[Coronary artery bypass grafting surgery in left ventricular dysfunction patients complicated with mitral regurgitation].

Objective: To evaluate the efficacy of coronary artery bypass grafting (CABG) surgery in left ventricular dysfunction patients complicated with different degrees of ischemic mitral regurgitation (IMR). Methods: The clinical data of 525 patients (428 males and 97 females) undergoing CABG in Fuwai Hospital Chinese Academy of Medical Sciences Shenzhen, and Tianjin Medical University General Hospital between January 2015 and December 2018 were collected. The average age was (61±7) years old. Among them, the patients with moderate to serve IMR and left ventricular ejection fraction(LVEF)≤40% were further selected, and the outcomes of CABG were analyzed. Results: In total, 67 patients (48 males and 19 females) with moderate to severe IMR and LVEF≤40% were enrolled, among which 52 patients had moderate IMR, with a LVEF of 38%(35%, 40%). Transesophageal echocardiography (TEE) of 52 cases displayed no damage of papillary muscles, and ventricular wall motion was improved after CABG. Therefore, no treatment on the mitral valve was performed in this group. Six patients were with moderate-severe mitral insufficiency, with a LVEF of 38%(35%, 39%). After surgery, TEE found that the ventricular wall motion and regurgitation were improved, and the mitral valve structures were well. Thus, mitral valves were not treated in these patients. Nine patients were with severe mitral regurgitation, with a LVEF of 38%(35%, 39%). Two of them received valve repair because the papillary muscle function and the ring were well. Another 7 patients received valve replacements because the valve ring was dilatated and the leaflet was prolapsed. All patients recovered well. The LVEF increased significantly at 6 months after surgery [47%(45%, 48%) vs 38%(35%, 39%), P=0.024], and the left ventricular end diastolic diameter also became smaller [57(56, 59) mm vs 61(59, 64) mm, P=0.002]. Conclusions: For patients suffered from left ventricular dysfunction complicated with IMR, TEE is crucial to evaluate the valve function. To those with moderate-severe regurgitation, if papillary muscle function and the ring were seriously affected by ischemia, the valve replacement could facilitate the improvement of postoperative cardiac function.

[Establishment and evaluation of a triple-color human papillomavirus pseudovirion neutralization assay].

Objective: To establish a triple-color pseudovirion-based neutralization assay (PBNA) and evaluate its capability of detecting immunogenicity of the sera generated by the immunization of HPV 9-valent vaccine. Methods: HPV pseudovirus (PsVs) 6/11/16/18/31/33/45/52/58 with the encapsidated fluorescence expressing red fluorescent plasmid N31-MCHREEY, green fluorescent N31-EGFP or blue fluorescent N31-mTagBFP were generated. The concentration of HPV PsVs and the infection titers of HPV PsVs were detected by double-antibody sandwich ELISA and TCID(50), respectively. The single- and triple color HPV 16/33/45 PsVs were used to detect the neutralization titers of mice sera immunized with HPV 9-valent vaccine and confirmed the accuracy and specificity of the triple-color PBNAs. Then, the single- and triple color HPV 6/11/18/31/33/45/52/58 PsVs were employed to detect the neutralization titers of cynomolgus macaques sera immunized with HPV 9-valent vaccine and determined whether the triple-color PBNAs could be applied to evaluate the immunogenicity of the sera generated by the immunization of HPV9-valent vaccine. Results: The concentration of HPV16 PsVs encapsulating green, red or blue fluorescent plasmid was 5.0 to 6.0 µg/ml and HPV6/11/18/31/33/45/52/59 triple-color HPV PsVs was about 1.0 to 3.0 µg/ml. 9 types HPV PsVs containing EGFP, Mcherry or mTagBFP reporter plasmid were obtained and the concentration can meet the need of neutralization detection. 9 types single-color fluorescent HPV PsVs had similar infectivity against 293FT cells with the infection titer values between 1×10(4) and 1×10(5). The results of PBNAs showed that there was no significant difference in the anti-HPV neutralization titers of mice sera induced by HPV 9-valent vaccine between single-color and triple-color HPV16/33/45 PsVs (P>0.05). Similarly, there was also no significant difference in the anti-HPV neutralization titers of cynomolgus macaques sera induced by HPV 9-valent vaccine between single-color and triple-color HPV6/11/18/31/33/45/52/58 PsVs (P>0.05). Conclusion: We successfully established the triple-color PBNAs and verified the accuracy and specificity of triple-color PBNAs consistent with single-color PBNAs. The triple-color PBNAs can be applied to evaluate the immunogenicity of HPV 9-valent vaccine's immune serum.

