Development of systemic immunity to glioblastoma multiforme using tumor cells genetically engineered to express the membrane-associated isoform of macrophage colony-stimulating factor.

We investigated the ability of Fischer rat T9 glioblastoma cells transduced with cDNA genes for the secreted (s) or membrane-associated (m) isoform of M-CSF to elicit an antitumor response when implanted into syngeneic animals. Intracranial (i.c.) implantation of 1 x 10(5) T9 cells expressing mM-CSF (T9/mM-CSF) resulted in 80% tumor rejection. Electron microscopy of the T9/mM-CSF tumor site, 2-4 days postimplantation, showed marked infiltration by macrophages, many of which were in physical contact with the T9/mM-CSF cells. Animals that rejected T9/mM-CSF cells were resistant to i.c. rechallenge with T9 cells, but not syngeneic MadB106 breast adenocarcinoma cells, suggesting that T9-specific immunity can be generated within the brain via the endogenous APCs. Intracranial injection of parental T9, vector control (T9/LXSN), or T9 cells secreting M-CSF (T9/sM-CSF) was 100% fatal. Subcutaneous injection of 1 x 10(7) T9/sM-CSF, T9/LXSN, or parental T9 cells resulted in progressive tumors. In contrast, T9/mM-CSF cells injected s.c. were destroyed in 7-10 days and animals developed systemic immunity to parental T9 cells. Passive transfer of CD3+ T cells from the spleens of immune rats into naive recipients transferred T9 glioma-specific immunity. In vitro, splenocytes from T9/mM-CSF-immunized rats specifically proliferated in response to various syngeneic glioma stimulator cells. However, only marginal T cell-mediated cytotoxicity was observed by these splenocytes in a CTL assay against T9 target cells, regardless of restimulation with T9 cells. Subcutaneous immunization with viable T9/mM-CSF cells was effective in eradicating i.c. T9 tumors.

Expression of surface antigens during the differentiation of human dendritic cells vs macrophages from blood monocytes in vitro.

High expression of MHC antigens and adhesion/costimulation molecules is considered as one of the major characteristics qualifying macrophages (M) and dendritic cells (DC) as professional antigen presenting cells. Since accessory activity of M is known to be weaker than that of DC but both M or DC can differentiate from blood monocytes (MO) depending on culture conditions (i.e. GM-CSF vs GM-CSF/IL-4), we investigated the kinetics of expression of MHC antigens and several adhesion/costimulation molecules during the differentiation of DC or M from blood MO. Blood MO cultured with GM-CSF consistently induced M that showed adherence to plastic and CD14 expression. In contrast, MO cultured with GM-CSF/IL-4 rapidly became nonadherent, acquired DC morphology and lost CD14 expression. M but not DC proliferated as demonstrated by [H3]thymidine incorporation. MHC Class I was highly expressed in both M and DC. In contrast, MHC Class II molecules were significantly higher on DC compared to M. CD80 was upregulated on both DC and M but only on a subset of cells. CD80 expression peaked at day 3 on M and declined thereafter, while on DC expression increased significantly until day 10. CD86 was upregulated on the majority of DC and M. However, while M maintained stable expression of CD86 after day 3, DC progressively upregulated CD86 throughout the culture period. CD1a expression was initially low in both cell types and peaked at day 3 in M declining thereafter, while expression remained stable on DC until day 10. ICAM-1 expression was significantly upregulated on M when compared to DC at day 3. However, on M, ICAM-1 expression became undetectable by day 5 while on DC it increased through day 10. Similarly, CD40 was transiently expressed on M until day 5, while on DC it continuously increased until day 10. Finally, in contrast to other antigens, LFA-3 was always more strongly expressed on M than DC at all culture periods. Taken together, these data suggest that M showed a rapid but transient upregulation in the expression of adhesion/costimulation molecules, suggesting that maximal accessory ability is reached by M at an earlier time point than DC. Significant differences in surface antigen expression DC vs M were recognizable for MHC class II, CD86, CD80, CD1a, CD40 and ICAM-1. Specifically, major differences occurred for MHC class II, CD86, CD40 and ICAM-1. Therefore, the higher accessory ability of DC compared to M in naive T cell priming may be related to qualitative and quantitative differences in expression of these immunologically important surface molecules.

