Expression of C Ⅱ TA Gene in Five Human Malignant Hematological Cell Lines and Its Significance

The role of MHC class Ⅱ transactivator (C Ⅱ TA) in constitutive or IFN-γ inducible expression of HLA molecules in human malignant hematological cell lines was investigated. The expression of HLA molecules and C Ⅱ TA protein was detected by Western blot, immunohistochemistry and flow cytometry. The expression of C Ⅱ TA gene was determined by RT-PCR. The capability of peripheral blood T cell reaction stimulated by tumor cells was monitored by mixed lymphocyte reaction. It was found that the HLA Ⅱ-positive tumor cells expressed the C Ⅱ TA quite well, and the expression of HLA Ⅰ + Ⅱ was increased in the tumor cells with constitutive or inducible expression of C Ⅱ TA after induced by IFN-γ. The tumor cells which did not express C Ⅱ TA after induced by IFN- γ were not response to the expression of HLA Ⅱ promoted by IFN- γ. It suggests a correlatior between the inability of some malignant hematological cell lines in response to IFN-γ for HLA expression and the deficiency in the inducible expression of C Ⅱ TA, indicating C Ⅱ TA might take part in the regulation of HLA Ⅰ + Ⅱ expression in the tumor cells, which might play an important role in tumor immunologic escape.     

CⅡTA基因在五种人类恶性血液病细胞株细胞中的表达及意义

目的:探讨5种血液病细胞株细胞的HLA-Ⅱ类抗原表达,以及对IFN-γ诱导HLA分子表达的反应性与MHCⅡ类分子反式激活因子 (CⅡTA) 表达的关系.方法:采用流式细胞术和免疫组化法检测肿瘤细胞HLA分子及CⅡTA 蛋白的表达,RT-PCR检测肿瘤细胞CⅡTA基因表达.混合淋巴细胞反应检测肿瘤细胞刺激外周血T细胞反应的能力.结果:肿瘤细胞HLAⅡ类分子表达与CⅡTA表达一致; 结构型或诱导型表达CⅡTA的肿瘤细胞,经IFN-γ作用后其HLAⅠ、Ⅱ类抗原表达增高;IFN -γ诱导后仍不表达CⅡTA的肿瘤细胞,其对IFN-γ促HLAⅡ表达的作用不反应.Jurkat诱导后刺激T细胞表达高水平的IL-2 mRNA.结论:某些恶性血液病细胞株细胞对IFN-γ不能诱导HLA分子表达与CⅡTA诱导型表达缺陷有关,表明CⅡTA参与调控肿瘤细胞 HLAⅠ、Ⅱ类抗原表达,可能在肿瘤免疫逃逸中起重要作用.     

<Emphasis Type="Italic">In vitro</Emphasis> biological activity of anti-C II TA hammerhead ribozyme—A novel approach for autoimmune diseases

Summary This study investigated the feasibility of using an hammerhead ribozyme against C II TA, a major regulator of MHC II antigens, to repress the expression of MHC II molecules on Hela cells. A hammerhead ribozyme (Rz464) specific to 463–465 GUC triplet of C II TA and its target gene were transcribed, then mixed up and incubatedin vitro. The cleavage products were analyzed by PAGE and silver-staining. Rz464 was then inserted into the pIRES2-EGFP vector (pRz464). Stable transfectants of Hela with pRz464 were tested for class II MHC induction by recombinant human interferon-gamma (IFN-γ). mRNA of C II TA was measured by RT-PCR. Our results showed that Rz464 could exclusively cleave C II TA RNA. When induced with IFN-γ, the expression of HLA-DR,-DP,-DQ on pRz464⁺ Hela was induced, and the mRNA content of C II TA decreased too. It is concluded that Rz464 could inhibit C II TA and thus the family of genes was regulated by C II TA: MHC II molecules. These results provided insight into the future application of Rz464 as a new nucleic acid drug against auto-immune diseases. LIU Fang, female, born in 1976, Postgraduate Student This project was supported by the Key Sub-projects of Science and Technology Development Fund of Shanghai Municipality (No. 00DJ14001-8).     