Modeling and predicting optimal treatment scheduling between the antiangiogenic drug sunitinib and irinotecan in preclinical settings.

We present a system of nonlinear ordinary differential equations used to quantify the complex dynamics of the interactions between tumor growth, vasculature generation, and antiangiogenic treatment. The primary dataset consists of longitudinal tumor size measurements (1,371 total observations) in 105 colorectal tumor-bearing mice. Mice received single or combination administration of sunitinib, an antiangiogenic agent, and/or irinotecan, a cytotoxic agent. Depending on the dataset, parameter estimation was performed either using a mixed-effect approach or by nonlinear least squares. Through a log-likelihood ratio test, we conclude that there is a potential synergistic interaction between sunitinib when administered in combination with irinotecan in preclinical settings. Model simulations were then compared to data from a follow-up preclinical experiment. We conclude that the model has predictive value in identifying the therapeutic window in which the timing between the administrations of these two drugs is most effective.

Theoretical investigation of the efficacy of antiangiogenic drugs combined to chemotherapy in xenografted mice.

Antiangiogenic drugs were developed with the aim to inhibit the formation of intratumoral blood vessels and in consequence the growth of solid tumors. As these drugs are generally combined with classical cytotoxic drugs in the treatment of cancer patients, finding the optimal combinations remains a complex challenge due to possible interactions of the antiangiogenic compound with the hemodynamic property of the treated tumor. To analyze this problem, we developed a multi-scale model of vascular tumor growth combining a molecular model of VEGF signaling pathways and a tissue model of the tumor expansion including the dynamics of cellular and tissue processes of tumor growth and response to treatments. We addressed the potential impact of antiangiogenic drug by defining a new index of vasculature quality which depends on the balance between stable and unstable vessels within the tumor mass. Our goal was to investigate the interactions between a chemotherapy and a antiangiogenic treatment, and, by simulating the model, to identify the optimal delay of chemotherapy delivery after the administration of the antiangiogenic compound. This theoretical analysis could be used in the future to optimize antiangiogenic drug delivery in preclinical settings and to facilitate the translation from preclinical to clinical studies.

Antisense anti IGF-I cellular therapy of malignant tumours: immune response in cancer patients.

The treatment of cancer by antisense anti-IGF-I cellular therapy inducing immune response has evoked interest among many promising strategies. Here, we reported some results obtained from patients with cancer, mainly glioblastoma treated by this strategy, which was also extended to patients with colon carcinoma, ovary cystadenocarcinoma and prostate adenocarcinoma. It was shown that, in the phase I of clinical trial, patients vaccinated with their own tumour cells treated by antisense IGF-I presented a slight increase of temperature. Their peripheral blood lymphocytes showed a shift in the percentage of CD8 effector cells as judged by expression of cell surface markers CD8+ CD28+. Particularly, in two treated patients with glioblastoma, the survival time was 19 and 24 months respectively in comparison to the range of 12 to 15 months observed in the case of classical treatment such as surgery, radiation or chemotherapy. These results, although preliminary, gave indication that the reported strategy could deserve consideration owing to its safety. Furthermore, the increase in the percentage of peripheral blood monomorphonucleated cells (PBMNCs) with effector phenotype, i.e., CD8+ CD28+ in vaccinated patients might explain their prolonged survival time.

Serologic prevalence of Toxoplasma gondii in field mice, Microtus fortis, from Yuanjiang, Hunan Province, People's Republic of China.