Effects of retinoic acid combined with irradiation on the expression of major histocompatibility complex molecules and adhesion/costimulation molecules ICAM-1 in human cervical cancer.

OBJECTIVES: Radiation treatment is one of the most standardized and effective modalities for contemporary cervical cancer therapy. In addition, the radiation-potentiating effects of retinoic acid have been recently described. In order to investigate whether enhanced immunogenicity might be responsible for such potentiation, we have evaluated the effects of retinoic acid combined with high doses of gamma-irradiation on the expression of major histocompatibility complex (MHC) Class I and II and intercellular adhesion molecule-1 (ICAM-1) in human cervical carcinoma cell lines. METHODS: The expression of surface antigens (MHC Class I and II and ICAM-1) was evaluated by FACS analysis in untreated control cells and in cells following their exposure to retinoic acid, high doses of gamma-irradiation (i.e., 5000 and 10,000 cGy), or the combination of the two procedures. RESULTS: HT-3 and SiHa cervical cancer cells expressed variable levels of MHC Class I and ICAM-1 antigens while Class II surface antigens were not detectable. Exposure to either 5000 or 10,000 cGy completely inhibited cell replication in both cell lines and significantly and consistently increased the expression of all surface antigens present on the cells prior to irradiation. Irradiation was unable to induce neoexpression of antigens previously not expressed by these cells (i.e., MHC Class II). In a similar fashion, retinoic acid was also able to significantly increase the expression of MHC Class I and ICAM-1 antigens when compared to untreated tumor cells but was not able to induce the expression of HLA Class II surface antigens. Exposure to the combination of radiation plus retinoic acid significantly upregulated HLA Class I and ICAM-1 molecules in an additive manner when compared to the levels obtainable with the exposure to radiation or retinoic acid alone. CONCLUSIONS: These data indicate that the combination of these two treatments could induce an additive effect on the expression of immunologically important surface antigens in human cervical cancer cells. These findings, together with the powerful antiproliferative effect of retinoids and irradiation on tumor cells, suggest that the combined regimen may be a promising and more effective combination for the treatment of cervical cancer.

The effects of irradiation on the expression of a tumour rejection antigen (heat shock protein gp96) in human cervical cancer.

PURPOSE: Studies were designed to analyse the effects of high doses of gamma-irradiation on the expression of a tumour rejection antigen (heat shock protein gp96) in human cervical carcinoma cell lines. MATERIALS AND METHODS: The expression of heat shock protein gp96 was evaluated at the transcriptional (Northern blot) and post-transcriptional levels (Western blot) in two human cervical carcinoma cell lines following exposure to high doses of gamma-irradiation. RESULTS: Doses of gamma-irradiation ranging from 25 to 100 Gy significantly and consistently increased the expression of heat shock protein gp96 on CaSki and HT-3 cervical cancer cells. The increase in the amount of protein was due to transcriptional up-regulation of this gene. Radiation doses unable to inhibit completely cell replication in the totality of tumour cells (i.e. 25 Gy), as well as higher (fully lethal) doses of irradiation (i.e. 50 to 100 Gy), were shown to up-regulate significantly the expression of heat shock protein gp96 mRNA in a dose-dependent manner. CONCLUSIONS: Recently, gp96 molecules have been implicated in the presentation of endogenous and viral antigens. A number of key elements in this pathway, including major histocompatibility complex (MHC) class I molecules as well as adhesion/co-stimulation molecules such as ICAM-1, are known to be sensitive to irradiation effects. The results show that radiation can also increase the expression of other immunologically important cell molecules such as a tumour rejection antigen (heat shock protein gp96) in human cervical cancer. Such findings may partially explain the increased immunogenicity of tumour cells following irradiation and further support a role for local radiation therapy as a powerful biologic response modifier.

Effects of retinoic acid combined with interferon-gamma on the expression of major-histocompatibility-complex molecules and intercellular adhesion molecule-1 in human cervical cancer.

