A new hamster fibrosarcoma model for in vitro/in vivo evaluation of cancer chemotherapeutic agents

A hamster fetal cell clone has been developed for in vitro chemotherapeutic studies as well as in an in vivo fibrosarcoma model system. Highly reproducible quantitative in vitro chemotherapeutic data can be obtained with this cell line within 5 days, and as few as 10² cells produce rapidly growing fibrosarcomas when injected subcutaneously into adult hamsters. We found using these cells in vitro that 1-β-D-arabinofuranosylcytosine (ara-C) can antagonize the effect of 5-azacytidine (aza-C) if given simultaneously or if aza-C treatment is preceded by a 2-hr exposure to ara-c. Using the same cell line as in vivo model for chemotherapy it was also shown that ara-C and cyclocytidine significantly inhibited tumor growth. This hamster cell line may be quite useful as an in vitro/in vivo model system for the study of cancer chemotherapeutic agents.     

Intracellular retention of cytosine arabinoside triphosphate in blast cells from children with acute myelogenous and lymphoblastic leukemia

The importance of the cellular pharmacokinetics of cytarabine triphosphate (ara-CTP) with regard to therapeutic efficacy is well established. In vitro and in vivo monitoring of pharmacokinetic parameters of leukemic blast cells were initiated in order to contribute to the pharmacological basis of optimal ara-C treatment strategies. Peripheral or bone marrow blast cells from 66 leukemic patients [51 acute myelogenous leukemia (ALL), 15 acute lymphoblastic leukemia (AML) were separated and incubated with ara-C for 1 hour and in ara-C-free medium for another 3 hours, and the intracellular formation and retention of ara-CTP was measured. In eight children who received continuous ara-C infusion for induction treatment, the ara-CTP concentration in circulating blast cells was monitored in vivo. The in vitro values observed in this assay corresponded to the cellular levels monitored in vivo. The ara-CTP retention differed clearly among the individual groups, as classified by immunophenotype at the time of the initial diagnosis: non-T-ALL 67 ± 25% (× ± SD, n = 33), T-ALL 37 ± 15% (n = 8), and AML 34 ± 18% (n = 14). The difference in ara-CTP retention between non-T-ALL and AML (P < 0.05) as well as T-ALL (P < 0.05) was significant. There was a tendency toward lower ara-CTP retention in relapsed as compared with newly diagnosed ALL, but the difference was not significant. The maximal accumulation of ara-CTP (after 1 hour incubation) was comparable in AML, T-ALL, non-T-ALL, and blast cells from children in relapse. The observed similarity of cellular accumulation in all groups and the significantly more rapid decrease in T-ALL and AML provide the pharmacokinetic rationale supporting the prolonged infusion duration for ara-C in these subgroups as an alternative to the intensification by high-dose ara-C schedules with short-term infusion. © 1996 Wiley-Liss, Inc.     

Preclinical testing of tandutinib in a transgenic medulloblastoma mouse model

Overexpression of platelet-derived growth factor receptor alpha (PDGFR-A) has been documented in association with primary tumors and metastasis in medulloblastoma. Tumors from our genetically engineered sonic hedgehog-driven medulloblastoma mouse model overexpress PDGFR-A in primary tumors and thus this mouse model is a good platform with which to study the role of PDGFR-A in this central nervous system malignancy. We hypothesized that inhibition of PDGFR-A in medulloblastoma can slow or inhibit tumor progression in living individuals. To test our hypothesis, we targeted PDGFR-A mediated tumor growth in vitro and in vivo using the tyrosine kinase inhibitor, tandutinib (MLN-518), which strongly inhibits PDGFR-A. Although PDGFR-A inhibition by this agent resulted in reduced mouse tumor cell growth and increased apoptosis in vitro, and reduced tumor cell proliferation in vivo, tandutinib did reduce tumor volume at the doses tested (360 mg/kg) in vivo. Thus, tandutinib may be an agent of interest for sonic hedgehog-driven medulloblastoma if a synergistic drug combination can be identified.     

Action of cytosine arabinoside on human tumor cells isolated from malignant effusions: in vitro phosphorylation and inhibition of DNA synthesis.