Clinical Value of Vascular Endothelial Growth Factor Combined with Interferon-γ in Diagnosing Malignant Pleural Effusion and Tuberculous Pleural Effusion

In order to investigate the clinical value of vascular endothelial growth factor （VEGF） combined with interferon-γ （IFN-γ） in diagnosing malignant pleural effusion and tuberculous pleural effusion, 42 cases of malignant pleural effusion and 45 cases of tuberculous pleural effusion in Tongji Hospital, from March 2004 to May 2005, were included, The carcinoembryonic antigen （CEA）, VEGF and IFN-γ levels of pleural effusion were detected by using ELISA, and adenosine deaminase （ADA） activity was determined by using enzyme kinetic analytical method. The sensitivity, specificity, accuracy and area under the curve （AUCR^ROC） of CEA and VEGF, VEGF/IFN-γ ratio, ADA and IFN-γ were measured by receiver operating characteristic curve （ROC）, The results showed that CEA, VEGF levels and VEGF/IFN-γ ratio were significantly higher and the ADA and IFN-γ levels were significantly lower in malignant group than those in tuberculous group （P〈0,01）, The sensitivity, specificity, accuracy and AUCR^ROC of VEGF/IFN-γ ratio （88,7%, 99,8%, 94,4%, 0.96 respectively） were higher than those of CEA （67.8%, 96.1%, 82,4%, 0.78 respectively） and VEGF （81,5%, 84,3%, 82.9%, 0.79 respectively）. The sensitivity, specificity, accuracy and AUCR^ROC of IFN-γ （85.7%, 96,4%, 90.9%, 0.94 respectively） were higher than those of ADA （80,2%, 87,6%, 83.8%, 0,81 respectively）. It was concluded that VEGF/IFN-γ ratio and IFN-γ could be used as valuable parameters for the differential diagnosis of malignant pleural effusion and tuberculous pleural effusion.     

Relationship between Host Survival and the Type of Immune Response in Different Organs during Disseminated Candidiasis

To examine the relationship between host survival and the type of immune response in different organs during disseminated candidiasis, the murine model of disseminated candidiasis was established by injection with Candida albicans via tail vein. The survival time was observed for up to 60 days. And the expression levels of cytokines in the spleen and kidney, including IFN-γ and IL-4, were determined with RT-PCR. Our results showed that in the spleen, both non-fatal and fatal inoculum caused a type Ⅱ immune response with steady expression levels of IFN-γ and the obviously increased levels of IL-4. While in the kidney, non-fatal inoculum induced a type Ⅰ immune response with the obviously increased levels of IFN-γ and the steady expression levels of IL-4. However, fatal inoculum induced a type Ⅱ immune response with a constant expression of IFN-γ and the evidently increased levels of IL-4. It is concluded that in disseminated candidiasis, host survival is associated with the type of immune responses in the kidney, but not in the spleen.     

Enhanced expression of HLA class I molecules in human hepatocellular carcinoma cells transduced with gamma-interferon gene.

OBJECTIVE To investigate the expression of exogenous gamma-interferon gene in human hepatocellular carcinoma cells following retroviral transduction and the effect on the expression of surface HLA class I molecules.

METHODS Retroviral vector pLXSN was used to introduce human gamma-interferon (IFN-gamma) gene into four different human hepatocellular carcinoma cell lines (HCC). The G418-resistant colonies were isolated and cloned. The integration and expression of IFN-gamma gene were determined by PCR and RT-PCR analysis. A bioassay method was used to test the amount of IFN-gamma secreted by gene modified HCC cells. The expression of HLA class I molecules in HCC cells were analyzed by flow cytometry using indirect fluorescence staining.

RESULTS Four different HCC cell lines were successfully transduced with human IFN-gamma gene using retroviral vector. The integration and expression of IFN-gamma gene were shown only in the transduced cells. All four genetically modified HCC cells can secrete varied amount of IFN-gamma and demonstrate a significant up-regulation of surface HLA class I antigens. One specific HLA class I antigen, HLA-A2, has almost the same degree of increase as that of the total HLA class I molecules after transduction with IFN-gamma gene.

CONCLUSIONS Gene modification with IFN-gamma gene can significantly enhance the expression of HLA class I molecules in HCC cells and may increase its immunogenicity. These gene modified tumor vaccines can be helpful in tumor biotherapy.