Retinoids and interferons are important regulators of human epithelial cell differentiation and have been successfully used in the clinical treatment of HPV-involved cervical cancer. In this study, 2 HPV-positive human cervical-carcinoma cell lines were analyzed for their surface expression of MHC-Class-I, MHC-Class-II and ICAM-1 surface antigens before and after exposure to all-trans retinoic acid, interferon-gamma and the combination of the 2 compounds. In addition, the effects on HLA-Class-I-mRNA expression were evaluated after such treatments. Both cell lines expressed MHC-Class-I molecules, and their levels were markedly up-regulated after exposure to IFN-gamma. Similarly, MHC-Class-II and ICAM-1 antigens were either induced or significantly up-regulated by IFN-gamma. Exposure to all-trans retinoic acid was also able to significantly increase the expression of MHC-Class-I and ICAM-I antigens as compared with untreated tumor cells. However, unlike IFN-gamma, retinoids were not able to induce the expression of HLA-Class-II surface antigens. Exposure to the combination of IFN-gamma plus retinoic acid significantly up-regulated (in an additive manner) HLA-Class-I and ICAM-1 molecules as compared with the levels obtainable after exposure to IFN-gamma alone. Finally, Northern-blot analysis of HLA-Class-I-mRNA expression confirmed that the activity of both of these biologic response modifiers was at transcriptional level. These data indicate that the combination of these modalities could induce an additive effect on the expression of immunologically important surface antigens on human cervical-cancer cells. These findings, together with the known anti-proliferative effects mediated by retinoids and IFN-gamma on tumor cells, further support the combination of these agents in the treatment of pre-invasive and invasive human cervical cancer.

Effects of irradiation on the expression of major histocompatibility complex class I antigen and adhesion costimulation molecules ICAM-1 in human cervical cancer.

PURPOSE: We initiated studies to analyze the effects of high doses of gamma irradiation on the surface antigen expression of MHC Class I, Class II, and ICAM-1 on human cervical carcinoma cell lines. METHODS AND MATERIALS: The expression of surface antigens (MHC Class I, Class II, and ICAM-1) was evaluated by FACS analysis on two cervical cell lines at different time points, following their exposure to high doses of gamma irradiation (i.e., 25.00, 50.00, and 100.00 Gy). RESULTS: The CaSki and SiHa cervical cancer cells we analyzed in this study expressed variable levels of MHC Class I and ICAM-1 antigens, while Class II surface antigens were not detectable. Whereas irradiation doses of 25.00 Gy were not sufficient to totally block cell replication in both cell lines, exposure to 50.00 or 100.00 Gy was able to completely inhibit cell replication. Range doses from 25.00 to 100.00 Gy significantly and consistently increased the expression of all surface antigens present on the cells prior to irradiation but were unable to induce neoexpression of antigens previously not expressed by these cells (i.e., MHC Class II). Importantly, such upregulation was shown to be dose dependent, with higher radiation doses associated with increased antigen expression. Moreover, when the kinetic of this upregulation was studied after 2 and 6 days after irradiation, it was shown to be persistent and lasted until all the cells died. CONCLUSIONS: These findings may partially explain the increased immunogenicity of tumor cells following irradiation and may suggest enhanced immune recognition in tumor tissue in patients receiving radiation therapy.

In vivo fluorescence detection of ovarian cancer in the NuTu-19 epithelial ovarian cancer animal model using 5-aminolevulinic acid (ALA).

The purpose of this study was to determine whether in vivo fluorescence detection of protoporphyrin IX (PpIX) could be used to identify intraperitoneal micrometastases of epithelial ovarian carcinoma after application of 5-aminolevulinic acid (ALA). ALA was applied intraperitoneal at different concentrations (25, 50, and 100 mg/kg) and iv (100 mg/kg) to immunocompetent Fischer 344 rats bearing a syngeneic epithelial ovarian carcinoma. At different time intervals after ALA administration (1.5, 3, and 6 hr) the peritoneal cavity was illuminated with ultraviolet (uv) light. In vivo fluorescence of PpIX initially was determined by direct visualization. Subsequently ex vivo measurements were made with a slow-scan, thermoelectrically cooled CCD camera. Red in vivo fluorescence was observed in ovarian micrometastases smaller than 0.5 mm in 100% of the ALA-administered animals independent of time interval, drug concentration, or route of administration. The intensity of the fluorescence was concentration dependent as strong fluorescence was consistently found only above 25 mg/kg ALA. Ex vivo tumor to peritoneum fluorescence yield peaked 3 hr after administration of a 100 mg/kg intraperitoneal dose. Direct visualization of in vivo fluorescence after ALA application may improve the detection of intraperitoneal ovarian cancer micrometastases.