Tumor cells were isolated from malignant effusions of three patients with disseminated solid tumors of different origin. Intracellular accumulation of nondiffusible cytosine arabinoside (ara-C) nucleotides was used to measure phosphorylation. Mouse leukemia L 1210 and L 1210/CA, and ara-C-resistant subline, were used as reference cells. Phosphorylation activity was similar in the cells from all three solid tumors and showed a linear increase with drug concentrations of 0.1--100 micograms/ml. In contrast to activity in L 1210 cells, the in vitro activity was not saturable at drug levels up to 100 micrograms/ml. Ara-C inhibited the incorporation of thymidine into DNA 84%--90% in the solid tumor cells at a concentration of 10 micrograms/ml. Higher drug concentrations did not result in further inhibition. In one patient, DNA synthesis of tumor cells isolated before and after intraperitoneal instillation of 1,000 mg ara-C was measured. The in vivo inhibition was found to correspond to the in vitro data. Solid tumor cells isolated from malignant effusion have no greatly reduced capacity for cellular formation of ara-C/nucleotides, but higher drug levels than achieved with conventional therapy are necessary for sufficient ara-C nucleotide synthesis.     

A New in Vitro Screening Method for Teratogens Using Human Embryonic Palatal Mesenchymal Cells

Abstract In order to establish an in vitro screening assay system for cleft palate-inducing teratogens, we tested 31 teratogenic and 10 tionteratogenic compounds using human embryonic cultured cells. We examined whether cleft palate-inducing ability can be detected by differential growth inhibition between human embryonic palatal mesenchymal (HEPM) cells and human embryonic fibroblasts (MRC-5). Thirty one compounds with proven cleft palate-inductive effects in vivo preferentially inhibited the proliferation of HEPM cells. The average of the relative resistant rates (rate of IC50 value for HEPM cells to MRC-5 cells) of teratogens was 0.53. In contrast, almost all nonterato-gens identically inhibited the proliferation of both cell lines and the average of the relative resistant rates was 1.01. These results show that teratogens which induce cleft palate in vivo preferentially inhibit the proliferation of embryonic palatal mesenchymal cells.
The data indicated that in vitro screening using HEPM and MRC-5 cells is useful for detecting the cleft palate-inducing ability of chemicals.     

Action of cytosine arabinoside on human tumor cells isolated from malignant effusions: In vitro phosphorylation and inhibition of dna synthesis

Tumor cells were isolated from malignant effusions of three patients with disseminated solid tumors of different origin. Intracellular accumulation of nondiffusible cytosine arabinoside (ara-C) nucleotides was used to measure phosphorylation. Mouse leukemia L 1210 and L 1210/CA, and ara-C-resistant subline, were used as reference cells. Phosphorylation activity was similar in the cells from all three solid tumors and showed a linear increase with drug concentrations of 0.1–100 μg/ml. In contrast to activity in L 1210 cells, the in vitro activity was not saturable at drug levels up to 100 μg/ml. Ara-C inhibited the incorporation of thymidine into DNA 84%–90% in the solid tumor cells at a concentration of 10 μg/ml. Higher drug concentrations did not result in further inhibition. In one patient, DNA synthesis of tumor cells isolated before and after intraperitoneal instillation of 1,000 mg ara-C was measured. The in vivo inhibition was found to correspond to the in vitro data. Solid tumor cells isolated from malignant effusion have no greatly reduced capacity for cellular formation of ara-C/nucleotides, but higher drug levels than achieved with conventional therapy are necessary for sufficient ara-C nucleotide synthesis.     