     

Enhanced expression of HLA class Ⅰ molecules in human hepatocellular carcinoma cells transduced with γ-interferon gene

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Effects of intrinsic nitric oxide on the expression of interleukin-4 and IFN-γ mRNA in the bronchial and lung tissues of sensitized rats

Summary To investigate the effects of intrinsic nitric oxide (NO) on the expression of interleukin-4 (IL-4) mRNA and interferon-γ (IFN-γ) mRNA in the airway inflammation of asthma, the rat models of asthmatic inflammaiton were established by sensitizing and then challenging the animals with ovalbumin. The 24 animals were randomly divided into control group, sensitized group, sensitized and L-Arg-treated group as well as L-NAME-treated group equally. By using in situ hybridization combined with compute physiological quantitative imaging analysis techniques, the influence of intrinsic NO on the expression of IL-4 mRNA and IFN-γ mRNA in the airway inflammatory cells was observed. In situ hybridization study demonstrated that IL-4 mRNA expression was obviously increased as compared with that in the control group, mainly distributed in the inflammatory cells in the submucous of airways in the sensitized group. The increase of intensity of IL-4 mRNA expression was positively correlated with the numbers of eosinophil (Eos) and lymphocyte (both with P < 0.05) in the sensitized group. There was no statistically difference in IFN-γ expression between the control group and the sensitized group. Imaging analysis showed that L-NAME could inhibit the expression of IL-4 mRNA (P < 0.05) and increase the expression of IFN-γ mRNA (P < 0.05), while L-Arg could increase the expression of IL-4 mRNA in inflammatory cells (P < 0.05). It was indicated that a suitable levels of intrinsic NO can influence the expression of IL-4 mRNA of Th2 lymphocytes and the expression of IFN-γ mRNA of Thl lymphocytes and in turn, promote the development of asthmatic airway inflammation.     

Effects of Baicalin on Expression of Inducible Nitric Oxide Synthase in Cultured Fibroblasts Stimulated by Cytokines

Objective: To investigate the effects of baicalin on expression of inducible nitric oxide synthase (iNOS) in fibroblasts and its mechanisms in treating psoriasis. Methods: Fibroblasts cultured in vitro were stimulated with tumor necrosis factor-α(TNF-α), interferon-γ (IFN-γ), interleukin-8 (IL-S) in different groups. iNOS was detected by western blot and immunocytochemistry assay, and in addition, the effects of baicalin on its expression were investigated. Results: Fibroblasts did not express iNOS without cytokine stimulation. When treated for 24 h with 1. 0× 106 U/L TNF-α, 0.2× 106U/L IFN-γ, 0.2× 106 pg/L IL-8 alone or in combinations indicated, fibroblasts produced iNOS when stimulated by TNF-α alone while neither IFN-γ nor IL-8 could induce the production of iNOS. The combination of TNF-α and IL-8 induced a strong expression of iNOS, the combined exposure of three kinds of cytokines showed an even stronger effects. The strongly stained area was in the cytoplasm near the nuclei. Expression of iNOS induced by TNF-α and IL-8 was inhibited by 50 μg/ mi of baicalin. Conclusion: Fibroblasts might express iNOS when stimulated by certain cytokines. Baicalin decreased production of nitric oxide through inhibiting the expression of iNOS, furthermore it reduced inflammation, which might be part of its mechanisms in treating psoriasis.     

Inhibitory Mechanism of Inteferon-gamma on Human Fibroblasts from Tenon＇s Capsule

The inhibitory mechanism of interferon-gamma (IFN-γ) on the fibroblasts from Tenon‘s capsule was studied. By using immunohistochemical SP method and pathological image system, the inhibitory effects of IFN-γ on the expression of transforming growth factor beta receptor I in the in vitro cultured fibroblasts from Tenon‘s capsule were quantitatively analyzed. The results showed that IFN-γ could reduce the expression of transforming growth factor beta receptor I in the fibroblasts with the following dose-effect relationship: Y= 1937.5—134.2 Igx (r=- 0. 971, P＜0.01).It was concluded that IFN-γ could inhibit the expression of transforming growth factor beta receptor I in the fibroblasts from Tenon‘s capsule. The modulation of the transforming growth factor beta receptor I expression by IFN-γ, may be beneficial to the alleviation of the hyperplasia of scar after trabeculectomy.     

