Overexpression and immunosuppressive functions of transforming growth factor 1, vascular endothelial growth factor and interleukin-10 in epithelial ovarian cancer.

OBJECTIVE: Transforming growth factor-1 (TGF-ß1), vascular endothelial growth factor (VEGF), and interleukin-10 (IL-10) may be critical cytokines in the microenvironment of a tumor, playing roles in immune suppression. This study was conducted to elucidate the roles and immunosuppressive functions of these cytokines in epithelial ovarian cancer (EOC). METHODS: The expression levels of TGF-ß1, VEGF and IL-10 in malignant tissue were evaluated by immune- histochemistry and compared with corresponding borderline, benign, and tumor-free tissues. Moreover, relationships among the levels of these cytokines and correlations between expression and the prognosis of EOC were analyzed by Pearson rank correlations and multi-factor Logistic regression. The roles of TGF-ß1, VEGF, and IL-10 in the immunosuppressive microenvironment of ovarian cancer were studied through dendritic cell (DC) maturation and CD4+CD25+FoxP3+ Treg generation in vitro experiments. RESULTS: TGF-ß1, VEGF, and IL-10 were expressed in 100%, 74.69%, and 54.96% of EOC patients, respectively. TGF-ß1 was an independent prognostic factor for EOC. IL-10 was significantly co-expressed with VEGF. In vitro, VEGF and TGF-ß1 strongly interfered with DC maturation and consequently led to immature DCs, which secreted high levels of IL-10 that accumulated around the tumor site. TGF-ß1 and IL-10 induced Treg generation without antigen presentation in DCs. CONCLUSIONS: TGF-ß1, VEGF and IL-10 play important roles in EOC and can lead to frequent immune evasion events.

Up-regulation of mitochondrial antioxidation signals in ovarian cancer cells with aggressive biologic behavior.

OBJECTIVE: Recently, a high frequency of mutations in mitochondrial DNA (mtDNA) has been detected in ovarian cancer. To explore the alterations of proteins in mitochondria in ovarian cancer, a pair of human ovarian carcinoma cell lines (SKOV3/SKOV3.ip1) with different metastatic potentials was examined. METHODS: Cancer cells SKOV3.ip1 were derived from the ascitic tumor cells of nude mice bearing a tumor of ovarian cancer cells SKOV3. SKOV3.ip1 exhibited a higher degree of migration potential than its paired cell line SKOV3. The proteins in the mitochondria of these two cells were isolated and separated by 2-D gel electrophoresis. The differently expressed proteins were extracted and identified using matrix assisted laser desorption ionisation/time-of-flight/time-of-flight (MALDI-TOF/TOF), and finally a selected protein candidate was further investigated by immunohistochemistry (IHC) method in nude mice bearing tumor tissues of these two cells. RESULTS: A total of 35 spots with different expressions were identified between the two cells using 2D-polyacrylamide gel electrophoresis (PAGE) approach. Among them, 17 spots were detected only in either SKOV3 or SKOV3.ip1 cells. Eighteen spots expressed different levels, with as much as a three-fold difference between the two cells. Twenty spots were analyzed using MALDI-TOF/TOF, and 11 of them were identified successfully; four were known to be located in mitochondria, including superoxide dismutase 2 (SOD2), fumarate hydratase (FH), mitochondrial ribosomal protein L38 (MRPL38), and mRNA turnover 4 homolog (MRTO4). An increased staining of SOD2 was observed in SKOV3.ip1 over that of SKOV3 in IHC analysis. CONCLUSIONS: Our results indicate that the enhanced antioxidation and metabolic potentials of ovarian cancer cells might contribute to their aggressive and metastatic behaviors. The underlying mechanism warrants further study.

[Variation feature of receptor binding sites of H1N1 influenza hemagglutinin in different hosts].

Recent outbreak of H1N1 virus worldwide has caused 16 226 deaths in over 213 countries and districts. Binding between the virus and the receptor on the host cell surface is the key initial event for the infection, which results in the fusion of viral host cell membrane. Hemagglutinin (HA) is the viral protein that mediates the receptor binding and membrane fusion. The receptor binding sites (RBSs) are located at the membrane-distal part of each subunit of the HA trimer and are formed by three secondary structure elements, 190 helix (residues 190 to 198), 130 loop (residues 135 to 138), and 220 loop (residues 221 to 228). HA1 with 327 amino acid sequences in length was collected from 1221 H1N1 viruses between 1918 and 2009, and bioinformatic studies were carried out through sequence comparison, entropy calculation for each amino acid residue, and 3D structure modeling. The results showed that the RBSs of different viruses with different hosts have different entropies, and the RBSs in HA1 with different hosts have different favorite amino acid sequences. The 3D modeling indicates the subtly conformation changes in the 190 helix region between different HA1s in H1N1. This study explores new characters of the RBS structure in different HA1s, and provides new information for the further investigation of the infection mechanism.

[Measurement of serum human epididymis secretory protein 4 combined with CA125 assay in differential diagnosis of endometriosis cyst and ovarian benign and malignant tumors].