Sequential oral hydroxyurea and intravenous cytosine arabinoside in refractory childhood acute leukemia: a pediatric oncology group phase 1 study

At concentrations >0.1 mM, hydroxyurea (HU) enhances the accumulation of cytosine arabinoside (ara-C) in leukemia cells in vitro. This study of children with refractory acute leukemia was designed to take advantage of this biochemical modulation. A fixed dose of HU and an escalating dose of ara-C were used. Oral HU (1200 mg/m2) was followed 2 hours later by ara-C (250-3100 mg/m2) intravenously in 15 minutes. The combination was given on days 1, 2, 3 and 8, 9, 10. Thirty-three children [26 acute lymphocytic leukemia (ALL), 7 acute nonlymphocytic leukemia] were treated; 29 received at least 1 full course. All patients developed grade 4 cytopenias. Other grade 3 to 4 toxicities included hyperbilirubinemia (2), elevated transaminases (3), transient gait disturbance (1), stomatitis (3), typhlitis (1), nausea/vomiting (9), and marrow aplasia >4 weeks (1). Three patients had intracranial bleeds while thrombocytopenic. Only liver toxicities and nausea/vomiting exhibited any dosage effect. The maximum tolerated dose of ara-C was 2400 mg/m2. There were 6 complete responses (5 ALL), 5 partial responses (3 ALL), and 19 patients with no response or progressive disease. There was no dosage effect for response with 2 complete responses occurring at the lowest ara-C level. Responses were transient (1 to 3 mo). Twenty of twenty-six patients achieved a peak serum HU level >0.5 mM by 2 hours after the HU dose. The mean level at 2 hours was 0.57 mM (range: 0.21 to 0.99 mM). This combination of HU and ara-C is tolerable and has efficacy in refractory leukemias. Responses at the lowest ara-C dose level suggests synergism.     

Neuroblastoma cells provoke Schwann cell proliferation in vitro

BACKGROUND: A subset of human neuroblastomas (NBs) has the capacity to mature completely, imitating sympathetic ganglia. Previously, we showed that the neuronal population in spontaneously maturing NBs usually has a near-triploid DNA content without 1p deletions, and we concluded that the constantly diploid Schwann cells (SCs) do not belong to the neoplastic component of these tumours. We therefore hypothesised that NB cells are able to stimulate SC proliferation, and that SCs trigger NB differentiation. PROCEDURE: We performed in vitro experiments to test this model and to test whether SCs can also influence the growth of aggressive NBs. Human SCs were co-cultivated with NB tumours and cell lines, and were harvested after defined time intervals. Proliferative activity of the SCs and the NB cells was determined by visualisation of 5-bromo-2'-deoxyuridine (BrdU) incorporation or Ki-67 staining. Neurite outgrowth and neurofilament (NF) expression were analysed immunocytochemically and apoptotic rate was determined by a terminal deoxynucleotidyl transferase-mediated dUTP-X fluorescein nick end labelling (TUNEL) assay. RESULTS: Human NB tumours or cell lines unequivocally increased the proliferation of SCs in vitro. In cocultivated NB cells, the proliferative activity was not altered in the first days of cocultivation, although neurite outgrowth and NF expression were enhanced. However, after 10 days, the mitotic rate of neuroblastic cells decreased and the apoptotic rate showed a marked increase. CONCLUSIONS: The results of the cocultivation experiments provide an experimental hint that the in vivo growth of SCs in NBs is caused by the neoplastic neuroblasts, and they also indicate that cells from peripheral nerves can influence the growth of aggressive NB cells if cocultivated.     

In vitro and in vivo evaluation of photodynamic techniques for the experimental treatment of human hepatoblastoma and neuroblastoma: preliminary results

Purpose  The purpose of this study was to test the susceptibility of human hepatoblastoma and neuroblastoma cells to photodynamic diagnostics (PDD) and photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) as a photosensitizer. Methods  Cell cultures of human hepatoblastoma (HuH6) and neuroblastoma (MHH-NB-11) were incubated with 5-ALA at increasing concentrations to measure the cellular kinetics of photosensitization. After optimizing incubation parameters, the cell cultures were then irradiated with increasing light doses and cell viability was measured by CTB assay. Human fibroblastic cells served as controls. So far, only the hepatoblastoma cell line has been tested in vivo. After injection of HUH6 cells in immunoincompetent rats, the efficacy of PDT was assessed. Photosensitization was achieved by intraperitoneal injection of 5-ALA. The pharmacokinetics of different tissues was studied. In a second study, a PDT of implanted hepatoblastoma, liver and peritoneum was performed. The irradiated areas were excised 48 h after treatment and studied by microscopy. Results  Cell culture experiments demonstrated a selective fluorescence for both tumor lines compared to controls. The photosensitized tumor cells demonstrated marked reductions in cell viability at significantly lower irradiation doses than the fibroblasts under PDT. The specificity of fluorescence was confirmed in vivo for hepatoblastoma, and all the sensitized and irradiated tumors showed marked phototoxic necrosis. Conclusion  Human hepatoblastoma and neuroblastoma demonstrate marked and specific fluorescence after the application of 5-ALA, making PDD possible. Cell death occurred in both cell lines after PDT in vitro. Additionally, hepatoblastoma was susceptible to PDT in an animal model. Further studies will be necessary to determine the role of PDT and PDD in a clinical setting.     

