牛津生物医学研究所的化验服务部门拥有超过 25 年的生物医学研究和化验开发经验。该公司已获得 40 多项 NIH 资助和合同来开发新的检测方法，我们的能力涵盖广泛的学术和制药需求。
我们为研究人员提供使用夹心和竞争性免疫检测进行细致分析的途径，因为以及使用最先进的比色法、荧光法和化学发光法进行的一系列酶和分析物测定。这些服务定期提供给学术、制药和政府科学家，以支持体外研究以及从使用实验室动物和人类受试者的研究中提取的样本。我们的主要专长是使用基于微孔板的测定系统测量抗氧化能力、氧化应激和炎症的生物标志物。我们的回头客包括 NIH、主要制药公司公司和学术研究中心。
我们了解您的研究是独一无二的，甚至在获取样本进行分析之前就需要花费大量时间和费用。我们可以进行试点研究，帮助您调整和优化参数，然后再将您的工作扩展到提供最高统计显着性的水平。我们的技术代表随时准备与您合作，设计最佳的检测组合以满足您的需求，并根据您的具体项目报价服务。我们协助确定样本采集的最佳条件、所需样本的数量以及适当的储存和运输程序。
为确保最高程度的可靠性，典型样本的分析使用多次重复稀释。结果的准确度和精密度至关重要。每个检测都包含多个对照，以确保报告的结果是在标准曲线的最佳范围内获得的。在报告之前验证所有结果的准确性和可靠性。
案例研究
开发了一种多级层析纯化方案，用于从复杂的生物来源中分离天然产物，并测试了生物效应体外和体内动物模型。优化了一组新型蛋白质分析物的酶免疫测定方案，并将生产从最初的测试批次扩大到规模不断扩大的持续生产。 Oxford Biomedical Research 优化了客户的原型免疫测定，能够提高灵敏度和保质期，并为批次间的可重复性实施适当的质量保证。客户每月都会收到完整的待售套件。分析患者样本中的氧化应激生物标志物。医学院实验室需要分析一组 60 名受试者的氧化应激。该研究需要测量血清和尿液样本中的五种生物标志物。牛津生物医学研究所提供了这些服务，并能够在不到一周的时间内提供一份包含统计分析的最终报告。返回顶部显示第 1 - 4 件，共 4 件产品CAT.#SizePriceCUPRACFood and Beverage Antioxidant AssayFS021 Kit$395.00Histamine EIA Kit for Food and BeveragesFS351 Kit$610.00Oil Polarity AssayFS621 Kit$300.00TBARS Assay for Food and BeveragesFS501 Kit$245.00返回顶部显示 1 - 10 件，共 43 件产品CAT.#SizePrice12-LOX 白细胞型（活性）LX12L100 U$220.0012-LOX 血小板型（非活性）LX12PS0.2 mL$245.0015-LOX form-2 StandardLX26100 ng$220.00AACOCF3FA3050 mg$305.00抗 cPLA2，小鼠多克隆抗体 PL051 .0 mg$570.00Anti-13(S)-HODE，山羊多克隆 H01100 ug$325.00Anti-15-LOX 形式 2，多克隆 LX250.1 mL$395.00Anti-6-keto-PGF1-alpha，绵羊多克隆 PG401.0 mL$200.00Anti-8- LOX，兔多克隆抗体LX080.1 mL$395.00Anti-9(S)-HODE，山羊多克隆抗体H070.1 mL$325.00分页当前页1页2页3页4页5下一页下一页›最后一页最后»返回顶部显示 1 - 10 件，共 488 件产品CAT.#SizePriceAc-TVASSSTA OctapeptidePI145.0 mg$600.00Active Human PAI-1 functional assay kitPI90Kit$615.00Active Human uPA Functional Assay KitPL91Kit$615.00Active Mouse tPA (recombinant)PA560.1 mg$430.00Active小鼠 uPA 功能检测试剂盒 PL92Kit $615.00活性小鼠尿激酶，Alexa Fluor 700 标记 VA560.05 mg$615.00活性鼠 PAI-1 功能测定试剂盒 PI91 试剂盒$615.00活性猪 PAI-1 功能测定试剂盒 PI95 试剂盒$615.00活性猪 tPA 功能测定 UP60 试剂盒$615.00活性猪 tPA，重组 UP59 0.1 mg$565.00分页当前页1页2页3页4页5页6页7页8页9…下一页pageNext ›Last pageLast »返回顶部显示第 1 - 2 个，共 2 个产品CAT.#SizePriceHuman Therapeutic IgG1 ELISA KitNF011 Kit$595.00Human Therapeutic IgG4 ELISA KitNF041 Kit$595.00返回顶部显示 1 - 10 件，共 77 件产品CAT.#SizePriceAndrostenedione EIA KitEA68Kit$245.00Anti Human PKC Panel of Polyclonal AntibodysPK50Panel$1,090.00Anti Human PKC-beta I Polyclonal AntibodyPK150.1 mL$395.00Anti Human PKC-beta II, Polyclonal AntibodyPK 201.0 毫升 395.00 美元抗蛋白激酶共有序列 PK751.0 mL$250.00抗蛋白丝氨酸/苏氨酸磷酸酶 2A/CPP220.1 mg$415.00抗蛋白丝氨酸/苏氨酸磷酸酶 2B AlphaPP160.1 mg$415.00抗兔蛋白丝氨酸/苏氨酸磷酸酶 X/CPP170.1 mg$415.00抗兔蛋白丝氨酸/苏氨酸磷酸酶 2A/BaPP200.1 mg$415.00抗兔蛋白丝氨酸/苏氨酸磷酸酶 2CabPP180.1 mg$415.00分页当前页1页2页3页4页5页6页7页8下一页下一页最后一页最后» Potent antitumor effects mediated by local expression of the mature form of the interferon-纬 inducing factor, interleukin-18 (IL-18) AbstractIL-18 is produced during the acute immune response by macrophages and immature dendritic cells. IL-18 receptors are induced on T cells and NK cells by IL-12 and together they enhance a cellular immune response. We constructed retroviral and adenoviral vectors encoding the mature bioactive murine IL-18 in order to examine their immune and antitumor effects in murine tumor models. Secretion of bioactive IL-18 from murine tumor cells was facilitated by transfecting them with recombinant viral vectors carrying the prepro leader sequence of human parathyroid hormone fused to the 5鈥?end of the mature murine IL-18 cDNA. Direct injection of the IL-18 recombinant adenoviral vector (Ad.PTH.IL-18) into an established MCA205 murine fibrosarcoma completely eradicated tumor in all animals with concomitant induction of protective systemic immunity. Co-administration of systemic IL-12 provided synergistic antitumor effects when combined with peritumoral injections of Ad.PTH.IL-18 without apparent side-effects as we observed with systemic administration of IL-18. Depletion of asialo GM-1+ cells completely abrogated the antitumor effects of Ad.PTH.IL-18, suggesting a major role for NK cells in mediating the anti-tumor effects of IL-18. Peritumoral injection of Ad.PTH.IL-18 was also associated with reduced numbers of CD8+ cells found鈥墂ithin the tumor (HBSS versus Ad.PTH.IL-18, P鈥?lt;鈥?.0001). this suggests that il-18 could be utilized as an alternative cancer gene therapy especially when combined with systemic administration of il-12. IntroductionInitiation of the adaptive immune response is believed to be a result of local necrotic death of tissues and release of inflammatory cytokines by resident macrophages, mast cells and recruited polymorphonuclear leukocytes. The initial cytokines released at the site of an acute inflammatory