COMBINED IL-2/IL-3 GENE THERAPY FOR G422 MOUSE GLIOBLASTOMA BY INTRATUMORAL INJECTION OF RECOMBINANT ADENOVIRUSES

Recombinant adenoviruses encoding murine IL 2 gene or IL 3 gene were directly injected into established subcutaneous tumor model of G422 glioblastoma cells. After treatment, the tumor size and survival of the glioblastoma bearing mice were observed. The splenic NK and CTL cytotoxicities were detected by standard 4 hour 51 Cr release assay. We also examined the histopathological changes of tumor by hematoxylin and eosin staining. The results showed that intratumoral injection of adenoviruses encoding murine IL 2 gene or IL 3 gene significantly inhibited the growth of G422 glioblastoma and prolonged the survival period of glioblastoma bearing mice. The CTL cytotoxicity of the gene therapy groups was significantly higher than that of the control groups, but NK activity remained unchanged, indicating that specific immunity contributes to the in vivo antitumor effect of the direct gene therapy. There were much more tumor necrosis and inflammatory cell infiltration in the tumor of the gene therapy groups. Combined IL 2/IL 3 gene therapy could induce higher level of CTL and enhance the therapeutic potential further. The results suggest that intratumoral injection of recombinant adenoviruses encoding certain kind of cytokines may be a useful approach in the treatment of a malignancy of the central nervous system.

Effects of Cimetidine on IL－2 and T Suppressor Cell Function in Rats with Obstructive Jaundice

Susceptibility to infection in patients with obstructive jaundice is much more higher than non-jaundiced patients. The reasons for this are not completely understood. It is postulated that this may have some relation to changes of patients′immune function. This article reported the changes of splenocyte IL-2 production and T Suppressor cell activity in rats with obstructive jaundice. Meanwhile, we also investigated effects of cimetidine on immune function in rats with bile duct ligation. The results show that IL-2 production in obstructive jaundiced rats significantly decreased and T suppressor cell activity markably increased. Cimetidine could remarkably enhance IL-2 production and suppress T Suppressor cell activity. Abmormaility of immune function may be one reason for high susceptibility to infection in patients with obstructive jaundice in perioperative period. Cimetidine, which could clearly improve immune function in rats with obstructive jaundice, might be a valuable agent for strengthening the capacity of fighting infection in patients with obstructive jaundice.

局限性硬皮病患者血清中可溶性白介素-2受体、肿瘤坏死因子-α及T淋巴细胞的表达

目的观察局限性硬皮病患者血清对T淋巴细胞增殖的影响和患者血清中可溶性白介素-2受体的表达变化。方法应用免疫学方法对39例局限性硬皮病患者血清中的sIL-2R、肿瘤坏死因子(TNF)-α表达和淋巴细胞增殖率进行检测,并与对照组比较。结果局限性硬皮病患者血清可明显降低T淋巴细胞增殖率;局限性硬皮病患者外周血中sIL-2R、TNF-α含量增高,其中活动期增高更加明显;差异均有统计学意义(P<0.05)。结论 T淋巴细胞增殖率的降低和血清中sIL-2R、TNF-α水平表达异常,这些免疫相关细胞和细胞因子可能参与了局限性硬皮病的发生和发展。

EFFICIENT ACTIVATION OF ANTITUMOR IMMUNITY BY IL-6 GENE-MODIFIED LEUKEMIA VACCINE IN COMBINATION WITH LOW DOSE CYCLOPHOSPHAMIDE AND LOW DOSEIL-2

Objective: To investigate the antitumor effect of the IL 6 gene modified erythroleukemia cells combined with low dose cyclophosphamide (Cy) and low dose IL 2 Methods: Mice inoculated with FBL 3 IL 6 in combination with low dose IL 2 and low dose cyclophosphamide (Cy) Results: Mice received combined therapy of FBL 3 IL 6, IL 2 and Cy developed tumors most slowly and survived much longer when compared with mice in control groups, with 5 out of 8 leukemia bearing mice being tumor free 100 days after the combined treatment To further explain the mechanism of the antitumor effects by the combined therapy It was found that combined therapy with low dose Cy, low dose IL 2 and FBL 3 IL 6 achieved maximal cytotoxic effects of nature killer cells and specific cytotoxic T lymphocytes, increased production IL 2, TNF and GM CSF from spleen lymphocytes in tumor bearing mice Vaccination with the FBL3 IL 6 also enhanced the cytotoxic activity of the peritoneal macrophages The results demonstrated that administration of low dose Cy and low dose IL 2 in combination with IL 6 gene modified leukemia vaccine could elicit potent anti leukemia effects, and the mechanisms involved in the antitumor process may include the induction of specific and nonspecific antitumor immunity, reversal of T suppressor cells that mediated local immuno suppression in tumor bearing mice Conclusion: The combined therapy with cytokine gene modified tumor vaccine, low dose of Cy and IL 2 might be a promising approach for the treatment of leukemia