Antibodies to Toxoplasma gondii were investigated in serum samples of field mice, Microtus fortis, from Yuanjiang, Hunan Province, People's Republic of China. The modified agglutination test (MAT) incorporating formalin-fixed whole tachyzoites and mercaptoethanol was used to determine antibodies. Antibodies to T. gondii (MAT > or = 1:20) were found in 36 (29%) of 124 trapped mice. The antibody titers of positive sera (percentage in parentheses) were 1:20 (8.9), 1:40 (3.2), 1:80 (3.2), 1:160 (1.6), 1:320 (1.6), 1:640 (1.6), 1:1,280 (1.6), 1: 2,560 (0.8), and > 1:2,560 (6.5). No antibody to T. gondii was found in 104 sera of laboratory-bred M. fortis infected with Schistosoma japonicum between 1 and 45 days after infection.

Presence of MHC-I in rat glioma cells expressing antisense IGF-I-receptor RNA.

The supposed immunogenic character of glioma cells transfected with antisense IGF-I-Receptor (IGF-I-R) expression vector was tested for the presence of MHC-I currently present in cells of IGF-I antisense type. C6 rat glioma cell line was comparatively transfected in vitro with IGF I antisense (pMT-Anti-IGF I) or IGF I Receptor antisense (pMT-Anti-IGF I R) expression vectors. The wild and transfected cells were examined for the presence of IGF-I and MHC-I molecules. Using RT PCR technique, the transfected "antisens" cells showed total inhibition of IGF-I. The both transfected cultures of IGF-I and of IGF-I-R type were positively stained for MHC-I. Moreover "antisense IGF-I-R" cells as compared to "IGF-I antisense" cells showed slightly higher expression of MHC-I. The transfected cells showed also the feature of apoptosis in 60% of cells. The immunogenicity of IGF-I-R antisense glioma cells is related to MHC-I presence; therefore both approaches of antisense IGF-I and of antisense IGF-I-R could be use in paralel for cellular therapy of glioblastoma.

IGF-I triple helix gene therapy of rat and human gliomas.

PURPOSE: IGF-I anti-gene technology was applied in treatment of rat and human gliomas using IGF-I triple helix approach. MATERIAL AND METHODS: CNS-1 rat glioma cell and primary human glioblastoma cell lines established from surgically removed glioblastomas multiforme were transfected in vitro with IGF-I antisense (pMT-Anti-IGF-I) or IGF-I triple helix (pMT-AG-TH) expression vectors. The transfected cells were examined for immunogenicity (immunocytochemistry and flow cytometry analysis) and apoptosis phenomena (electron microscopy). 3 x 10(6) transfected cells were inoculated subcutaneously either into transgenic Lewis rats or in patients with glioblastoma. The peripheral blood lymphocytes (PBL) derived from "vaccinated" patients were immunophenotyped for the set of CD antigens (CD4, CD8 etc). RESULTS: Using immunocytochemistry and Northern blot techniques, the transfected "antisense" and "triple-helix" cells showed total inhibition of IGF. Transfected cultures were positively stained either for both MHC-I and B7 antigens--60% of cloned lines, or for MHC-I only--40% of cloned lines. Moreover "triple helix" cells as compared to "antisense" cells showed slightly higher expression of MHC-I or B7. Transfected cells also showed the feature of apoptosis in 60%-70% of cells. In in vivo experiments with rats bearing tumors, the injection of "triple helix" cells expressing both MHC-I and B7 interrupted tumor growth in 80% of cases. In contrast, transfected cells expressing only MHC-I stopped development in 30% of tumors. In five patients with surgically resected glioblastoma who were inoculated with "triple helix" cells, PBL showed an increased percentage of CD4 + CD25+ and CD8 + CD11b-cells, following two vaccinations. CONCLUSIONS: The anti-tumor effectiveness of IGF-I anti-gene technology may be related to both MHC-I and B7 expression in cells used for therapy. The IGF-I antigene therapy of human glioblastoma multiforme increases immune response of treated patients.

Prevalence of antibodies to Toxoplasma gondii in the sera of rare wildlife in the Shanghai Zoological Garden, People's Republic of China.