response include IL-1, IL-6, IL-12 and IL-18,1 as well as a number of chemokines. Tumor recognition3 also requires induction of an immune response, recruitment and maturation of dendritic cells,3 and IL-12 mediated expansion of T cells specific for tumor antigen.4 IL-18 is a recently identified monokine with important immunoregulatory functions which induces high levels of IFN-纬 secretion from both NK and T cells.5 IL-12 synergizes with IL-18 to enhance IFN-纬 production by up- regulating the expression of IL-18 receptors on T cells.6,7 IL-18 is produced by cells of monocyte lineage, augments NK cytolytic activity and enhances proliferation of T cells.5 We and others have reported significant antitumor effects of recombinant (r) IL-18 administration鈥塱n tumor-bearing mice.8,9 Systemic administration of rIL-18 suppressed the growth of MCA205, but complete eradication of tumor was not observed when treatment with rIL-18 was initiated on day 7 or later. Combination therapy with systemic rIL-12 and rIL-18 administration was associated with significantly enhanced antitumor effects when compared with that of rIL-12 or rIL-18 alone; however, lethal side-effects were observed concomitant with extremely high serum levels of IFN-纬. To overcome these problems, we examined the effectiveness and safety of IL-18 gene therapy employed as an alternative strategy for cancer immunotherapy.L-18, like IL-1, lacks a typical signal sequence for cytokines.5 It is only processed into the mature active form by Caspase-1, an IL-1尾 converting enzyme.10,11 In the case of IL-1尾, several reports indicate that the addition of a conventional signal sequence is necessary for extracelluar secretion of mature IL-1尾.12,13,14 Therefore, we fused a signal sequence derived from human prepro-parathyroid hormone14 to the 5鈥?end of mature IL-18 cDNA to construct vectors capable of expressing bioactive IL-18. In the present study, we demonstrated that direct injection of an IL-18 adenoviral vector into tumor elicited an effective immune response, as well as potent antitumor effects which could be further enhanced by concomitant systemic IL-12 administration. Sequential IL-12/IL-18 production may be necessary for efficient induction of a cellular immune response.ResultsFusion of the signal sequence from human parathyroid hormone to mature murine IL-18 cDNA fragment enables the production of biologically active IL-18MCA205 cells were transduced with retroviral vectors carrying the full length cDNA of IL-18 (MCA205 DFG.FL.IL-18.Neo) (Figure鈥?a) or a cDNA of mature IL-18 fused with the prepro sequence of human parathyroid hormone (MCA205 DFG.PTH.IL-18.Neo) (Figure鈥?b) (see Materials and methods). Conditioned medium was prepared from 106 of these transfectants, cultured for 24鈥塰, and measured for secreted IL-18 in the medium using an ELISA. Simultaneously, to confirm that the secreted IL-18 was biologically active, an aliquot of the conditioned medium was measured for the ability of these supernatants to induce IFN-纬 from cocultured splenocytes in 24鈥塰 (Figure鈥?a). Conditioned medium of MCA205DFG.FL.IL-18.Neo contained no detectable IL-18 (ND, not detectable/106 cells/24鈥塰) nor was it able to induce a significant amount of IFN-纬 from splenocytes (4.8鈥壜扁€?.6鈥塸g/106 cells/24鈥塰). Nevertheless, conditioned medium of MCA205DFG.PTH.IL-18.Neo contained a significant amount of IL-18 (406.8鈥壜扁€?.1鈥塸g/106 cells/24鈥塰) which could induce IFN-纬 (40.8鈥壜扁€?.3鈥塸g/106 cells/24鈥塰). Secretion of bioactive IL-18 was also detected in the conditioned medium of MCA205 cells transduced with a recombinant adenoviral vector carrying PTH.IL-18 gene (Ad.PTH.IL-18) (Figure鈥?c and 2b). MCA205 cells transduced with Ad.PTH.IL-18 at a multiplicity of infection (MOI) of 1000 and 100 produced IL-18 (57.0鈥壜扁€?.1 and 2.9鈥壜扁€?.1鈥塸g/106 cells/24鈥塰, respectively) which can induce a significant amount of IFN-纬 (32.8鈥壜扁€?.9 and 8.0鈥壜扁€?.5鈥塸g/106 cells/24鈥塰, respectively) from spleno- cytes.Figure 1Structure of IL-18 recombinant retroviral and adenoviral vectors. We prepared polycistrone retroviral vectors and adenoviral plasmids using the full length or mature IL-18 genes. (a) DFG.FL.IL-18.Neo; (b) DFG.PTH.IL-18.Neo; (c) Ad.PTH.IL-18 (SA, splice acceptor site; SD, splice donor site; 唯, packaging site; FL.IL-18, full length IL-18 cDNA; hPTH, prepro leader peptide of human thyroid hormone; M IL-18, mature IL-18 cDNA; CMV-P, cytomegalovirus promoter; An, polyadenylation signal; LTR, long terminal repeat; ITR, internal terminal repeat.Full size imageFigure 2MCA205 IL-18 transfectants secrete IL-18 which induces high levels of IFN-纬 