Differential transforming growth factor-beta secretion in adenocarcinoma and squamous cell carcinoma of the uterine cervix.

Tumor cells from eight freshly isolated cervical cancers (i.e., four adenocarcinomas and four squamous carcinomas) were analyzed for their production of the immune-inhibitory cytokine transforming growth factor-beta (TGF-beta) in vitro. All fresh adenocarcinomas secreted significant levels of TGF-beta (mean 397, range between 207 and 782 pg/ml/10(5) cells/48 hr). In contrast, no detectable TGF-beta was present in the supernatants from the four fresh squamous carcinoma cultures (P < 0.001). These data suggest that major differences in the secretion of the immunoinhibitory cytokine TGF-beta exist between squamous cell carcinomas and adenocarcinomas of the uterine cervix. Furthermore, these findings suggest that at least some of the differences in the natural biologic behavior, as well as in the response to radiation treatment, between these two histologic types of cervical cancer could be related to differences in secretion of this immune-inhibitory cytokine.

Lymphocyte and monocyte-induced motility of MCF-7 cells by tumor necrosis factor-alpha.

A potentially important tumor-host interaction is increased tumor-cell invasiveness in response to motility factors derived from stromal and lymphoid cells. Conditioned medium of IL-2-stimulated lymphocytes and fractions enriched in either T cells, natural killer (NK) cells, or monocytes induced motility in MCF-7 breast carcinoma cells. ELISA and antibody neutralization studies demonstrated that this effect was due to tumor necrosis factor-alpha (TNF-alpha) secretion by the lymphoid cells or the enriched fractions. Unstimulated leukocytes in direct contact with MCF-7 cells also induced motility that was inhibited by anti-TNF-alpha antiserum. Time-lapse video microscopy of cells exposed to 10 ng/ml TNF-alpha showed that motility was independent of its toxic effects. Immunoperoxidase showed that MCF-7 cells expressed both the 55-kDa and the 75-kDa TNF-alpha receptors (TNFR). Antiserum against the 55-kDa TNFR, like TNF-alpha, induced motility in MCF-7 cells. This was most likely due to cross-linking of the 55-kDa TNFR monomers, since the monomeric F(ab) did not produce this effect. Our results raise the possibility that TNF-alpha-induced motility is one mechanism by which tumor cells overcome the potential anti-tumor immune function of lymphocytes and macrophages in peri-tumoral infiltrates.

Development and characterization of a clinically useful animal model of epithelial ovarian cancer in the Fischer 344 rat.

OBJECTIVE: Our purpose was to develop and characterize a spontaneously arising, nonimmunogenic experimental animal model of epithelial ovarian cancer. STUDY DESIGN: NuTu-19 is a cell line derived from a poorly differentiated adenocarcinoma formed in a female athymic mouse after subcutaneous injection of spontaneously transformed Fischer 344 rat ovarian surface epithelial cells. This cell line was injected intraperitoneally into naive, immunocompetent Fischer 344 rats to determine tumor growth and animal survival. Immunogenicity of this cell line was determined by repetitive vaccination of naive rats with either mitomycin C-treated or irradiated (5000 cGy) NuTu-19 cells, followed by intraperitoneal rechallenge with viable tumor cells. Kaplan-Meier survival analysis was used to analyze survival data. Major histocompatibility complex class I and class II and intercellular adhesion molecule-1 cell surface antigens were determined by fluorescence-activated cell sorting analysis. RESULTS: NuTu-19 cells injected intraperitoneally grew progressively as numerous serosal nodules (peritoneum, omentum, diaphragm, liver, bowel), exhibited local tissue invasion and formed malignant ascites in a manner typical for human ovarian epithelial carcinomas. Animal survival was dosage dependent where as few as 10(4) cells were fatal when introduced intraperitoneally; mean animal survival was noted to be approximately 49 days when 10(5) cells were injected intraperitoneally. Repetitive immunizations of animals with large doses (10(7)) of inactivated NuTu-19 cells did not confer immunity to the animals, which all died on subsequent challenge with viable parental tumor cells. NuTu-19 cells expressed high levels of major histocompatibility complex class I and intercellular adhesion molecule-1 cell surface antigens and very low levels of major histocompatibility complex class II antigens. CONCLUSION: This is the first report of a reliable, spontaneously arising, nonimmunogenic epithelial ovarian cancer animal model. Because this model exists in an immunocompetent animal, it will be useful for studying the biologic and immunologic features of ovarian cancer.