Searching for Peptide Ligands of Interleukin-2 Receptor α Chain in Phage-displayed Peptide Library

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Production of Interleukin 2 like Growth Factor from the Mitogen Activated Peripheral Blood Lymphocytes of Indian Major Carp, Labeo rohita

Optimum conditions for in vitro production of interleukin 2 （IL 2） like activity from the Peripheral blood lymphocytes （PBL） of an Indian major carp, Labeo rohita were studied. Culture supernatants were generated by culturing the PBL in RPMI-1640 media supplemented with Glutamine and 10% fetal bovine serum （FBS） and stimulating with two different mitogens： concanavalin A （Con A） and Phytohaemagglutinin （PHA） at different concentrations separately. Significantly （P 〈 0.01） higher proliferation response was obtained from the culture supematant stimulated with concanavalin A （Con A） at a concentration of 10 lag mLl. The effect of phorbol myristate acetate （PMA） was also studied by co-stimulating PBL with Con A and PHA separately and it was found to synergistically enhancing the stimulation index with Con A whereas the stimulation index remain unchanged with PHA. The Con A （10 μg mLl） stimulated PBL were also cultured at different cell density, incubation period and incubation temperature in order to optimize the in vitro L. rohita IL2 production. The IL2 like activity was studied by lymphocyte proliferation assay on 72 h Con A blasts using WST based assay technique. Significantly （P 〈 0.01） higher stimulation indices were obtained when the PBL were cultured at a cell density of 1 × 10^6 cells mL^-1 for 30-36 h at an incubation temperature of 30 ℃. The IL2 like activity was purified by DEAE-Sepharose anion exchange chromatography and recorded between 70-130 mM NaCI with peak activity at 110 Mm NaCI. The molecular weight of the factor responsible for IL2 like activity was found to be 15-17 KD.

EFFECT OF ACUPUNCTURE ON IL 2-IFN-NKC IMMUNOREGULATORY NETWORK IN MICE WITH TRANSPLANTED HEPATOCARCINOMA

: To study the effect of acupuncture on interleukin 2 (IL 2)-interferon(IFN)-natural killer cells (NKC) immunoregulatory network of mice with transplanted hepatocarcinoma(HAC) in order to provide new evidence for acupuncture treatment of hypofunction of immune system. Methods:The 28 HAC-vaccinated BALB/C mice are randomly divided into control group (n=14) and acupunctrue group (n=14). In the latter group bilateral'Dazhui' (BL 11) and 'Zusanili' (ST 36) which are located according to the same positions indicated in Comparative Anatomy of Macro-animals, are needled once every day, twelve sessions altogether. Twenty-four hours after the needling treatment the mice are killed and the spleen is taken out to be made into cell suspension for assaying concentrations of IL2 (MTT method) and NKC (colorimetric method) respectively. Obital Serum IFN is determined by using (CPE microplate staining), and the tumor mass is taken out and balanced with an analytical balance (1/10000) to calculate the tumor inhibition rate according to the formula. Results: The tumor weight of the mice of aupuncture group is obviously decreased (inhibition rate 43.06%) while the activity of the IL2 and NKC, and the IFN tiler are increased greatly, which have significant differences compared with those of control group (P>0.01). Conclusion:The results of the study show that acupuncture can strengthen the positive immunoregulatory function of the IL2 -IFN-NKC network in immune hypofunction mice bearing HAC.

