ CD45RA~+).Conclusion:Tbere lies great differences of γδ T cells in quantity and subtypes between healthy person peripheral blood and neonatus cord blood.Peripheral blood γδ T cells can be activated and proliferated with IPP, while cord blood γδ T cells have the potential to deferentiate into director memory-type which can be used for experimental and clinical study with the synergy of corresponding cytokines.The immuno-regulation and effector function will be reported in other papers.

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Expression of HIF-1α and VEGF in synovium of knee joint in adjuvant-induce-arthritis rat

Yudong XU ; Xinmei SONG ; Hongtao JIN ; Shaojun ZHANG

Chinese Journal of Immunology.2010;26(4):360-362. doi:10.3969/j.issn.1000-484X.2010.04.016

Objective:To investigate the expression of HIF-1 and VEGF and the correlation with pathological score of synovium in adjuvant-induce-arthritis rat knee joint.Methods: Models of adjuvant arthritis(AA)in rats were made successfully, the arthral pathological score was calculated, production of HIF-1α and VEGF protein was assayed by histoimmunchemical staining at different stage after CFA inoculation.Results:The HIF-1α and VEGF concentration in experimental group were higher than that in control group, and there was significant difference (P＜0.01).And correlation between them was significantly(P＜0.01), similar with pathological score of synovium(P＜0.01).Conclusion:HIF-1α and VEGF play important roles in the development and progression of synovium of knee joint in AA rat.The interaction of them promote development and progression of neonatal vascular in synovium.

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The relationship of multi-locus gene polymorphisms, functional expression of fibrinogen Bβ-chain and the type of cerebral infarction

Xiaodong YUAN ; Shujuan WANG ; Yaru XU ; Jing LI ; Na YANG ; Hongfen LI

Chinese Journal of Immunology.2010;26(4):354-359. doi:10.3969/j.issn.1000-484X.2010.04.015

Objective:To study the correlation of β-fibrinogen-854G/A,-455G/A,-249C/T,-148C/T,448G/A and BcI-1G/A polymorphisms, functional expression of plasma fibrinogen concentration, molecular reactivity, and the type of cerebral infarction.Methods: A casecontrol study was used to analyze 54 patients with main-think cerebral infarction(MCI), 106 patients with penetrating-arterial cerebral infarction (PCI)and 160 heathy cases as control group in Kailuan Hospital between July 2002 and June 2003.Gene polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).Fg concentration, fibrin monomer polymerized velocity(FMPV), absorbance maximum(Amax), FMPV/Amax and biochemistry factors including TG were measured, Results: Fg concentration, FMPV, FMPV/Amax in the MCI group and TG, VLDL and FMPV in the PCI group were higher than in the control group(P＜0.05).The frequencies of854A and Bcl-1A alleles had significant difference among three groups,and the frequencies of GA and AA genotypes in the MCI and PCI groups were higher than in the control group(P＜0.05), however, no different genotypes and allele frequencies of the remaining sites were found in the three groups(P＞0.05).Fg concentration and FMPV of allele T carriers in the MCI group were less than that of-249C/C homozygous ones(P ＜0.05); FMPV/Amax of allele T carriers in the PCI group was higher than that of-148C/C homozygous ones(P＜0.05);with allele A carriers, Fg concentration of control group and FMPV of PCI group were higher than that of Bcl-1 wild homozygote(P＜0.05).Conclusion: Bβ-249 C/T polymorphism in the 5-flanking promoter region can influence the expression of plasma FMPV, Bβ-148 locus is the main regulation location of Fg molecular conglomerate function.Bcl-1 locus in the 3-flanking region is an important gene regulator of plasma Fg concentration, moreover,people with its mutated genotypes are susceptible to MCI.The abnormal plasma Fg concentration, FMPV/Amax and FMPV simultaneously are important risk factors for MCI, and only abnormal FMPV and TG are prone to PCI.

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The inhibitory effect of CCR5Delta32 protein on cell surface expression of the HIV-1 coreceptor CCR5 and CXCR4

Cuiying LI ; Qunxing AN ; Xinyu GAN

Chinese Journal of Immunology.2010;26(4):345-347. doi:10.3969/j.issn.1000-484X.2010.04.013

Objective: To demonstrate that expression of the CCRSDelta32 protein in PBMCs able to down-regulate surface expression of the HIV-1 coreceptor CCR5 and CXCR4.Methods:CCR5Delta32 gene was amplified from human peripheral blood mononuclear cells(PBMCs)genomic DNA by using PCR, and then cloned into lentiviral vector pLenti6/V5-D-TOPO.Recombinant lentiviral particles were produced by packaging using 293T cells.Human PBMCs were transfected with the constructed recombinant lentiviral particles and the expression of CCR5Delta32 was detected by Western blot.The level of CCR5 and CXCR4 expression on transfected PBMCs was detected by FACS analysis.Results: The recombinant lentiviral vector pLenti-CCR5Delta32 was constructed successfully, and the target protein was expressed in PBMCs.FACS analysis showed that CCR5Delta32 protein expressed in PBMCs was able to down-regulate cell surface expression of CCR5 and CXCR4.Conclusion: This study is expected to be used for the gene therapy on AIDS, which deserves further study.

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Establishment of a hepatic stem cell line stably expressing human interleukin-2

Heming ZHENG ; Xiujun CAI

Chinese Journal of Immunology.2010;26(4):341-344. doi:10.3969/j.issn.1000-484X.2010.04.012

Objective:Establishing the hepatic stem cell(WB-F344)line that expresses human interleukin-2 stably and effectively.Methods: We constructed the retroviral vector Plpcx-il-2 containing human interleukin-2 gene, and transfected it into PT67 packaging cells.The infectious viruses were used to infect the hepatic stem cell WB-F344,then the puromycin resistant clones were acquired after puromycin selection.The expression of IL-2 was detected using RT-PCR,immunocytochemistry, Western blot and indirect immunofluorescence, respectively.Results: The retroviral vector containing human IL-2 gene was constructed.The titer of virus in the supernatant reached 10~5 CFU/ml after transfecting PT67 packeting cells.The puromycin resistant clones were acquired after infection of WB-F344 and the cell line WB-F344/Plpcx-IL-2 expressing human interleukin-2 stably was established.Conclusion: By retroviral-mediated transfecting human interleukin-2 gene, the hepatic stem cell stably and effectively expressing interleukin-2 are obtained.This provides a good basis for further research on immuno-gene therapy of liver cancer.

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