Differential effects of high-dose gamma irradiation on the production of transforming growth factor-beta in fresh and established human ovarian cancer.

Tumor cells from five freshly isolated ovarian tumors and four established human ovarian carcinoma cell lines were analyzed for the production of the immunoinhibitory cytokine transforming growth factor-beta (TGF-beta) before and after exposure to gamma irradiation and/or the cytokines TNF-alpha plus IFN-gamma. All fresh tumors secreted high levels of TGF-beta when compared to the levels produced by the established ovarian carcinoma cell lines. TGF-beta produced by fresh tumors was significantly reduced after high doses of gamma irradiation (10,000 cGy). In contrast with the established cell lines, irradiation significantly increased TGF-beta secretion. Exposure of fresh tumor cells to cytokines followed by irradiation caused significant reduction of TGF-beta released when compared to the amount released after exposure to cytokines only. However, in the established cell lines, cytokines followed by irradiation again significantly increased TGF-beta production. These data indicate that high doses of irradiation in fresh ovarian tumors, unlike established ovarian carcinoma cell lines, can significantly reduce the local production of this potent immunoinhibitory cytokine. This effect could work to further amplify weak immunological responses within the tumor. In addition, these findings indicate major differences between fresh tumor samples and established cell lines and warn against the sole use of continuous cell lines as models for tumors growing in vivo.

Effects of irradiation on the expression of surface antigens in human ovarian cancer.

Tumor cells from four established human ovarian carcinoma cell lines were analyzed for their expression of surface antigens including MHC Class I, Class II, ICAM-1, and the tumor-associated antigens CA 125 and Her2-neu before and after exposure to high doses of gamma irradiation. All four ovarian cell IInes expressed variable levels of MHC Class I and Her2-neu. ICAM-1 antigens were expressed in only two cell lines and Class II and CA 125 surface antigens were absent in all the cell lines evaluated. Exposure to high doses of gamma irradiation (i.e., 5000 to 10,000 cGy) significantly and consistently increased the expression of all surface antigens present on the cells prior to irradiation. Importantly, the irradiation-induced upregulation was persistent and lasted until all the cells died. Irradiation was unable to induce neoexpression of antigens previously not expressed by the cells (i.e., MHC Class II or ICAM-1). These findings may partially explain the increased immunogenicity of tumor cells following irradiation and may suggest enhanced immune recognition in tumor tissue in patients receiving radiation therapy.

Effects of cytokines combined with high-dose gamma irradiation on the expression of major histocompatibility complex molecules and intercellular adhesion molecule-1 in human ovarian cancers.

Tumor cells from 7 freshly isolated human ovarian tumors and 2 continuous human ovarian cancer cell lines were analyzed for their surface expression of MHC class-1, class 11 and ICAM-1 surface antigens before and after exposure to gamma-irradiation and/or the cytokines TNF-alpha plus IFN-gamma. All 7 fresh tumors expressed high levels of MHC class 1 and 1CAM-1 antigens, and levels were markedly up-regulated after exposure to TNF-alpha plus IFN-gamma Similarly, class-11 antigens were either induced (3 out of 7 tumors) or significantly up-regulated by TNF-alpha plus IFN-gamma. Exposure to high doses of gamma-irradiation also increased the expression of MHC class-1 and ICAM-1 antigens, albeit to a modest degree. MHC class 1 and ICAM-1 antigens expression was much lower on continuous human ovarian cell lines than on the fresh tumors. Exposure of these cells to TNF-alpha plus IFN-gamma markedly up-regulated antigen expression to levels comparable to those expressed on the freshly isolated tumors. With the established ovarian cell lines, removal of cytokines caused a rapid down-regulation of antigen expression to basal levels within 6 days, while in the fresh tumors a low level of up-regulation was still present at this time. In contrast, exposure to cytokines followed by high-dose gamma-irradiation resulted in a highly significant and long-lasting expression of each surface antigen which was either up-regulated or induced by the cytokines. These data indicated that the combination of these modalities may be beneficial in generating optimal antigen expression for use of tumor cells in vaccine studies.