The prevalence of antibodies to Toxoplasma gondii in the sera of rare wildlife in the Shanghai Zoological Garden, PR China, was examined using a modified agglutination test (MAT) and an enzyme-linked immunosorbent assay (ELISA). Forty-one (35%) of 117 animals belonging to two classes, 10 orders, 18 families, 37 genera and 52 species (including sub-species) were sero-positive for MAT. By MAT, T. gondii antibodies were found in 11.1% (4/36) of birds, in 25% (4/16) of primates, in 69.4% (25/36) of carnivores and in 27.6% (8/29) of herbivores. Thirty-three (33.7%) of 98 animals tested by protein A ELISA were sero-positive. By ELISA, T. gondii antibodies were found in none of 36 birds, in 33.3% (4/12) of primates, in 87.1% (27/31) of carnivores and in 10.5% (2/19) of herbivores.

[Qualitative and quantitative comparison of three agglutination tests for detecting Toxoplasma gondii antibodies].

OBJECTIVE: To evaluate the diagnostic value of three agglutination tests used in three countries for detection of antibodies to Taxoplasma gondii. METHODS: A total of 288 human serum samples were assayed using modified agglutination test (MAT-1, using selfmade antigen), latex agglutination test (LAT, using Japanese Kit) and modified agglutination test (MAT-2, using French antigen). RESULTS: The positive rates of MAT-1 (> or = 1:20), LAT (> or = 1:32) and MAT-2 (> or = 1:20) were 9.7% (28/288), 8.9% (10/112) and 8.1% (17/210), respectively. No significant statistical difference was found among these positive rates (chi 2 = 0.392, P > 0.05). High agreements were found between MAT-1 and LAT (93.7%), LAT and MAT-2 (94.5%), and MAT-2 and MAT-1 (97.3%). Significant correlation were demonstrated in MAT-1 and LAT (r = 0.613), LAT and MAT-2 (r = 0.551), and MAT-2 and MAT-1 (r = 0.841), p < 0.001. CONCLUSION: The detection efficiency of the three agglutination tests is in good agreement and could alternatively be used for the diagnosis of toxoplasmosis.

Suicide gene therapy of chemically induced mammary tumor in rat: efficacy and distant bystander effect.

We examined the efficacy of suicide gene therapy for nitrosomethylurea-induced mammary tumors in rats. Individual tumors were directly injected with a retrovirus-producing cell line that releases retroviral vectors that transduce the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene. HSV1-TK specifically converts the nucleoside analogue ganciclovir (GCV) into a toxic metabolite. Compared to control rats receiving saline, we observed a significant tumor regression of the injected tumors following GCV administration, accompanied by a stromal inflammation and an extensive lymphocyte infiltration invading the tumor epithelium. It is noteworthy that the neighboring uninjected tumors also regressed, demonstrating the occurrence of a distant bystander effect. This is the first demonstration that HSV1-TK/GCV can efficiently treat multiple solid tumors directly generated from an epithelial tissue.

Effects on brain tumor cell proliferation by an adenovirus vector that bears the interleukin-4 gene.

A recombinant adenovirus vector bearing the IL-4 gene (AD-IL-4) was used to infect rat glioma C6 cells in culture at multiplicity of infections (MOI) from 50 to 1800. C6 cell proliferation was not altered significantly by adenoviral infection. However, IL-4 production increased in a dose-dependent manner. To ascertain effects on in vivo cell proliferation, a subcutaneous tumor model was used. Rat C6 glioma cells alone or C6 mixed with the control virus bearing the LacZ gene (Ad-LacZ) produced tumors that measured an average of approximately 3000 mm3 35 days after implantation. In contrast, C6 cells mixed with Ad-IL-4 produced significant inhibition of tumor growth (P=0.035 compared to C6 tumor; P=0.023 compared to C6+Ad-LacZ tumor. Student's ttest). IL-4 levels in mice serum were measured by ELISA and reached a peak of approximately 700 pg/ml at 14 days. These preliminary results showed that adenovirus-mediated delivery of the IL-4 gene may result in a significant inhibition of rat C6 cell tumor growth. Further studies will be necessary to refine this anti-tumor effect for as a potential therapy for cancer.