production from splenocytes. (a) Tranfectants prepared by retroviral vectors (b) Transfectants prepared by adenoviral vectors. IFN-纬 secretion was induced from 106 murine splenocytes by 50鈥壩糽 of the supernatant of 4鈥壝椻€?06 of transfectants or rIL-18 in 24鈥塰 and then determined in the supernatant of cell cultures and expressed as the amount of IFN-纬. Values are expressed as mean鈥壜扁€塻.d. *Level of IL-18 secretion was determined in the supernatant of cell cultures and expressed as the amount of IL-18 per 106 transfectants in 24鈥塰.Full size imageDirect injection of the IL-18 recombinant adenoviral vector into tumor induces regression of an established MCA205 sarcomaTo examine the antitumor effects of Ad.PTH.IL-18, groups of five mice received intradermal (i.d.) inoculation of 1鈥壝椻€?05 of MCA205 cells into the right flank on day 0 and treatment was administered on day 9 and 12. Each group was treated with peritumoral (p.t.) injection of Hanks balanced salt solution (HBSS), p.t. injection of AdLacZ (1鈥壝椻€?09 plaque-forming unit (p.f.u.)) (AdLacZ p.t.), p.t. injection of Ad.PTH.IL-18 (1鈥壝椻€?09 p.f.u.) (AdIL-18 p.t.) or i.d. injections of Ad.PTH.IL-18 (1鈥壝椻€?09 p.f.u.) in the left flank distant from the tumor (c.l. AdIL-18) (Figure鈥?a). Although treatment with contralateral injection of Ad.PTH.IL-18 did not affect the growth of tumor (similar to HBSS treatment (HBSS versus c.l. AdIL-18, P鈥?鈥?.37鈥?.99 on day 3 to 30)), p.t. injection of Ad.PTH.IL-18 initiated tumor regression immediately after treatment (HBSS versus AdIL-18 p.t. , P鈥?lt;鈥?.01 on day 12 to 30; AdLacZ p.t. versus AdIL-18 p.t., P鈥?lt;鈥?.05 on day 12 to 15, 21 and P鈥?lt;鈥?.01 on day 18, 24 to 30) and led to complete eradication of tumor in all animals by day 21. Interestingly, p.t. injection of AdLacZ slightly retarded the growth of tumor with the eventual tumor regression in one mouse. However, the difference in the mean tumor area from the HBSS-treated group did not reach statistical significance (P鈥?鈥?.07鈥?.61 on days 3 to 30). To investigate if the mice free of palpable tumor due to injection of Ad.PTH.IL-18 acquired specific immunity against MCA205 cells, mice were rechallenged with 1鈥壝椻€?06 of MCA205 cells on day 30, and five of six rechallenged mice were immune, rejecting the injected tumor.Figure 3Direct injection of an IL-18 adenovirus into tumor eradicated MCA205 tumors in a synergistic manner with co-administration of systemic rIL-12. Groups of five mice received 1鈥壝椻€?05 of MCA205 cells i.d. inoculation into the right flank on day 0 followed by the injection of HBSS, AdLacZ or Ad.PTH.IL-18 into tumor, or Ad.PTH.IL-18 into the contralateral left flank on day 9 and day 12. Data represent the mean鈥壜扁€塻.d. (a) Treatment with peritumoral injection of Ad.PTH.IL-18 alone (1鈥壝椻€?09 p.f.u.) completely abrogated the growth of the MCA205 tumor, whereas injection of Ad.PTH.IL-18 into the contralateral flank did not. Successfully treated animals were capable of rejecting a subsequent challenge with tumor in five of six instances. (b) Systemic administration of rIL-12 enhanced the antitumor effects of Ad.PTH.IL-18. Animals received intra- peritoneal administration of HBSS or suboptimal doses (0.05鈥壩糶) of rIL-12 daily from day 7 to day 14 combined with peritumoral injection of 3鈥壝椻€?08 p.f.u. of adenovirus vector on days 9 and 12. Treatment with rIL-12 alone suppressed the growth of tumor. Peritumoral injection of Ad.PTH.IL-18 alone retarded tumor growth. Combination therapy with peritumoral injection of Ad.PTH.IL-18 along with systemic administration of rIL-12 effectively suppressed tumor growth, completely eradicating tumor in all animals. Mice free of palpable tumor subsequently received inoculation with 5鈥壝椻€?05 of MCA205 cells with four of five mice rejecting this subsequent rechallenge.Full size imageSynergistic antitumor effects with systemic administration of rIL-12 and peritumoral injection of Ad.PTH.IL-18While we observed synergistic antitumor effects of systemic administration of rIL-18 combined with rIL-12,9 mice receiving combination therapy died with extraordinarily high serum levels of IFN-纬. Therefore, we examined the antitumor effects associated with p.t. injection of Ad.PTH.IL-18 in combination therapy with rIL-12. Mice were inoculated with 1鈥壝椻€?05 of MCA205 cells i.d. on day 0 and received daily intraperitoneal (i.p.) injection of HBSS or 0.05鈥壩糶 of rIL-12 on days 7鈥?4 combined with p.t. injection of HBSS (鈭?HBSS and 鈭?IL-12), 3鈥壝椻€?08 p.f.u. of AdLacZ (LacZ p.t./HBSS and LacZ p.t./IL-12), 3鈥壝椻€?08 p.f.u. of Ad.PTH.IL-18 (IL-18 p.t./HBSS and IL-18 p.t./IL-12) or i.d. injection of 3鈥壝椻€?08 p.f.u. of Ad.PTH.IL-18 in the left flank distant from the site of tumor (c.l. IL-18/HBSS and c.l. IL-18/IL-12) (Figure鈥?b). While p.t. injection of AdLacZ alone or contralateral injection of Ad.PTH. IL-18 alone did not suppress the growth of tumor (鈭?HBSS versus LacZ p.t./HBSS, P鈥?