The effect of vitamin a on secretion of IFN-γ and IL-4 in A549 cells induced by <Emphasis Type="Italic">Mycoplasma Pneumoniae</Emphasis>

In order to investigate the effect of vitamin A （VA） on the secretion of IFN-γ and IL-4 in Mycoplasma Pneumoniae （MP）-induced A549 cells, A549 cells were co-cultured with MP for different time lengths and then the levels of IFN-γ and IL-4 in the cell culture supernatants were detected before and after treatment with different concentrations of VA by using the enzyme-linked immu-nosorbent assay （ ELISA）. The results showed that the level of IFN-γ and IL-4 in the supernatants of MP-induced A549 cells was much higher than that in non-induced cells （P〈0.01）. After application of VA, IL-4 level was not increased until the concentration of VA was up to 0.5×10-5 mol/L （P〈0.01）. However, with concentration of VA increased up to 1×10-4 mol/L, IL-4 was significantly suppressed （P〈0.01）. It was concluded that MP could induce the secretion of IFN-γ and IL-4 in A549 cells. VA could inhibit the secretion of IFN-γ and increase the IL-4 level in MP-induced A549 cells. However, high concentration of VA had an inhibitory effect on the secretion of IL-4 as well as on the IFN-γ. These data provided a theoretical basis for the application of VA in MP pneumonia in the clinical practice.     

Effects of concurrent use of rh-IFN-γ and curcumin on the antiproliferative capacity of HL-60 cells

Summary To understand the effect of rh-IFN-γ on the ability of curcumin to kill HL-60 cellsin vitro, the myeloid leukemic cell line HL-60 was studied by using cell culture. BrdU incorporation rate was examined by SABC, DNA content was determined by flow cytometry and apoptotic cell percentage was determined by TUNEL method. The results showed that curcumin inhibited proliferation of leukemic cells in a dose-dependent manner. When HL-60 cells were treated with 25 μmol curcumin for 24 h, the proliferative inhibitory rate was 43. 75±2. 00 %. This effect could be enhanced obviously by IFN-γ, the combined proliferative inhibitory rate increased to over 80 %. The 5-BrdU incorportion rate and the distribution of DNA content indicated that curcumin could arrest cells in the G₁,/G₀ and G₂/M phase of cell cycle. At the same time, the sub-G₁ peak (apoptotic peak) appeared. After IFN-γ combined with curcumin DNA synthesis rate decreased further. It showed a significant difference when compared with single drug group (P < 0. 05). Meanwhile, sub-Gi peak also increased. The percentage of TUNEL positive cells was aslo increased. It is concluded that IFN-γ can enhance the antiproliferative ability of curcumin against HL-60 cells.     

Effect of polypeptide CH50 on macrophage activationin vivo and antitumor function

Surgery,chemotherapy and radiotherapy aremajor methods used in tumor therapy.After the re-moval of large tumor tissue by surgery,the mainproblem to be faced with is the inhibition and elimi-nation of the residual tumor cells in vivo. Ifmacrophages can be effectively activated,the residualtumor cells could be inhibited and eliminated as earlyas possible,the conditions favorable to the induce-mentof specificimmune response againsttumorcouldalso be created. Recombinant polypeptide CH50 wasprepar…     

Peroxisome proliferator-activated receptor-γ activated by ligands can inhibit human lung cancer cell growth through induction of apoptosis

Summary To study the expression of peroxisome proliferator-activated receptor-γ(PPAR-γ) in lung cancer cells, and to testify if the PPAR-γ agonists can inhibit human lung cancer cell growth through induction of apoptosis, PPAR-γ was detected in two lung cancer cell lines by RT-PCR and immunohistochemistry, the inhibition of human lung cancer cell growth was investigated by MTT and cell counts, and the apoptosis was assessed by TUNEL. The results showed that: (1) PPAR-γ expressed on two lung cancer cell lines; (2) PPAR-γ activated by ligands could inhibit human lung cancer cell growth remarkably; (3) PPAR-γ agonists could induce apoptosis to inhibit lung cancer cell growth. It was concluded that PPAR-γ expressed in lung cancer cell can be activated by ligands and can inhibit lung cancer cell growth through induction of apoptosis. Zhang Min, female, born in 1977, Doctor in Charge This project was supported by a grant from Natural Sciences Foundation of Hubei Province (Serial No. 98J102).     