Establishment of an in vivo model for pediatric Ewing tumors by transplantation into NOD/scid mice

Ewing tumors are a clinically heterogeneous group of childhood sarcomas that represent a paradigm for understanding solid tumor biology, as they are the first group of sarcomas for which a chromosome translocation has been characterized at the molecular level. However, the biologic organization of the tumor, especially the processes that govern proliferation, differentiation, and metastasis of primitive tumor stem cells is poorly understood. Therefore, to develop a biologically relevant in vivo model, five different Ewing tumor cell lines and primary tumor cells from three patients were transplanted into immune-deficient mice via intravenous injection. NOD/scid mice that carry a complex immune deficiency and thus nearly completely lack the ability to reject human cells were used as recipients. Overall, 26 of 52 mice (50%) transplanted with VH-64, WE-68, CADO-ES1, TC-71, and RM-82 cells and 4 of 10 mice (40%) transplanted with primary tumor cells engrafted. Moreover, primary cells that did not grow in vitro proliferated in mice. The pattern of metastasis was similar to that in patients with frequent metastases in lungs (62%), bone marrow (30%), and bone (23%). Using limiting dilution experiments, the frequency of the engraftment unit was estimated at 1 Ewing tumor-initiating cell in 3 x 10(5) VH-64 cells. These data demonstrate that we have been able to establish an in vivo model that recapitulates many aspects of growth and progression of human Ewing tumors. For the first time, this model provides the opportunity to identify and characterize primitive in vivo clonogenic solid tumor stem cells. This model will, therefore, be instrumental in studying many aspects of tumor cell biology, including organ-selective metastasis and tumor angiogenesis.     

Clinical and in vitro antiproliferative properties of recombinant DNA-derived human interferon-alpha 2

The properties of the recombinant DNA-derived human leukocyte interferon, HuIFN alpha 2, were studied in patients with advanced leukemia or lymphoma. In vitro, HuIFN alpha 2 induced an increased activity of 2-5A synthetase in leukemic and in control cells indicating cellular responsiveness to IFN. HuIFN alpha 2 also produced a dose-responsive decline in marrow leukemia blast progenitor colony growth, and in normal hematopoietic colony formation in vitro, confirming its antiproliferative effect. A course of intravenous therapy given to a lymphoma patient produced a modest decline in peripheral white blood cell (WBC) and neutrophil counts; higher, more frequent doses in a second patient induced a profound drop in WBC's, neutrophils, and platelets. When the leukemia patients were given an intravenous course of HuIFN alpha 2 as a sole agent, blast cytoreduction was seen in peripheral blood in three patients, and in marrow of one patient with acute myeloblastic leukemia (AML). Elevated 2-5A synthetase levels could be detected after therapy. No modulation of leukemic cell markers was seen after in vitro or in vivo treatment with HuIFN alpha 2, implying that the cytoreduction was not linked to blast cell differentiation. These studies suggest that this subtype of recombinant DNA-derived IFN has antileukemic properties, and indicates the possibilities for IFN as an adjunctive form of therapy in leukemia.     