鈥?.06 to 0.76; 鈭?HBSS versus c.l. IL-18/HBSS, P鈥?鈥?.10 to 0.91), p.t. injection of Ad.PTH.IL-18 alone (IL-18 p.t./HBSS) retarded the growth of tumor (鈭?HBSS versus IL-18 p.t./HBSS, P鈥?lt;鈥?.01 on day 22 to 26) at a dose substantially lower than what was applied in previous experiments (Figure鈥?a). Systemic administration of rIL-12 alone (鈭?IL-12), combined with AdLacZ (LacZ p.t./IL-12) or contralateral injection of Ad.PTH.IL-18 (c.l. IL-18/IL-12) retarded the growth of tumor (鈭?HBSS versus 鈭?IL-12, LacZ p.t./IL-12 and c.l. IL-18/IL-12, P鈥?lt;鈥?.05 on days 13 to 30). However, administration of rIL-12 contributed to complete eradication of tumor in all animals only when combined with p.t. injection of Ad.PTH.IL-18 (IL-18 p.t./IL-12) (鈭?HBSS versus IL-18 p.t./IL-12, P鈥?lt;鈥?.01 on days 13 to 30; 鈭?IL-12 versus IL-18 p.t./IL-12, P鈥?lt;鈥?.05 on days 16 to 30).All mice completed the full treatment cycle without apparent side-effects. Blood samples were taken from two mice in each group before and 2 days after the first injection of Ad.PTH.IL-18 and serum levels of IFN-纬 were measured by ELISA. Serum IFN-纬 levels of all samples before treatment were鈥塨elow limit of detection ( 15.6鈥塸g/ml). Serum IFN-纬 levels of the mice treated with systemic administration of HBSS were also lower than 15.6鈥塸g/ml regardless of the i.d. injection (鈭?HBSS, LacZ p.t./HBSS, IL-18 p.t./HBSS and c.l. IL-18/HBSS). Treatment with i.p. injection of rIL-12 moderately elevated serum IFN-纬 levels even in the AdIL-18 treatment groups (57.8鈥壜扁€?9.6鈥塸g/ml in IL-18 p.t./IL-12 group and 27.9鈥壜扁€?.5鈥塸g/ml in c.l. IL-18/IL-12 group) which were similar to the control groups (170.5鈥壜扁€?0.4鈥塸g/ml in 鈭?IL-12 group and 63.7鈥壜扁€?8.6鈥塸g/ml in LacZ p.t./IL-12 group).In vivo depletion of NK cells by anti-ASGM1 antibody completely abrogates the antitumor effects mediated by Ad.PTH.IL-18, whereas depletion of CD4+ or CD8+ cells only partially inhibitsTo analyze the effector cell population in antitumor effects of Ad.PTH.IL-18, asialo (AS) GM1+, CD4+ or CD8+ cells were depleted using specific antibodies (Abs) administered before and during treatment with Ad.PTH.IL-18 (Figure鈥?). Mice received p.t. injection of either HBSS or 1鈥壝椻€?09 p.f.u. of Ad.PTH.IL-18 on day 7 and 10 after i.d. inoculation of 1鈥壝椻€?05 of MCA205 cells. Treatment with Ad.PTH.IL-18 significantly suppressed the growth of tumor (鈭?HBSS versus 鈭?IL-18; P鈥?lt;鈥?.01 on days 12 to 30) and caused total tumor rejection in four of five mice. However, depletion of NK cells with anti-ASGM1 Ab administration completely abrogated the growth inhibitory effects of IL-18 (control versus ASGM1; P鈥?lt;鈥?.01 on days 12 to 30). Tumor growth in mice treated either with anti-CD4 Ab (CD4/HBSS) or anti-CD8 Ab (CD8/HBSS) was more rapid when compared with mice not treated with Abs (鈭?HBSS). The growth inhibitory effects of IL-18 were only partially abrogated following treatment with anti-CD4 Ab (CD4/IL-18) or treatment with anti-CD8 Ab (CD8/IL-18) and remained statistically significant (control versus CD4; P鈥?lt;鈥?.01 on days 12 to 18 and P鈥?lt;鈥?.05 on days 21 to 30, control versus CD8; P鈥?lt;鈥?.01 on days 12 to 30).Figure 4Antitumor effects of Ad.PTH.IL-18 were completely abrogated by administration of anti-asialo GM1 antibody (Ab) and partially abrogated by administration of anti-CD4 or anti-CD8 Abs. Animals were pretreated with specific antibodies to abrogate NK, CD4+ or CD8+ cells before tumor inoculation with 1鈥壝椻€?05 of MCA205 cells. Peritumoral injection of either HBSS or 1鈥壝椻€?09 of Ad.PTH.IL-18 was performed on days 7 and 10. Treatment with Ad.PTH.IL-18 was associated with significant inhibition of tumor establishment (鈭?IL-18) and completely eradicated tumors in four of five animals. Depletion of NK cells completely abrogated the antitumor effects of Ad.PTH.IL-18 (ASGM1/IL-18). Growth of the tumor inoculated in mice treated either with anti-CD4 (CD4/HBSS) or anti-CD8 Ab (CD8/HBSS) was more rapid when compared with that of untreated mice. Treatment with anti-CD4 Ab (CD4/IL-18) or anti-CD8 Ab (CD8/IL-18) partially abrogated the growth inhibitory effects of administration of Ad.PTH.IL-18.Full size imageAdministration of Ad.PTH.IL-18 was associated with a reduced number of CD8+ cells but not with CD4+ cells infiltrated into tumorTo analyze the effects of local