Effect of sublingual immunotherapy on level of cytokines in PBMCs of patients with allergic asthma

This study examined the possible mechanism of sublingual immunotherapy(SLIT) in the treatment of allergic asthma.Forty asthma patients allergic to dust mite were enrolled.They received SLIT with dermatophagoides farinae(Der.f) drops for one year.Thirty healthy subjects served as controls.The levels of IL-4 and IFN-γ of peripheral blood mononuclear cells(PBMCs) were determined in allergic asthma patients before and after the SLIT as well as the healthy subjects.The results showed that the level of IL-4 was substantially increased and that of IFN-γ remarkably decreased in the patients before the SLIT as compared with those in the healthy subjects(P<0.05).After the SLIT,the level of IL-4 was significantly reduced and that of IFN-γ elevated in these allergic asthma patients.It was concluded that sublingual immunotherapy is effective for patients with allergic asthma.And it may work by regulating the balance of Th1/Th2 through changing the expression of IL-4 and IFN-γ in PBMCs.     

Local immune regulatory effects of Bangdeyun on the endometrium of mice with embryo implantation dysfunction during the implantation time

This study examined the effects of Bangdeyun on the expressions of nuclear factor-kappaB （NF-κB）, interferon-gamma （IFN-y） and interleukin-10 （IL-10） in the endometrium of mice with embryo implantation dysfunction （EID） during the implantation time （namely on pregnancy day 5, 6, 7 and 8） and explored the local immune regulatory effects of Bangdeyun. The gestational mice were randomly divided into normal group, model group and Bangdeyun-treated group. EID models of mice were established by using indomethacin. The endometrial expression of NF-κB was detected by immunohistochemistry and Western blotting. IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay （ELISA）. The results showed that in the normal group, NF-κB and IFN-γ were weakly expressed and IL-10 was strongly expressed in the endometrium during the whole implantation period. In the model group, the expressions of NF-κB and IFN-T were increased on pregnancy day 5, 6 and 7, and IL-10 expression decreased during the whole implantation time when compared with those in the normal group （P〈0.01 for all）. In the Bangdeyun-treated group, little amount of NF-κB and IFN-γ was expressed and IL-10 expression was strong, much the way they were expressed in the normal group （P〉0.05）. The expressions of NF-κB and IFN-T were much lower in the Bangdeyun-treated group than those in the model group on pregnancy day 5, 6 and 7 （P〈0.01 for all）, while the expression of IL-10 was much higher than in the model group during the whole implantation time （P〈0.01）. It was suggested Bangderun may favor a shift from Thl- to Th2-type immune response, therefore inhibiting the maternal immune rejection, inducing the immune tolerance and improving the fetal implantation.     

Correlation between the Expression of Fas, Bcl-2 in Peripheral Blood Lymphocytes and the LeveL of IFN-γ,IL-4 in Setum of Patients with Condyloma Acuminata

Summary In order to investigate the correlation between the expression of the apoptotic regulatory proteins (Fas, Blc-2) in peripheral blood lymphocytes (PBLC) and the level of IFN-γ and IL-4 in serum of the patients with condyloma acuminata (CA) in the immune pathogenesis of CA, indirect immunofluorescence labeling method of flow cytometer and solid sandwich ELISA method were performed for detecting the expression of Fas, Bcl-2 in PBLC and the level of IFN-γ and IL-4 in serum of 60 cases of CA. The results showed the expression level of Fas in PBLC of CA was significantly higher than in the normal control group, but the expression level of Bcl-2 was significantly lower (bothP<0.01). The level of IFN-γ in serum of CA was significantly lower than in the normal control group (P<0.01), but IL-4 was significantly lower (bothP<0.01). The expression of Fas in PBLC had a negative correlation with the level of IFN-γ in serum of patients with CA, but had a positive correlation with the level of IL-4: The expression of Bcl-2 had a positive correlation with the level of IFN-γ, but had a negative correlation with the level of IL-4. All the correlation coefficients had significant differerce byt test (P<0.01). It was suggested abnormal apoptosis in PBLC, the suppressed secretion of the TH1-associated cytokines (eg: IFN-γ) and the increased secretion of the TH2-associated cytokines (eg: IL-4) existed in the patients with CA and might play an important role in the immune pathogenesis of CA. LIU Houjun, male, born in 1963, Associate Professor     