Estabishment of A Human Liver Cancer Cell Line Transfected with IL-2 cDNA and Its Biologic Activity

Objective To obtain IL 2 gene transfected human liver cancer cells and study IL 2 expression and its biologic activity in vivo. Methods\ Human liver cancer cells SMMC 7721 were cocultured with recombinant retroviral vector LNC IL 2,and screening was performed in G418 medium.The exogenous IL 2 cDNA at the DNA,RNA,and protein levels were determined by using dot hybridization,PR PCR and MTT methods respectively.The tumorigenesis and antitumorigenesis of the screened liver cancer cell with subcutaneous injection in nude mice were observed. Results and Conclusion\ The IL 2 cDNA was successfully integrated into SMMC 7721 cell genomic DNA and continuously expressed for more than 88 days.Subcutaneous vaccination of the nude mice with transfected cells revealed an obvious suppression of its tumorigenicity,and could induce antitumor activity in vivo. \ \

Cellular immune function and liver damage in post hepatitic cirrhosis

AIM To study the cellular immune function in patients with post hepatitic cirrhosis (PHC) and its relation with different liver damages.

CD4+ T cell responses in hepatitis C virus infection

Hepatitis C virus (HCV) infection is a major cause of liver damage, with virus-induced end-stage disease such as liver cirrhosis and hepatocellular carcinoma resulting in a high rate of morbidity and mortality worldwide. Evidence that CD4+ T cell responses to HCV play an important role in the outcome of acute infection has been shown in several studies. However, the mechanisms behind viral persistence and the failure of CD4+ T cell responses to contain virus are poorly understood. During chronic HCV infection, HCV-specific CD4+ T cell responses are rela- tively weak or absent whereas in resolved infection these responses are vigorous and multispecific. Persons with a T-helper type I profile, which promotes cellular effec- tor mechanisms are thought to be more likely to experi- ence viral clearance, but the overall role of these cells in the immunopathogenesis of chronic liver disease is not known. To define this, much more data is required on the function and specificity of virus-specific CD4+ T cells, especially in the early phases of acute disease and in the liver during chronic infection. The role and possible mechanisms of action of CD4+ T cell responses in deter- mining the outcome of acute and chronic HCV infection will be discussed in this review.

The Effect of Astragapolysaccharide on the Lymphocyte Proliferation and Airway Inflammation in Sensitized Mice

Summary: In order to investigate the regulating role of Astragapolysaccharide (APS) in the mice model of asthmatic airway inflammation, the airway eosinophil number, spleen T lymphocyte proliferation, level of IL 2 production and their relationships were studied in sensitized mice and sensitized mice treated with different concentrations of APS. The results showed that the number of eosinophils as well as lymphocytes in the airway of the sensitized animals were significantly increased, and a marked positive correlation between the inflammation cells and spleen T lymphocyte proliferation was found. Moreover, there was a positive correlation between inflammation cells and the level of IL 2 production. The APS of given dosage could significantly reduce the number of eosinophils in the airway of the sensitized animals. At the same time the level of IL 2 secreted by spleen T lymphocytes stimulated with ConA was also significantly decreased and there was a marked positive correlation between them. Our results suggested that APS of given dosage could prevent antigen induced the number of eosinophils infiltrating into the airway of sensitized mice and inhibit the proliferation and activation of lymphocyte and IL 2 production. Through its immuno regulating effect, APS can be helpful in the treatment of asthma.

Characterization of colonic dendritic cells in normal and colitic mice

AIM： Recent studies demonstrating the direct involvement of dendritic cells （DC） in the activation of pathogenic T cells in animal models of inflammatory bowel disease identify DC as important antigen presenting cells in the colon. However, very little is known about the properties of colonic DC. METHODS： Using immunohistochemistry, electron microscopy and flow cytometry we have characterized and compared colonic DC in the colon of healthy animals and interleukin-2-deficient （IL2-/-） mice that develop colitis. RESULTS： In the healthy colon, DC resided within the lamina propria and in close association with the basement membrane of colonic villi. Type i myeloid （CD11c^＋, CD11b^＋, B220, CD8） DC made up the largest （40-45%） population and all DC expressed low levels of CD80, CD86, and CD40, and had high endooltic activity consistent with an immature phenotype. In colitic IL2-/- mice, colonic DC numbers increased four- to five-fold and were localized within the epithelial layer and within aggregates of T and B cells. They were also many more DC in mesenteric lymph nodes （MLN）. The majority （〉85%） of DC in the colon and MLN of IL2./ mice were type 1 myeloid, and expressed high levels of MHC class II, CD80, CD86, CD40, DEC 205, and CCR5 molecules and were of low endocytic activity consistent with mature DC. CONCLUSION： These findings demonstrate striking changes in the number, distribution and phenotype of DC in the inflamed colon. Their intimate association with lymphocytes in the colon and draining lymph nodes suggest that they may contribute directly to the ongoing inflammation in the colon.