IL-18 expression on the population of cells infiltrating into tumor, tissue samples of MCA205 tumor were excised from animals 2 days following injection of HBSS, 1鈥壝椻€?09 p.f.u. of AdLacZ or 1鈥壝椻€?09 p.f.u. of Ad.PTH.IL-18 into tumor. Samples were immunostained with anti-CD4, anti-CD8, anti-CD11b, anti-CD11c, anti-CD16 or anti-NLDC145 Abs. A dense infiltration of CD11b+ or CD16+ cells (data not shown) and a sparse penetration of CD4+ (Figure鈥?a, b, c) and CD11c+ or NLDC145+ cells (data not shown), were observed within tumor regardless of the treatment. Treatment with HBSS or AdLacZ were associated with a moderate infiltration of CD8+ cells within tumor (d and e, respectively). Interestingly marked reduction in the number of CD8+ cells within tumor was observed following treatment with Ad.PTH.IL-18 (f).Figure 5Ad.PTH.IL-18 injection was associated with a decreased number of CD8+ cells found within tumor. Mice received i.d. inoculations of 1鈥壝椻€?06 of MCA205 cells in the right flank on day 0 and peritumoral injection of HBSS, 1鈥壝椻€?09 p.f.u. of AdLacZ or 1鈥壝椻€?09 p.f.u. of Ad.PTH.IL-18 on day 9. Tumors were excised from mice on day 11 and examined for infiltrating cells using immunohistochemistry. Each sample was immunostained with anti-CD4 (a, b, c) or anti-CD8a (d, e, f) antibodies, respectively. The stained cells were counted in five fields at a magnification of 脳400 in each sample. Nonparametric Wilcoxon rank test was used in the statistical analysis of the number of immunostained cells found in a tumor in individual groups. Representative photographs are shown with the number of the counted immunostained cells infiltrating in tumor. HBSS or AdLacZ-treated tumor showed a sparse infiltrate with CD4+ (a, 49.7鈥壜扁€?.1, and b, 67.0鈥壜扁€?1.2, respectively) and a moderate infiltrate with CD8+ cells (d, 125.3鈥壜扁€?.1, and e, 103.0鈥壜扁€?0.1, respectively). While no significant changes in the number of CD4+ cells (c, 38.3鈥壜扁€?.2; HBSS versus Ad.PTH.IL-18, P鈥?鈥?.524) were observed following treatment with Ad.PTH.IL-18, a significant reduction of the number of infiltrating CD8+ cells was found (f, 18.7鈥壜扁€?.0; HBSS versus Ad.PTH.IL-18, P鈥?lt;鈥?.0001; AdLacZ versus Ad.PTH.IL-18, P鈥?lt;鈥?.002). Original magnification 脳200. Similar results were observed in two animals in each group.Full size imageDiscussionWe have constructed viral vectors capable of producing mature IL-18 by fusing a leader sequence of the human parathyroid hormone gene to mature IL-18 cDNA. Peri- tumoral injection of an IL-18 adenoviral vector (Ad.PTH.IL-18) caused complete elimination of tumors in vivo. Injection of Ad.PTH.IL-18 synergized with systemic administration of rIL-12 to induce antitumor responses without apparent side-effects. The antitumor effects of IL-18 were completely abrogated by depletion with anti-ASGM1 Ab but only partially abrogated by depletion of CD4+ cells or CD8+ cells. Furthermore, immunohistological analysis revealed that CD4+ cells, but not CD8+ cells were the predominant cells infiltrating tumors treated with IL-18.IL-18 gene has similarities with IL-1 genes in amino acid sequence (12% homology with IL-1伪 and 19% with IL-1尾, respectively) and lacks a typical leader sequence.5 IL-18 as well as IL-1尾 is synthesized as a precursor, which is subsequently processed into a biologically active form by Caspase-1, the IL-1尾-converting enzyme.10,11,15,16 As a substrate IL-18 is more avidly cleaved by this enzyme than IL-1 itself. Cell lines transfected with full-length pro-IL-1尾 cDNA failed to process the IL-1尾 precursor or to secrete the mature protein.17 Several studies demonstrated that fusion of heterologous signal peptides to the region of mature IL-1尾 cDNA allowed secretion of mature IL-1尾 from transduced cells.12,13,14 Adopting this strategy, we constructed IL-18 recombinant viral vectors using the mature IL-18 cDNA ligated to the prepro leader sequence of the human parathyroid hormone gene (Figure鈥?b, c) and confirmed secretion of bioactive IL-18 from the cells transfected with these vectors carrying hybrid IL-18 gene (Figure鈥?). We also detected secretion of bioactive IL-18 from other murine cell lines, such as colon cancer, lung cancer and melanoma cell lines following infection with Ad.PTH.IL-18 (unpublished data).We have previously reported that significant antitumor effects of systemic administration of rIL-18 in tumor bearing mice.9 Systemic administration of 1鈥壩糶 per day of rIL-18 for 7 days suppressed the growth of MCA205 tumor; however, it was not sufficient to cause rejection of tumors when the treatment was initiated 7 days after tumor inoculation. A