The hollow fiber assay for drug responsiveness in the Ewing's sarcoma family of tumors

OBJECTIVE: To investigate the use of the National Cancer Institute's hollow fiber assay (HFA) to evaluate and prioritize novel treatment strategies for clinical trials in the Ewing's sarcoma family of tumors (ESFT). STUDY DESIGN: The growth and morphology of ESFT cell lines in hollow fibers (HFs) was characterized in vitro and in vivo. Reliability and reproducibility were evaluated using doxorubicin. RESULTS: The numbers of viable cells in all 6 ESFT cell lines increased with time in vitro (0 to 96 hours). The SKES-1 and A673 cell lines grew exponentially after implantation of HFs in nude mice at subcutaneous and intraperitoneal sites. ESFT cells formed highly organized distinctive morphology within the HFs in vitro and in vivo. The number of viable ESFT cells within the HFs decreased in a time-dependent (24 to 96 hours) and dose-dependent (1 to 10 mg/kg) manner after treatment with doxorubicin in vivo. CONCLUSIONS: The HFA is a versatile short-term in vivo model that may be exploited to predict efficacy of potential anticancer agents in ESFT cells. Tumor markers and pharmacodynamic endpoints may be quantified in the pure population of ESFT cells from within the HFs.     

Pharmacokinetics of cytosine arabinoside in cerebrospinal fluid and of its metabolite in leukemic cells

Concentrations of ara-CTP in leukemic cells isolated from CSF and of ara-C in lumbar CSF were measured following intraventricular ara-C administration in two girls with refractory meningeal leukemia. CSF samples were collected with a permanent intrathecal-lumbar catheter. In contrast to the comparatively short retention of ara-C in the CSF (t1/2 1.8 to 2.9 hours), there was a high accumulation and an extremely long retention of ara-CTP in the leukemic cells (t1/2 8.1 to 36 hours). The patients included in this study had an ara-C-resistant disease. No obvious relationship was seen between concentrations of ara-C in the CSF and of ara-CTP in the leukemic cells. Similar studies were performed after simultaneous intraventricular administration of hydrocortison and ara-C. Hydrocortison did not increase ara-CTP retention in the leukemic cells, nor did it effect CSF pleocytosis.     

Caspase-8和DR5在TRAIL诱导神经母细胞瘤细胞凋亡中的作用

目的：肿瘤坏死因子相关凋亡诱导配体(TRAIL)能诱导多种肿瘤细胞凋亡而对正常细胞无诱导凋亡作用。大多数神经母细胞瘤细胞株对TRAIL的诱导凋亡作用耐受与其Caspase8表达缺失有关,也与细胞表面TRAIL受体的表达和分布有关。该文主要探讨Caspase8及TRAIL受体DR5的表达在TRAIL诱导神经母细胞瘤细胞株SKNDZ凋亡中的作用及其发生机制。方法：应用RTPCR方法检测IFNγ作用前后SKNDZ细胞Caspase8的表达;应用WesternBlot方法检测化疗药作用前后SKNDZ细胞DR5的表达;应用四甲基偶氮唑蓝(MTT)比色法及流式细胞仪(FCM)检测TRAIL、IFNγ+TRAIL、化疗药+TRAIL及化疗药+IFNγ+TRAIL对SKNDZ细胞生长及凋亡的影响。结果：SKNDZ细胞不表达Caspase8,IFNγ作用后的SKNDZ细胞Caspase8表达明显增加。对照组未检测到DR5蛋白表达,而阿霉素和依托泊苷处理后检测到DR5蛋白表达。表达Caspase8的SKNDZ细胞对TRAIL的诱导凋亡作用仍不敏感,而同时表达Caspase8和DR5的SKNDZ细胞对TRAIL的诱导凋亡作用敏感。阿霉素/依托泊苷+IFNγ+TRAIL组早期凋亡率为:(17.9±3.6)%、(14.8±3.3)%,与IFNγ+TRAIL组(3.9±1.2)%比较,差异有显著性(F=26.233,P<0.01)。结论：同时表达Caspase8和DR5的SKNDZ细胞恢复了对TRAIL的敏感性,Caspase8和DR5在TRAIL诱导SKNDZ细胞凋亡中起着十分关键的作用。     

Retinoic acid-induced apoptosis of the CHP134 neuroblastoma cell line is associated with nuclear accumulation of p53 and is rescued by the GDNF/Ret signal