The Effect of Hydroxyapatite Ultrofine Powder on the Immunity Function of Tumor-bearing Mice

The inhibitory effect of hydroxyapatite ultrofine powder on tumor and the effect on the immunity function of body were investigated. The levels of IL 2 in the spleen cells and serum TNF levels in the tumor bearing mice at the 7th day and 14th after peritoneal injection of HAUFP were detected by using the methods of colorimetric analysis of MTT and crystal purple decoration, respectively. The disappearance of the ascites of the mice was observed. The results showed that the levels of IL 2 and TNF in the tumor bearing mice were higher obviously in the drug treated group than in the control group , the ascites growth was inhibited. It was suggested that HAUFP could increase the levels of IL 2 and TNF of the tumor bearing mice and improve the immune function of body.

AUGMENTATION OF IMMUNE FUNCTIONS AND AUTOLOGOUS TUMOR-KILLING ACTIVITY BY KAPPA-SELENOCARRAGEENAN IN MICE BEARING SARCOMA 180

Objective: To study the enhancement of the immune functions and autologous tumor killing (ATK) activity by kappa selenocarrageenan (KSC) in mice bearing sarcoma 180. Methods: To measure the effects of KSC and/or Cyclophosphamide (Cy) on natural killer (NK) activity, lymphokine activated killer (LAK) activity, the produc tion of interleukin 2 (IL 2), ATK activity and the growth of sarcoma 180 (S 180 ). Results: KSC promoted NK activity, LAK activity and ATK activity in vivo , increased IL 2 production at 40 mg/kg/d×9d. It also enhanced the antitumor action of Cy (20 mg/kg/d×9d) and offset the inhibition of Cy on immunocopetent cells. The ATK activity in splenocytes of S 180 bearing mice could be induced and increased by recombinant interleukin 2 (rIL 2) in vitro . Conclusion: KSC has an up regulating effect on the immune functions and ATK activity in tumor bearing mice. It can be used as a biological response modifier (BRM) in cancer biotherapy.

IL-2-loaded Polypeptide Nanoparticles for Enhanced Anti-cancer Immunotherapy

Interleukin 2 (IL-2) is widely used as an active immunotherapeutic agent in clinical metastatic cancers. However, its therapeutic concentrations do not last long due to its short half-life. Thus, only a transient proliferation of the anti-cancer CD8+ T cells can be achieved, resulting in poor efficacy. Therefore, the aim of this work was to create a system that promotes CD8+ T cell proliferation at the tumor site using IL-2 persistently present and activates an anti-cancer immune response. This goal was achieved by the design of the IL-2-loaded polypeptide nanoparticles (P-IL-2) where methoxy poly(ethylene glycol) block poly-[(N-2-hydroxyethyl)-aspartamide] phenylboronic acid was used to encapsulate IL-2 through boron-nitrogen coordination with poly(L-lysine). P-IL-2 significantly prolonged the circulation time of IL-2 and achieved a selective drug release at the tumor site in the presence of high levels of reactive oxygen species, thus activating an anti-cancer immune response and exerting a better anti-cancer effect. The half-life of P-IL-2 was 3.15-fold higher than that of IL-2, and the quantity of CD8+ T cells after using P-IL-2 was 1.89-fold higher than that after using IL-2. In addition, the combination of P-IL-2 and anti-CTLA-4 monoclonal antibody resulted in an enhanced immune activation. Hence, this work provides a new approach to improve the efficacy of IL-2 in anti-cancer immunotherapy.