Development and characterization of an interleukin-2-transduced human ovarian carcinoma tumor vaccine not expressing major histocompatibility complex molecules.

OBJECTIVE: We initiated studies to develop cytokine-secreting human ovarian carcinoma cells for the purpose of using these cells as vaccines for the treatment of advanced epithelial ovarian cancer. STUDY DESIGN: A human ovarian carcinoma cell line (UCI-107) was genetically engineered to secrete the cytokine interleukin-2 by retroviral-mediated gene transduction. RESULTS: One clone, termed UCI-107A IL-2 AS, constitutively secreted high levels of interleukin-2 (i.e., 2000 to 2300 pg/ml/10(5) cells per 48 hours) for > 55 passages and 8 months of study. Unlike parental- and vector-transduced cells, UCI-107A IL-2 AS cells were aneuploid and failed to express major histocompatibility complex class I and HER2/neu surface antigens. UCI-107A IL-2 AS cells were highly resistant to killing by gamma irradiation and continued to produce high levels of interleukin-2 even after irradiation with 10,000 cGy. Balb/C nude mice injected intraperitoneally with UCI 107-A IL-2 AS cells survived significantly longer than control animals, with 25% of the animals totally rejecting their tumors. UCI-107A IL-2 AS was totally resistant to killing by fresh allogeneic peripheral blood lymphocytes in four hour chromium 51 release assays but induced high levels of killing in 72-hour long-term cytotoxic assays. CONCLUSION: The potential use of these interleukin-2-secreting ovarian carcinoma cells as vaccines for women with advance ovarian cancer will be discussed.

The role of NK cell activity in the pathogenesis of poly I:C accelerated and spontaneous diabetes in the diabetes prone BB rat.

The development of insulin dependent diabetes mellitus (IDDM) and diabetes in the diabetes prone (DP) BB rat animal model of IDDM is thought to be due to an autoimmune process. Natural killer (NK) cells have been implicated but not proven to play a pathogenetic role in BB rats due to the increased NK cell number and activity found in these animals. We have recently reported that poly I:C, an inducer of cytokines and a potent enhancer of NK cell function, accelerates the development of diabetes in DP BB rats and induces diabetes in diabetes resistant (DR) BB rats. Since we have further demonstrated that poly I:C administration to BB rats increases NK cell number and levels of inducers of NK cell activity, interferon-alpha and IL-6 which is described therein, we tested the hypothesis that NK cell activity plays an important role in poly I:C accelerated disease. The role of NK cells in poly I:C accelerated diabetes and spontaneous diabetes was examined by determining whether selective depletion of NK cells using a rat NK cell specific antibody (anti-NKR-P1 antibody) alters the development of diabetes. The treatment of BB rats with anti-NKR-P1 antibody resulted in a significantly lower mean NK cell activity of splenic mononuclear cells than that found in control animals. However, the development of diabetes and degree of insulitis was not significantly different between treatment groups. BB rats administered anti-NKR-P1 antibody with poly I:C had a lower mean splenocyte NK cell activity and lower mean NK cell number within the peripheral blood and inflamed islets than rats administered poly I:C alone. However, anti-NKR-P1 antibody administration did not alter the accelerated development of diabetes or the degree of insulitis in poly I:C treated animals. These data document that NK cells do not play a major role in the pathogenesis of poly I:C accelerated diabetes or spontaneous diabetes in the DP BB rat.

Development and in vitro characterization of a GM-CSF secreting human ovarian carcinoma tumor vaccine.