Regression of experimental brain tumors with 6-thioxanthine and Escherichia coli gpt gene therapy.

The identification of transgenes with antitumor activity is critical to the development of gene therapy of cancer. Retrovirus-mediated transfer of the Escherichia coli gpt gene into rat C6 glioma cells without subsequent selection still inhibited the proliferation of this mixed polyclonal population upon addition of the prodrug, 6-thioxanthine, with an ID50 of 4.1 microM, whereas parental C6 cells were not affected at a concentration of 500 microM. In a time-course assay, effects of the prodrug on the mixed polyclonal cell proliferation required at least 10 days of exposure. In mixed co-cultures, a bystander effect was not present over the first 4 days of prodrug exposure, but required trypsinization of the co-cultures and replating at lower densities. This "modified" bystander assay thus revealed a 50% decrease in C6 cell proliferation, even when the initial ratio of gpt-expressing to parental C6 cells was as low as 1:19. In a nude mouse model of subcutaneous tumors, co-grafts of C6 glioma and gpt-retrovirus producer cells displayed retarded growth upon exposure to 6-thioxanthine (6-TX). In a nude mouse model of intracerebral tumors, grafting of the gpt-retrovirus producer cells leads to an 80% reduction in intracerebral tumor volumes after 6-TX treatment. This reduction results in a 28% increase in the mean time of survival of animals that harbor intracerebral tumors (p < 0.0005). These antitumor effects indicate that the gpt/6-TX enzyme/prodrug pair is a promising alternative to the thymidine kinase gene and ganciclovir combination in the gene therapy of cancer.

Establishment of a monkey kidney epithelial cell line with the BARF1 open reading frame from Epstein-Barr virus.

We previously reported that the BARF1 (BamH1-A right frame 1) gene product from Epstein-Barr Virus (EBV) may have oncogenic properties since injection into new-born rats of transfected cell lines resulted in the development of BARF1 expressing tumors, which were aggressive in the case of murine fibroblasts and transient in that of human B lymphocytes. As EBV has been associated with nasopharyngeal carcinoma (NPC) and evidence of BARF1 transcription in this cancer was emerging from our biopsy analyses, we examined the effects of BARF1 transfection into primate primary epithelial cells. The expression of the BARF1 open reading frame in primary monkey kidney epithelial cells led us to the establishment of continuously dividing lines. The BARF1 transfectants showed the major characteristics of immortalized cells: morphological change, short cell doubling time, ability to divide at low cell density and continuous growth over 50 passages. Injection of BARF1 transfectants into nude mice did not induce any tumor. Established subclones were shown to be epithelial cells expressing known keratins as well as the BARF1 coded mRNA and protein. This is the first report indicating that expression of the BARF1 gene product in primary epithelial cells may contribute to the establishment of cell lines.

Antibody and antibody-dependent cellular cytotoxicity responses against the BamHI A rightward open-reading frame-1 protein of Epstein-Barr virus (EBV) in EBV-associated disorders.

Antibody-dependent cellular cytotoxicity (ADCC) is an important antiviral effector mechanism. ADCC to the protein encoded by the Epstein-Barr virus (EBV) BamHI A rightward open-reading frame-1 (BARF1) was studied by transducing Raji-tk- cells with the BARF1 gene using a retroviral expression vector. The transduced Raji cells expressed BARF1 on the cell surface, as determined by flow cytometry. Sera from chronic and acute infectious mononucleosis and nasopharyngeal carcinoma patients were found to contain antibodies that react with the BARF1 protein. When BARF1-expressing Raji cells were used as targets for ADCC, sera from several nasopharyngeal carcinoma patients demonstrated significant ADCC reactivity, whereas sera from healthy EBV-seronegative and -seropositive persons lacked such reactivity. BARF1-specific ADCC activity could be competitively inhibited with recombinant BARF1 protein. The level of anti-BARF1 antibody activity in sera of patients with EBV-associated diseases suggests that the BARF1 protein may serve as a target on EBV-infected cells for ADCC.