Coexistence of Immune-Neuro-Endocrine Substances in the Rat Central Neurons

To investigate the expression of interleukin 2 (IL 2), metabotropic glutamate receptor subunit 1 (mGluR1) and estrogen receptor (ER) in neurons of the rat central nervous system (CNS) and identify the coexistence possibility of these immune neuro endocrine substances in the central neurons, the tri labeling immunocytochemical technique with different species specific primary antibodies (goat anti IL 2 antibody, rabbit anti mGluR1 antibody and mouse anti ER antibody ) were used to incubate two serial neighbor sections (one for demonstrating IL 2, another for mGluR1 and ER) of the cerebral cortex, medulla oblongata and spinal cord. There were IL 2 , mGluR1 and ER immunoreactivity (IR) positive labeled neurons in the above mentioned central areas. The IL 2 IR production showed brown color, located in the cytoplasm; In the neighbor serial section, the mGluR1 IR, production showed blue black color, located on the cell membrane; the ER IR production also showed brown color, located in the cytoplasm and nuclei. There were mGluR1/ER double labeled cells in the same section, which accounted for about 50 %-60 % of the total single and double labeled neurons. It was identified by projection check of serial neighbor sections that had mGluR1/ER/IL 2 tri labeled cells, which accounted for about 30 % of total mGluR1/ER double labeled neurons. The results indicate that mGluR1, ER and Il 2 can coexist in the same rat central neurons, therefore, providing morphological basis for the theory about immune neuro endocrine network at the cellular level for the first time.

The Effect of Tremella Fuciformis Spores Polysaccharides (TSP) on Immune Cellular Function of Mice in Vitro

TSP could markedly enhance the proliferative response of the murine splenocyte to LPS and induce the mitogenesis of the spleen cells.Furthermore,it was able to augment the activity of natural killer cell and ADGG;at a dosage of 25-250μg/ml,the ability of splenocytes to produce IL-2 induced by onA had been improved; at the concentration of 250μg／ml or more,TSP could inhibit the proliferative response of the murine lymphocyte to GonA and the ~3-HTdR spontaneous incorporation rate of thymocytes,and the inhibitory action ran in paralell with the increase in concentration of TSP.

Immunoregulatory Effects of Indomethacin on Rats with Trauma

In this article, we invertigated changes of immune functions and immunoregulatory effects of indomethacin on rats with trauma. The results show that spontaneous suppressor T cell activity of spleen significantly increased and Interleukin 2 production and DNA synthesis capacity of splenocytes markedly decreased in rats with trauma. Indomethacin could markedly improve immune function , decreased spontancous suppressor T cell activity and prompted Interleukin 2 production and DNA synthesis capacity of splenocytes.

Electrophoretic Analysis on RNA of Deltamethrin-resistant Culex Pipiens Pallens

TSP could markedly enhance the proliferative response of the murine splenocyte to LPS and induce the mitogenesis of the spleen cells.Furthermore,it was able to augment the activity of natural killer cell and ADGG;at a dosage of 25-250μg/ml,the ability of splenocytes to produce IL-2 induced by onA had been improved; at the concentration of 250μg/ml or more,TSP could inhibit the proliferative response of the murine lymphocyte to GonA and the ￣3-HTdR spontaneous incorporation rate of thymocytes,and the inhibitory action ran in paralell with the increase in concentration of TSP.

Growth inhibition of interleukin-2 receptor gene-transduced peripheral blood lymphocytes on human ovarian cancer cells

Objective To investigate the growth inhibition of interleukin 2 receptor (IL 2R) gene transduced peripheral blood lymphocytes (PBLs) on human ovarian cancer cells Methods Interleukin 2 (IL 2) and IL 2R genes were transfected into human ovarian cancer cell line 3AO and PBLs, respectively, using the same Fugene vector Twenty four hours later transfected and nontransfected PBLs were cocultured with transfected and nontransfected 3AO for 48 hours Cytotoxity of PBLs on 3AO was detected by the MTT assay Results The morphology of IL 2 transduced 3AO and IL 2R transduced PBLs remained unchanged 3AO cells could be transfected with the IL 2 gene and expressed IL 2 mRNA, and PBLs could be transfected with the IL 2R gene and expressed IL 2R mRNA IL 2 transduced 3AO cells enhanced their response to the cytotoxity of PBLs Furthermore, growth inhibition of PBLs to 3AO cells increased significantly when the IL 2R was transfected into PBLs and when the IL 2 gene was transfected into 3AO cells and the two were combined Conclusions IL 2R gene transduced PBLs are able to enhance their cytotoxity on IL 2 gene transduced ovarian cancer cells This method may be a new way to investigate IL 2 gene therapy for ovarian cancer