A human ovarian carcinoma cell line (UCI-107) was genetically engineered to secrete the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF), by retroviral medicated gene transduction. This line was transduced with the LXSN retroviral vector containing the human GM-CSF gene and the neomycin resistance selection marker. Numerous GM-CSF secreting clones were randomly isolated and one clone, termed UCI-107M GM-CSF-MPS, extensively characterized. This clone was shown to constitutively secrete high levels of GM-CSF (ie 420-585 pg ml-1 105 cells-1 48 h-1 for over 35 passages and 6 months of study. Like the parental cell line UCI-107, UCI-107M GM-CSF-MPS cells expressed MHC class I and Her2/Neu surface antigens but did not express detectable MHC class II, ICAM-1 or CA-125. No change in the expression of these surface proteins was noted between the parental cells and this GM-CSF secreting clone. The morphology of UCI-107M GM-CSF-MPS did not differ from that of the parental or LXSN vector control cells; however, parental cells had a slightly faster growth rate than the transductants. UCI-107M GM-CSF-MPS was sensitive to gamma irradiation, since as little as 2500 rads killed the cells within 10 days of irradiation. However, even after higher doses of irradiation (ie 10000 rads), GM-CSF secretion continued in vitro until about day 8. Interestingly, irradiation induced up-regulation of the surface antigens previously expressed, and they remained up-regulated for as long as the cells remained viable. The potential use of these GM-CSF secreting ovarian carcinoma cells as vaccines for women with advanced ovarian cancer will be discussed.

Development and characterization of an IL-4-secreting human ovarian carcinoma cell line.

Human ovarian carcinoma cell lines were genetically engineered to secrete the cytokine interleukin-4 (IL-4) by retroviral-mediated gene transduction. These cells were transduced with the LXSN retroviral vector containing the human IL-4 gene and the neomycin resistance selection marker. Numerous IL-4-secreting clones were isolated from different papillary serous carcinoma cell lines, including SKOV-3, UCI-101, and UCI-107, and one clone derived from UCI-107 extensively characterized. This clone, termed UCI 107E IL-4 GS, was shown to constitutively express high levels of IL-4 (i.e., 900 to 1300 pg/ml/10(5) cells/48 hr) for over 35 passages and 6 months of study. Like the parental cell line (UCI-107), UCI 107E IL-4 GS cells expressed MHC class I and Her-2/neu surface antigens but did not express detectable MHC class II, ICAM 1, CA 125, or IL-4 receptors. No increase in expression of surface proteins was noted between parental and UCI 107E IL-4 GS. The morphology of this clone did not differ from that of the parental or LXSN vector control cells; however, parental cells had a faster growth rates than transductants. UCI 107E IL-4 GS was sensitive to gamma irradiation since as little as 2500 rad killed most of the cells within 10 days of irradiation. However, after irradiation, IL-4 secretion continued until about Day 8. The potential use of these IL-4-secreting ovarian carcinoma cells as vaccines for woman with advanced ovarian cancer will be discussed.

A comparison of blood lipid and lipoprotein values in young adults who die suddenly and unexpectedly from atherosclerotic coronary artery disease with other noncardiac deaths.

Postmortem blood lipid and lipoprotein analyses were conducted in a case-controlled study of young adults (ages 22-43) who died suddenly and unexpectedly of atherosclerotic coronary artery disease in Allegheny County, Pennsylvania. None of the individuals in the study group (n = 28 cases) had a significant medical or cardiac history except in the immediate antemortem period. The control group (n = 31) consisted of age- and sex-matched cohorts who died of noncardiac related fatalities and who had no evidence of CAD. The results indicated a male-to-female ratio of nearly 30:1 with a marked predominance of young white men. Mean total cholesterol (241 mg/dL), triglycerides (583 mg/dL), and low-density lipoproteins (LDL) (107 mg/dL) were all significantly elevated in the study group as compared to controls (p < 0.001, p < 0.018, and p < 0.001 for the three parameters, respectively). Mean Apolipoprotein B (98.7 mg/dL) was also significantly elevated compared to control values (p < 0.001). By contrast, mean high-density lipoprotein values (36 mg/dL) were not significantly different from control values (p = 0.35), and mean values of Apolipoproteins A1 (121 mg/dL) and A2 (37.6 mg/dL) were essentially identical to control values (p = 0.44 and p = 0.64, respectively). The differences in these biochemical markers between the two groups could not be explained by differences in postmortem interval or by the presence of recently ingested food. These findings indicate that elevations of plasma cholesterol, triglycerides, LDL, and Apolipoprotein B are important biochemical markers for the development of early and apparently clinically silent yet life-threatening coronary artery disease.