Preliminary study on the pathological model of Piyinxu in rats.

AIM: To study the method of constructing a pathological model of Piyinxu (spleen-yin deficiency) using Sprague-Dawley rats and to make preliminary observations. METHODS: Folium sennae (0.8 g) and tabellae thyroidei (80 mg) were given daily by stomach feeding to each rat for 12 d to establish the pathological model of Piyinxu. The activity of the rats and the characteristics of their stool were observed. RESULTS: We observed not only the symptoms of Pixu, such as diarrhea, poor appetite, abdominal distention, weight loss, but also Yinxuneire (endogenous heat), such as increase in water drinking, nervousness, and restlessness. These symptoms were all different from that of the control group of Piyangxu (spleen-yang deficiency). CONCLUSION: Combining folium sennae and tabellae thyroidei is an effective method for establishing a pathological model of Piyinxu.

Characteristics of saliva secreted by patients with TCM-Piyinxu.

AIM: To investigate various characteristics of saliva secreted by patients with TCM-Piyinxu (Spleen-yin deficiency). METHODS: Twenty-five individuals with Piyinxu (15 males and 10 females; age range 26-70 years, mean age = 45 years) diagnosed based on criteria used in traditional Chinese medicine, were compared with 20 individuals with Shenyinxu (Kidney-yin deficiency) (11 males, 9 females; age range 35-75 years, mean age = 50) and 30 normal individuals (17 males, 13 females; age range 35-65 years, mean age = 49 years). After acid stimulation, the saliva flow in each group was measured, and the levels of amylase and protein in saliva were determined using an automatic biochemical analyzer. The resultant data were analyzed using the Kruskal-Wallis test and one-way factorial ANOVA test. RESULTS: The flow rates of saliva and amylase in Piyinxu patients (0.27 ± 0.016 mL/min and 2134.13 ± 343.51 IU/min, respectively) were lower than those in normal subjects (0.46 ± 0.027 mL/min and 3501.63 ± 1099.63 IU/min, respectively, P < 0.01), but higher than those in the Shenyinxu group (0.13 ± 0.051 mL/min and 951.62 ± 383.17 IU/min, respectively, P < 0.01). The three groups showed no significant difference in their level of total salivary protein (Piyinxu group, 3.07 ± 0.60 g/L; Shenyinxu group, 3.01 ± 0.90 g/L, and control group, 2.94 ± 1.13 g/L, P = 0.869), amount of amylase per saliva volume, or their ratio of amylase to protein in secreted saliva (P = 0.173 and P = 0.436, respectively). CONCLUSION: Piyinxu patients showed altered rates of saliva and amylase secretion when compared with those parameters in patients with Shenyinxu and normal subjects.

Escherichia coli gpt gene sensitizes rat glioma cells to killing by 6-thioxanthine or 6-thioguanine.

Genes that encode enzymes that convert inactive "prodrugs" into anticancer metabolites may be therapeutically useful against brain tumors. Unlike other genes tested to date in brain tumor models, the Escherichia coli gpt gene is unique in that it not only sensitizes cells to the prodrug 6-thioxanthine (6TX) but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus providing a means to select for gpt-positive cells. In the present study, rat C6 glioma cells were infected with a retrovirus vector that transduces this gene. A clonal line (C6GPT-7) was derived that exhibited significant 6TX susceptibility in vitro with an ID50 of 2.5 mumol/L, whereas 50% growth inhibition of parental C6 cells was not achieved at concentrations tested (up to 50 mumol/L). This line also exhibited significant sensitivity to 6-thioguanine (6TG), with an ID50 of 0.05 mumol/L, whereas 50% growth inhibition of parental C6 cells was achieved at 0.5 mumol/L. In a "bystander" assay, C6GPT-7 tumor cells efficiently transferred 6TX sensitivity to C6 cells at ratios as low as 1:9 (C6GPT-7:C6). This in vitro bystander effect was abrogated when C6GPT-7 and C6 cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites. In vivo both 6TX and 6TG significantly inhibited the growth of subcutaneously transplanted C6GPT-7 cells but not that of C6 cells in athymic mice. In an intracerebral model, both 6TX and 6TG exhibited significant antiproliferative effects against tumors formed by C6GPT-7 cells. These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the E coli gpt gene to provide chemosensitivity against 6TX and 6TG.