BACKGROUND: Neuroblastoma (NBL) is one of the most common solid malignancies in childhood and is derived from the sympathetic precursor cells. Although p53, a tumor suppressor, has been reported to be rarely mutated in NBLs, it is sequestered abnormally in the cytoplasm of the NBL cell. The mechanism and functional role of the abnormal intracellular localization of p53 remain unclear. PROCEDURE: Here, we established an in vitro system of apoptosis model using a NBL cell line CHP134 which also showed a cytoplasmic sequestration of p53. The treatment of the cells with 1 or 5 microM all-trans retinoic acid (RA) induced moderate neurite outgrowth followed by massive death of CHP134 cells by days 5 to 6. RESULTS: TUNEL staining showed that the cell death was due to apoptosis. Immunofluorescent stain demonstrated that p53 was strongly positive in the nucleus on day 5, which was accompanied with induction of p21WAF1. In addition, expression of caspase-3 was also increased during the cell death. Intriguingly, the RA treatment induced expression of Ret tyrosine kinase receptor in CHP134 cells. CONCLUSIONS: The addition of ligands, glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN), inhibited apoptosis as well as nuclear accumulation of p53 in the cell. The present results suggest that the RA-induced apoptosis of NBL cells is associated with activation of both the caspase cascade and the p53-mediated pathway with its nuclear translocation. The neurotrophic signal through the GDNF-Ret system may prevent the neuronal cell death.     

Humoral response to vaccination with interleukin-2-expressing allogeneic neuroblastoma cells after primary therapy

BACKGROUND: Immunotherapy using cytokine-expressing tumor cells has shown promise as an anticancer strategy. We have recently begun a trial of interleukin-2 (IL-2) gene-modified allogeneic neuroblastoma cells administered in a sequence of eight injections to patients with high-risk neuroblastoma following completion of primary therapy. Six patients to date have completed treatment. PROCEDURE: We examined humoral responses to the immunizing cell line and, when available, to the patients' autologous tumor cells using an in vitro binding assay. RESULTS: Five of six patients developed a rise in antitumor antibodies to the immunizing neuroblastoma cell line following vaccination. Two of these patients had autologous tumor available; both demonstrated a humoral response to these cells as well. CONCLUSIONS: Our results demonstrate that vaccination with IL-2-expressing allogeneic tumor cells after intensive primary therapy can elicit a humoral response to the immunizing line. These antibodies are cross-reactive with the patients' own tumor cells in the two cases in which autologous cells were available. This suggests that different patients' tumors may share common antigens that can be exploited in immunotherapy strategies and supports the continued exploration of allogeneic tumor cells as tumor vaccines.     

DOR-1, A novel CD10+ stromal cell line derived from progressive Langerhans cell histiocytosis of bone

BACKGROUND: Langerhans cell histiocytosis (LCH) is granulomatous proliferative disorder characterized by the presence of activated Langerhans cells admixed with macrophages, lymphocytes, and eosinophils. In an effort to obtain an LCH ex vivo model, we succeeded in establishing the DOR-1 cell line from an LCH lesion of bone in a 3-year-old girl. PROCEDURE: The DOR-1 cell line was established from a CD1a immunoreactive LCH lesion of bone maintained in long-term cell culture. The phenotypic characteristics were assessed by immuno-cytochemistry and fluorescence activated cell sorter (FACS) analysis. Cytogenetic analysis was performed by RHG-banding that was supplemented by fluorescence in situ hybridization (FISH). RESULTS: The DOR-1 cells grew in vitro as a poorly differentiated mesenchymal-like cells with a doubling time between 72 and 96 hr. The cells exhibited pleomorphism and consistent immuno-reactivity for CD10 (50%), CD13 (55%), CD68 (65%), and CD117 (70%) while CD1a, Langerin and HLA-DR were not detected. By RHG-banding, several aberrant chromosomes were detected including the t (9; 17) (p23; p13) translocation and a pair of long dicentric marker chromosomes indicating clonal abnormality. Functionally, exposure to 33 nM 12-O-tetradecanoyl phorbol mirystate-13-acetate (TPA) induced DOR-1 cell differentiation with appearance of cytoplasmic extensions. CONCLUSIONS: The DOR-1 cell line exhibits distinct immuno-cytochemical features and carries the t (9; 17) (p23; p13) translocation suggesting involvement of stromal-like cell lineage in LCH initiation and progression.     