NKR-P1+ cells in the rat uterus: granulated metrial gland cells are of the natural killer cell lineage.

An intriguing component of the maternal response to pregnancy is the differentiation of large numbers of large granular lymphocytes, termed granulated metrial gland (GMG) cells, in the decidua and then in the metrial gland, a structure in the mesometrial triangle unique to rodent pregnancy. We have used the monoclonal antibody 3.2.3 to NKR-P1, a surface molecule involved in triggering natural killer (NK) cells for lysis, to determine the numbers and distribution of NK cells in the nonpregnant rat uterus and during the dramatic changes at implant sites during pregnancy. NKR-P1+ cells were abundant in the nonpregnant uterus, especially in the endometrium. These cells also expressed CD8, CD2, and AsialoGM1. In the subepithelial stroma, the numbers were greatest during proestrus and estrus; in ovariectomized animals, they were severely decreased, but returned to normal with estrogen supplementation. At the time of blastocyst attachment (Day 6), NKR-P1+ cells were few around the implant and in the decidualizing stroma. However, on Day 8, substantial numbers were present in the mesometrial decidua only, and many of these cells expressed the cytolytic protein perforin. By Day 10, NKR-P1+ cells were common within the inner muscle at the base of the mesometrial triangle and in the developing metrial gland, often containing perforin. Larger numbers of perforin+ cells were present in the central decidua towards the ectoplacental cone, and many were weakly NKR-P1+ only. On Day 12, NKR-P1+ cells were almost completely restricted to the metrial gland, with few in decidua, and many were weakly positive. Substantially more perforin-containing cells were seen, indicating that many had lost detectable NKR-P1. This distribution pattern from Days 6-12 is similar to that described for GMG cells and demonstrates that in the rat they belong to the NK cell lineage. These cells were also CD8+ and AsialoGM1+ but negative for CD2 and class II histocompatibility antigens, which is very different from interleukin-2-activated NK cells which they resemble morphologically. The loss during differentiation of NKR-P1 and CD2, which are involved in target adhesion and triggering of NK cells, is consistent with the poor cytolytic capacity reported for these cells.

CD8+ T-cells are required for adoptive transfer of the BB rat diabetic syndrome.

LGLs with NK activity account for the majority of BB rat PBLs expressing CD8, and it has been suggested that these LGL/NK cells are involved in the pathogenesis of the BB rat diabetic syndrome. By using a recently developed mouse MoAb, 3.2.3, specific for rat LGL, we demonstrate that BB and WF rat LGLs are phenotypically and functionally similar. To directly assess the role of LGLs in the development of diabetes in vivo, an adoptive transfer of T-cells to young LGL/NK cell-depleted diabetes-prone BB rats was performed. CD4+8- and CD4-8+ T-cells (> 98.5% pure), isolated from diabetic BB rats, were activated in vitro and injected into 30-day-old diabetes-prone BB rats. Recipients were either chronically injected with 3.2.3 (n = 15) or received an isotype-matched irrelevant MoAB (n = 14). Secondary lymphoid organs of 3.2.3-treated recipients contained < 0.1% 3.2.3+ lymphocytes, and this depletion was associated with a major decrease in the NK activity of their splenocytes. Despite this, the incidence of diabetes in 3.2.3-treated animals (40%) was not significantly different from that observed in control recipients (57%). Thus, the BB rat diabetic syndrome can be adoptively transferred in the absence of LGL/NK cells, suggesting that BB rat CD8+ T-cells are involved in the diabetogenic process. To assess the pathogenic role of CD8+ T-cells, we compared the incidence of diabetes in three groups of diabetes-prone BB recipients after injection of T-cells isolated from diabetic donors.(ABSTRACT TRUNCATED AT 250 WORDS)