dose of 25鈥壩糶/day of rIL-18 protein can be administered in vivo, but antitumor effects of this high rIL-18 dose is not more potent in comparison with that of 1鈥壩糶 per day of rIL-18 (unpublished data). Although the expression of IL-18 obtained by transfection with Ad.PTH.IL-18 is relatively low, even at an MOI of 1000 (57.0鈥壜扁€?.1鈥塸g/106 of MCA205 cells/24鈥塰), when compared with recombinant adenoviral vectors carrying other cytokines,18 peritumoral injection of 109 p.f.u. of Ad.PTH.IL-18 alone successfully eradicated tumor in all animals (Figure鈥?a) and 3鈥壝椻€?08 p.f.u. of Ad.PTH.IL-18 (Figure鈥?b) suppressed the growth of an established MCA205 tumor even when treatment was initiated as late as day 9. These data suggest that limited production of IL-18 by Ad.PTH.IL-18 was enough to mediate significant antitumor effects. Intradermal injection of Ad.PTH.IL-18 into the contralateral flank at a site distant from the tumor did not have any apparent antitumor effects. Thus, it is likely that the antitumor effects of peritumoral injection with Ad.PTH.IL-18 is mediated not by the systemic circulation of IL-18, but rather by local high concentration of IL-18. Since most of the mice (four of five), which had complete regression of MCA205 cells following peritumoral injection of Ad.PTH.IL-18, rejected a subsequent challenge with tumor (Figure鈥?a), it appears that such an approach can indeed induce a potent and specific systemic immune memory against tumor. Destruction of tumor cells following injection of Ad.PTH.IL-18 could cause enhanced processing and presentation of tumor-associated antigen, increased activation of antigen-presenting cells, or increased T cell responses ultimately inducing a systemic immune response.In our previous study,9 systemic daily administration of 1鈥壩糶 of rIL-18 combined with 0.1鈥壩糶 of rIL-12 had the most significant antitumor effects on an established MCA205 tumor, but severe toxicity and death was noted in all animals by the 4th day of treatment. Side-effects included hemorrhagic diarrhea and weight loss accompanied by extremely high levels of serum IFN-纬 (17鈥?71.5鈥壜扁€?07.1鈥塸g/ml). To avoid these deleterious effects of combination therapy, we employed peritumoral injection of Ad.PTH.IL-18 as an alternative to systemic administration of rIL-18 and examined its antitumor and side-effects (Figure鈥?b). Systemic administration of rIL-12 at suboptimal dose enhanced antitumor effects of reduced doses (3鈥壝椻€?08鈥塸.f.u.) of peritumoral injection of Ad.PTH.IL-18 without apparent side-effects. The intradermal injection of Ad.PTH.IL-18 did not cause significant elevation of serum IFN-纬 even when combined with rIL-12. These data suggest that such an approach is not only an effective way to suppress tumor growth, but is also much safer (at least in mice) when combined with systemic administration of rIL-12.Depletion with anti-ASGM1 Ab completely abrogated the antitumor effects of Ad.PTH.IL-18 (Figure鈥?). Immunostaining of MCA205 tumor samples revealed a dense infiltration of CD11b+ and CD16+ cells which include macrophages and NK cells. Differences in the number of CD11b+, CD16+ were not significant in Ad.PTH.IL-18-treated tumors. NK cells appear to be the critical initial effector cells in the antitumor effects mediated by Ad.PTH.IL-18 therapy similar to what was observed in rIL-18 therapy.9 These characteristics of effector cell population in IL-18-treated animals are different from those observed in animals treated with IL-12. NK cells mediated only early phase of antitumor efficacy of IL-12,19 and did not appear to contribute to the suppression of tumor when treatment with IL-12 was initiated on day 14.20 Treatment with Ad.PTH.IL-18 reduced the number of CD8+ cells found within tumor but did not alter the number of CD4+ cells (Figure鈥?). These findings are consistent with results obtained from previous rIL-18 studies.9 Both anti-CD4+ and CD8+ cells partly, but not totally, mediate the antitumor effects of Ad.PTH.IL-18 (Figure鈥?). Further studies are necessary to determine the definitive roles of CD4+ and CD8+ cells in IL-18 antitumor effects. Since treatment with IL-18 induces Fas ligand expression on activated murine Th1 cells21 and on NK cell clones,22 Fas ligand expression on NK and CD4+ cells might play an important role in its antitumor effects and CD8+ cell elimination.In conclusion, we demonstrated significant antitumor effects of an IL-18 adenoviral vector which is capable of producing mature and bioactive IL-18. Concomitant systemic administration of rIL-12 showed synergistic effects with peritumoral injection of Ad.PTH.IL-18 without side-effects. These results indicate that IL-18 gene therapy may be evaluable in humans for its effects as a cancer therapy.Materials and methodsConstruction of retroviral vector and establishment of IL-18 transfectantsThe 818鈥塨p fragment of full length murine IL-18 cDNA (supplied from Hayashibara Biochemical Institute, Oka- yama, Japan) was used as a template to amplify and generate an NcoI site at the initiation codon and an EcoRI site at the 3鈥?end of this fragment by PCR. This fragment was designated as FL.IL-18. The sense and anti-sense primer sequences used were as follows: 5鈥测€塆AGCCATGGCTG- CCATGTCAGAAGAC鈥?