重组人白介素-11对脂多糖致新生大鼠空肠上皮细胞系-6损伤的影响

目的 探讨重组人白介素-11（rhIL-11）对新生大鼠空肠上皮细胞系-6（IEC-6）增殖和凋亡的影响。方法 通过脂多糖（LPS）作用于正常IEC-6细胞,建立NEC体外模型,为LPS处理组;IL-11治疗组在LPS作用后给予100 ng/mL rhIL-11处理;空白对照组在实验过程中加入等剂量的生理盐水替代。采用MTT比色法选择LPS作用的最佳浓度（5~200 μg/mL）及最佳时间（1~24 h）;MTT 比色法检测rhIL-11 加入后3、6、9和12 h各组细胞的增殖活力;流式细胞术检测各组的细胞凋亡率和坏死率。结果 LPS可降低IEC-6细胞的增殖活力,且根据不同浓度LPS作用于IEC-6细胞不同时间结果,LPS的最佳作用浓度为100 μg/mL,最佳作用时间为3 h。100 ng/mL rhIL-11作用于LPS 处理过的细胞9 h后,IL-11治疗组细胞增殖活力较LPS组显著提高（PP>0.05）;且IL-11治疗组的总凋亡和坏死率较LPS处理组降低（PP结论 rhIL-11可促进由LPS致IEC-6细胞损伤后的细胞增殖,有助于IEC-6细胞增殖活力的恢复,降低凋亡和坏死率,提示rhIL-11对LPS致IEC-6细胞损伤具有保护作用。     

Immunotherapeutic strategies in neuroblastoma: antitumoral activity of deglycosylated Ricin A conjugated anti-GD2 antibodies and anti-CD3xanti-GD2 bispecific antibodies

BACKGROUND: The antigen GD2 is selectively expressed on the surface of neuroblastoma cells, and is detected by the monoclonal antibody BW704. In this study, we describe the antitumoral capacity of the immunotoxin BW704dgA (BW704 conjugated to deglycosylated ricin A), and of anti-CD3xanti-GD2 bispecific antibodies that are capable of redirecting cytotoxic T cells towards neuroblastoma cells. We further investigate the in vivo activity of BW704dgA immunotoxins in a human neuroblastoma model in SCID mice. PROCEDURE: BW704dgA immunotoxins were injected i.p. as a single close (48 microg/mouse) on day 4 or divided into three doses on day 4, 5, and 6 after i.v. inoculation of the human neuroblastoma cell line IMR5-75. RESULTS: The mean survival time (MST) of BW704dgA treated animals was significantly increased (MST 49 days) compared to the control animals treated with irrelevant immunotoxin, unconjugated BW704, or control buffer (MST 33 to 39 days, P < 0.0001), without differences in the application schedules. Anti-CD3xanti-NP antibodies and NP-conjugated GD2-antibodies (BW704-NP) were used in a cytotoxicity assay with cytotoxic T-cells as effectors, and tracer labeled neuroblastoma cell line IMR5 as target cells. Anti-CD3xanti-NP antibodies, together with BW704-NP, showed increased cytotoxic activity compared to the incubation with CD3xanti-NP antibodies alone or with unconjugated anti-GD2. Additionally, a dose-dependent effect of NP-conjugated anti-GD2-antibodies upon the lysis of the target cells could be demonstrated. In this report, we describe two immunotherapeutic approaches using GD2 binding BW704 antibodies, modified as immunotoxin and a bispecific antibody, for the targeting and elimination of neuroblastoma cells. CONCLUSIONS: We envisage a combined immunotherapeutic regimen consisting of BW704dgA mediated stem cell purging, followed by a systemic treatment with anti-CD3xanti-GD2 bispecific antibodies in neuroblastoma.