Transgene inheritance and retroviral infection contribute to the efficiency of gene expression in solid tumors inoculated with retroviral vector producer cells.

One strategy to achieve efficient gene delivery into brain tumors employs the stereotactic implantation of fibroblasts that express a foreign gene and produce a retroviral vector bearing that gene. Another method involves the grafting of fibroblasts genetically engineered to produce a foreign gene product of interest. It is not clear to what extent retrovirus production in vivo provides an advantage over the grafting of genetically engineered cells for the purpose of achieving transgene expression. These two methods of gene delivery were compared in vivo by using the following cell lines: CRIP-MFG-LacZ cells, which express the lacZ gene and produce retrovirus vectors that bear this gene, and CRIP-LacZ cells, which express the lacZ gene, but do not produce retrovirus. Gene delivery was assessed in C6 gliomas established in the righ frontal lobe of athymic mice. CRIP-MFG-LacZ or CRIP-LacZ cells were inoculated stereotactically into these tumors. When CRIP-MFG-LacZ cells were used, a relatively elevated level of lacZ gene expression was present in cells scattered throughout the tumor. Using a computerized imaging system, this expression occurred in approximately 10% of the tumor area at 1 week, 42% at 2 weeks, and 32% at 3 weeks. In contrast, with CRIP-LacZ cells, lacZ gene expression was much weaker and occurred in a more focal area within the tumor. This expression occupied approximately 5% of the tumor area at 1 and 2 weeks and had almost disappeared at 3 weeks. In both cases there was no notable expression of the transgene in normal brain cells. In conclusion, transgene expression in brain tumors was achieved in more cells, at higher levels, and for longer time periods with retroviral vector-producing cells than with genetically engineered fibroblasts. This efficiency of gene delivery likely results from direct in situ delivery of the transgene to tumor cells with subsequent inheritance of the reporter gene to progeny tumor cells.

Diffusible cytotoxic metabolites contribute to the in vitro bystander effect associated with the cyclophosphamide/cytochrome P450 2B1 cancer gene therapy paradigm.

Tumor cells become sensitive to the inert prodrug cyclophosphamide (CPA) after transfer of the gene encoding cytochrome P450 2B1. This enzyme activates CPA into 4-hydroxycyclophosphamide, which ultimately degrades into acrolein and phosphoramide mustard, the anticancer and DNA-alkylating metabolite. It is imperative that any prodrug-activating gene therapy strategy against cancer possess the capacity to affect the proliferation of tumor cells even when they do not express the transgene (bystander effect), because current methodologies cannot achieve gene transduction in all tumor cells. Prodrug-activating gene therapy schemes described to date exhibit a bystander effect that is not mediated by conditioned medium in culture and may depend on cell contact. In contrast, we find that CPA-sensitized, P450-expressing C6 glioma cells (C6-P450) transfer cytotoxicity to nonexpressing cells by releasing diffusible metabolites through the medium. A 3-h exposure to the prodrug is necessary and sufficient to achieve killing of the transfected cells, and medium conditioned by these cells can kill untransfected cells with similar potency. This bystander effect occurs in the presence of CPA even when only 10% of cells in culture express the P450 2B1 gene, and it is not reproduced by cells that have been irradiated. In an animal model of intracerebral brain tumors, expression of the P450 2B1 gene within the neoplastic cells enhanced significantly the antitumor effect of CPA, even when it was administered systemically. This study shows that CPA/P450 2B1 gene therapy represents a novel tumor-killing strategy that displays an expanded range of cytotoxic action both spatially and temporally within tumor cells and significantly potentiates the anticancer action of CPA when administered i.v.