鈥?and 5鈥测€塆AGGAATTCAGGCG- AGGTCATCAC鈥?鈥?(amplification cycles: 30; annealing temperature: 65掳C). To allow positive selection for retrovirally transfected cells, IRES-Neo fragment with an EcoRI site at the 5鈥?end and a BamHI site at the 3鈥?end was prepared as previously described.23 FL.IL-18 and IRES-Neo fragments were ligated between the NcoI and BamHI sites of the proviral plasmid termed MFG.24 This retroviral vector was designated as DFG.FL.IL-18.Neo (Figure鈥?a). To facilitate efficient secretion of bioactive IL-18, the fragment encoding the prepro leader peptide (amino acid 1鈥?1) of human thyroid hormone14 was fused to the sequence encoding mature IL-18 cDNA, and this fragment was termed PTH.IL-18. The PTH.IL-18 fragment and the IRES-Neo fragment were ligated to the MFG retroviral vector plasmid and this vector was designated as DFG.PTH.IL-18.Neo (Figure鈥?b). Retroviral supernatant was generated by transfecting these proviral plasmids into BING cell lines.25 To obtain IL-18 transfectants, MCA205 (a gift from Dr SA Rosenberg, National Cancer Institute, Bethesda, MD, USA) were transduced with retroviral supernatant of DFG.FL.IL-18.Neo. and DFG.PTH.IL-18.Neo. and underwent selection with G418 as previously described.19Construction of IL-18 adenoviral vectorThe PTH.IL-18 fragment was inserted into a shuttle plasmid termed pAdlox to make an E1 and E3-substituted recombinant adenoviral vector26 and this plasmid was designated as Adlox.PTH.IL-18 (Figure鈥?c). A recombinant adenoviral vector encoding PTH.IL-18 was made through Cre-lox recombination as previously described26 and termed Ad.PTH.IL-18. All of the necessary materials for constructing Ad.PTH.IL-18 were kind gifts from Dr Stephen Hardy (Cell Genesys, Alameda, CA, USA).Bioassay and ELISA for cytokinesBiologic activity of IL-18 produced from MCA205 transfectants was measured by their ability to induce IFN-纬 from murine splenocytes as described elsewhere.27 Secretion of IFN-纬 and IL-18 in the culture media from the IL-18 transfectants or in the serum of mice treated with Ad.PTH.IL-18 and/or rIL-12 was determined using ELISA (Pharmingen, San Diego, CA, USA, for IFN-纬 and Hayashibara Biochemical Institute, Okayama, Japan for IL-18, respectively) and is expressed as the amount from 106 of splenocytes or transfectants in 24鈥塰. The IL-18 ELISA used here detects both the precursor and mature protein of IL-18. The lower limits of the detection of IFN-纬 and IL-18 by ELISA were 15.6鈥塸g/ml.Animal experiments and statistical evaluationC57/BL6 mice were purchased from Taconic Farms (Germantown, NY, USA) and were used for experiments when they were 7 to 10-weeks-old. All the animals were ear-tagged and randomized before the experiments and treated in a blinded fashion. 1鈥壝椻€?05 of MCA205 wild-type cells were inoculated i.d. in the flank of C57BL/6 mice on day 0. Each group consisted of five animals. Mice received daily i.p. injections of rIL-12 (a gift from Hoffman La-Roche Inc, Nutley, NJ, USA) or HBSS (GIBCO BRL, Grand Island, NY, USA) as a control. The tumor size was measured every 3鈥? days and expressed as the product of the perpendicular diameters of individual tumors. Each animal experiment was repeated at least twice and representative results are shown. Blood samples from two mice per group were obtained by tail vein phlebotomy 1 day before and 2 days after adeno- virus injection. Serum was separated by centrifugation and stored at 鈭?0掳C until assayed. Nonparametric Wilcoxon rank test was used in the statistical analysis of the size of tumor and the number of immunostained cells infiltrated in tumor in individual groups. The difference was considered statistically significant when P value was less than 0.05.Antibody depletion of immune cellsTo deplete NK cells in mice, anti-ASGM1 antiserum (Wako Bioproducts, Richmond, VA, USA) was administered i.p. as previously described.19 Monoclonal Abs against CD4+ cells (GK1.5) and CD8+ cells (53鈥?.72) (all purchased from American Tissue