KBv200细胞株耐药逆转前后HLA、B7抗原的表达

目的 探讨肿瘤多药抗性细胞的免疫逃避机制。方法 利用特异性切割mdr1的核酶为工具,以表达Mdr1的耐药细胞株KBv200为靶细胞,采用脂质体转染技术,将含核酶的质粒pHβApr-1neo/5mR3及空载体pHβApr-1neo导入KBv200及其亲本KB细胞内,运用Northern-blotting、免疫组化方法观察核酶对mdr1 mRNA及P-gp的影响,运用流式细胞仪技术检测各种不同细胞亚株HLA-Ⅰ、HLA-Ⅱ、B7-1、B7-2的表达。 结果 含核酶的质粒pHβApr-1neo/5mR3及空载体pHβApr-1neo可以在KB、KBv200细胞中稳定表达,核酶可以特异性地切割mdr1,导致KBv200/5mR3的mdr1 mRNA含量下降,P 糖蛋白（P-gp）表达减低,各种不同细胞亚株均表达较强的HLA-Ⅰ类抗原,而HLA-Ⅱ、B7-1、B7-2的表达较低。各亚株HLA-Ⅰ表达无明显差异,但HLA-Ⅱ、B7-1、B7-2的表达变化较大,KB的HLA-Ⅱ、B7-1、B7-2的表达较KBv200强,经化疗药物作用后KB的HLA-Ⅱ、B7-2进一步表达增强,核酶逆转后,KBv200/5mR3 HLA-Ⅱ、B7-1、B7-2的表达趋于接近KB水平。 结论 mdr1-核酶在细胞内具有一定的逆转肿瘤多药抗性的生物学效应;多药耐药细胞和敏感株细胞有着不同的免疫逃避特点,与敏感株相比,KBv200较易逃避机体的免疫反应。     

Dysregulation of the TGF-β Postreceptor＇Signaling Pathway in Cell Lines Derived from Primary or Metastatic Ovarian Cancer

Summary Transforming growth factor-beta (TGF-β) may cause cell cycle arrest, terminal differentiation, or apoptosis in most normal epithelial cells, whereas most malignant cell lines are resistant to TGF-β. Mechanisms of resistance to TGF-β caused by modulation of cell cycle regulators and/or inactivation of components of the TGF-β signaling transduction pathway such as C-myc and Smad4 are not well understood. To investigate the potential association between loss of sensitivity to TGF-β and expression status of transforming growth factor receptor II (TβRII), Smad4, CDC25A and C-myc in 14 cell lines derived from ovarian cancer, the expression levels of these genes were detected by semi-quantitative RT-PCR. Normal ovarian surface tissues were used as controls. The expression of TβR II was detectable in all of 14 cell lines. The expression of Smad4 was decreased in 10 cell lines and 9 cell lines overexpressed CDC25A, as compared to normal controls. CDC25A gene was overexpressed with 88% (8/9) in tumorigenic cell lines as determined by xenografts in nude mice, and only in 20% (1/5) of non-tumorigenic cell lines (P<0.05). C-myc was not overexpressed in any of these cell lines. The loss of sensitivity to TGF-β of cell lines derived from ovarian cancers may be related to a decreased expression of Smad4, which mediates TGF-β induced growth inhibition, and/or an overexpression of CDC25A. This overexpression of CDC25A correlates with increased tumorigenicity of ovarian cancer cell lines. The loss of sensitivity to TGF-β is not associated with a lack of TβRII. XI Ling, female, born in 1972, M.D., Ph.D., Doctor in Charge This project was supported by grants from the National Crackajack Youth Foundation (30025017) and the National Emphasis Basis Research Project of China (2002CB513100).