Culture Collection, Rockville, MD, USA) were prepared and injected to mice to deplete subsets of immune cells 3 days before and once a week after the inoculation of tumor. These treatments are confirmed to deplete 95% of specific cell subsets in the spleen after the administration of Abs.Immunohistological analysisMice received peritumoral injection of HBSS, 1鈥壝椻€?09 p.f.u. of AdLacZ or 1鈥壝椻€?09 p.f.u. of Ad.PTH.IL-18 7 days after the i.d. inoculation of 5鈥壝椻€?05 of MCA205 cells into the flank. Tumors were harvested 2 days after adenovirus injection. Tumors were immediately frozen and embedded in OCT compound. Serial 5-渭m sections were made from these tumors using cryostat and underwent immunochemical staining using Abs to CD4, CD8a, CD11b, CD11c, CD16 (all from Pharmingen, San Diego, CA, USA) and NLDC145 (Serotec, Oxford, UK) with the Vectastain ABC kit (Vector, Burlingame, CA, USA). The stained cells were counted in five fields at a magnification of 脳400 in each sample. Evaluation of the results was performed in a blinded fashion.AcknowledgementsWe thank Drs Toru Kitagawa, Yasuhiko Nishioka, Muneo Numasaki, Takuya Takayama, Motohiro Hirao, Shusuke Moriuchi, Hideho Okada, Kazumasa Hiroishi, Hiromune Shimamura and Levent Balkir for their valuable assistance and suggestions. We are also grateful to Ms Loraine R McKenzie and Susan F Schoonover for their excellent technical assistance. This work was supported in part by Career Development Award of American Society of Clinical Oncology 鈥?4 (HT) and a National Institutes of Health Grant 1PO1 CA 48047鈥?1 (HT, PDR, MTL). References1Antonysamy M et al. Interleukin 16, 17, 18. In: Thomson AW (ed) . The Cytokine Handbook, Third edition Academic Press:London 1998 465鈥?69 Google Scholar聽 2Maeure MJ, Lotze MT . Tumor recognition by the cellular immune system: new aspects of tumor immunology Int Rev Immunol 1997 14: 97鈥?32Article聽Google Scholar聽 3Lotze MT et al. 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A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock Infect Immun 1995 63: 3966鈥?972CAS聽 PubMed聽 PubMed Central聽Google Scholar聽 Download referencesAuthor informationAuthor notesT OsakiPresent address: Department of Medicine III, Osaka University Medical School, 2鈥? Yamada-oka, Suita, Osaka, 565鈥?871, JapanAffiliationsDepartment of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USAT Osaki,聽W Hashimoto,聽M T Lotze聽 聽H TaharaDepartment of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USAA Gambotto,聽P D Robbins,聽M T Lotze聽 聽H TaharaDivision of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Hyogo, JapanH OkamuraFujisaki Institute, Hayashibara Biochemical Laboratories, Inc, Okayama, JapanM KurimotoAuthorsT OsakiView author publicationsYou can also search for this author in PubMed聽Google ScholarW HashimotoView author publicationsYou can also search for this author in PubMed聽Google ScholarA GambottoView author publicationsYou can also search for this author in PubMed聽Google ScholarH OkamuraView author publicationsYou can also search for this author in PubMed聽Google ScholarP D RobbinsView author publicationsYou can also search for this author in PubMed聽Google ScholarM KurimotoView author publicationsYou can also search for this author in PubMed聽Google ScholarM T LotzeView author publicationsYou can also search for this author in PubMed聽Google ScholarH TaharaView author publicationsYou can also search for this author in PubMed聽Google ScholarCorresponding authorCorrespondence to H Tahara.Rights and permissionsReprints and PermissionsAbout this articleCite this articleOsaki, T., Hashimoto, W., Gambotto, A. et al. Potent antitumor effects mediated by local expression of the mature form of the interferon-纬 inducing factor, interleukin-18 (IL-18). Gene Ther 6, 808鈥?15 (1999). https://doi.org/10.1038/sj.gt.3300908Download citationReceived: 15 October 1998Accepted: 23 December 1998Published: 14 May 1999Issue Date: 01 May 1999DOI: https://doi.org/10.1038/sj.gt.3300908KeywordsIL-12gene therapyvirus vectorleader sequence Maryam Moossavi, Negin Parsamanesh, Afsane Bahrami, Stephen L. Atkin Amirhossein Sahebkar Molecular Cancer (2018) Agnieszka Kami艅ska, Katarzyna Winkler, Aneta Kowalska, Evelin Witkowska, Tomasz Szymborski, Anna Janeczek Jacek Waluk Scientific Reports (2017) J-N Zheng, D-S Pei, L-J Mao, X-Y Liu, F-H Sun, B-F Zhang, Y-Q Liu, J-J Liu, W Li D Han Cancer Gene Therapy (2010) Yong Seok Kim, Jae Youn Cheong, Sung Won Cho, Kee Myung Lee, Jae Chul Hwang, Bermseok Oh, Kuchan Kimm, Jung A. Lee, Byung Lae Park, Hyun Sub Cheong, Hyoung Doo Shin Jin Hong Kim Digestive Diseases and Sciences (2009)