OBJECTIVE: To investigate the value of human epididymis secretory protein 4(HE4) combined with CA125 assay in differential diagnosis of endometriosis cyst and ovarian malignant tumor. METHODS: The level of HE4 and CA125 were measured by enzyme-linked immunosorbent assay (ELISA) in the serum specimens of 46 cases in endometriosis cyst group, 36 cases in malignant ovarian tumor group, 60 cases in benign ovarian diseases and 50 women in healthy women group. Those results were shown with median level. The normal range were 0-150 pmol/L in HE4 and 0-35 kU/L, which either one was more than the threshold value defined as positive index. The sensitivity of assay was evaluated by receiver operating characteristic (ROC) curve, the relation and value of HE4 or CA125 alone and combination assay in diagnosis of endometriosis was analyzed by Mann-Whitney U test and correlation analysis. RESULTS: (1) HE4: the median levels of HE4 were 52.4, 51.0, 50.0 pmol/L in group of endometriosis, normal control and benign ovarian tumor, which didn't show statistical difference. However, HE4 was 507.5 pmol/L in ovarian cancer group, which was significantly higher than those of 3 groups (P<0.05). (2) CA125: there were significant different in median level of CA125 was observed as 743.0 kU/L in ovarian cancer, 84.9 kU/L in endoemtriosis, 15.4 kU/L in benign ovarian disease, and 11.5 kU/L in healthy women (P<0.05). (3) The single assay: when compared with that in endometriosis group, receiver operating characteristic area under the curve (ROC-AUC) were 0.933 in HE4 alone and 0.821 in CA125 alone assay in ovarian cancer group. The specificity was 95% and the sensitivity was 79.6% and 49.0%. (4) The combination assay: when compared with those in endometriosis group, the ROC-AUC was 0.936, the specificity was 95% and the sensitivity was 81.0% in ovarian cancer. CONCLUSIONS: Measurement of HE4 could be used in differential diagnosis of endometriosis cyst. And the combination of HE4 and CA(125) assay could discriminate ovarian endometriosis cysts from ovarian malignant tumors effectively.

Screening for genes associated with ovarian cancer prognosis.

BACKGROUND: Human epithelial ovarian cancer cell line SKOV3.ip1 is more invasive and metastatic compared with its parental line SKOV3. A total of 17 000 human genome complementary DNA microarrays were used to compare the gene expression patterns of the two cell lines. Based on this, the gene expression profiles of 22 patients with ovarian cancer were analyzed by cDNA microarray, and screened the 2-fold differentially expressed genes compared with the normal ones. We screened genes relevant to clinical prognosis of serous ovarian cancer by determining the expression profiles of ovarian cancer genes to investigate cell receptor and immunity-associated genes, and as groundwork, identify ovarian cancer-associated antigens at the gene level. METHODS: Total RNA was extracted from 22 patients with ovarian cancer and DNA microarrays were prepared. After scanning, hybridization signals were collected and the genes that were differentially expressed twice as compared with the normal ones were screened. RESULTS: We screened 236 genes relevant to the prognosis of ovarian cancer from the 17 000 human genome cDNA microarrays. According to gene classification, 48 of the 236 genes were cell receptor or immunity-associated genes, including 2 genes related to the International Federation of Gynecology and Obstetrics (FIGO) stage, 4 genes to histological grade, 18 genes to lymph node metastasis, 11 genes to residual disease, and 13 genes to the reactivity to chemotherapy. Several functionally important genes including fibronectin 1, pericentriolar material 1, beta-2-microglobulin, PPAR binding protein were identified through review of the literature. CONCLUSIONS: The cDNA microarray of ovarian cancer genes developed in this study was effective and high throughput in screening the ovarian cancer-associated genes differentially expressed. Through the studies of the cell receptor and immunity-associated genes we expect to identify the molecular biology index of ovarian cancer-associated antigens.

[Specific immune cell therapy against ovarian cancer in vivo and in vitro].

BACKGROUND & OBJECTIVE: 6B11minibody (6B11mini), an anti-idiotypic vaccine against human ovarian cancer, has been proven to induce specific humoral and cellular immunity against ovarian cancer in vivo and in vitro. This study was to investigate the safety and efficacy of using 6B11mini as an antigen to treat ovarian cancer. METHODS: After being loaded with purified 6B11mini, dendritic cells (DCs) were co-cultured with peripheral blood mononucleocytes (PBMNC) and stimulated by various cytokines, including CD3 monoclonal antibody,interleukin-2, interferon-gamma, to obtain 6B11mini-ovarian-cytokine-induced-killer cells (6B11-O-CIK). Tumor-forming ability was determined using soft agar colony-forming assay in vitro and nude mice xenografts in vivo. The acute toxicity of 6B11-OCIK at different doses was observed in BALB/c mice. Cytotoxicity of 6B11-OCIK to different target cells was detected using 51Cr release test in vitro. The ovarian tumor model was established using severe combined immune deficiency (SCID) mice transplanted with human ovarian cancer cell line SKOV3. The tumor growth was detected after injection of 6B11-OCIK into SCID mice. Injection of CIK, PBMNC and physiological saline were used as controls. RESULTS: After a cultural period of 14 days in soft agar, SKOV3 cell clones were well formed with a ratio of 50%; while 6B11-OCIK, CIK and PBMNC did not form any clones. All nude mice injected with human cervical carcinoma cell line HeLa (positive control) grew tumors after 14 days, and mice injected with 6B11-OCIK, CIK, PBMNC and normal human fetal lung fibroblast WI-38 cells did not form tumors after 13 weeks. BALB/c mice did not show any abnormal response half an hour after the administration of 6B11-OCIK cells at different doses. Mice were sacrificed 13 days after treatments, but no distinct abnormality of the main organs were found. 6B11-OCIK exerted specific cytotoxicity against tumor cells with positive OC166-9, which was related to the limitation of MHC. The tumor weights of SCID mice transplanted with SKOV3 cells were significantly lighter in 6B11-OCIK treatment group than in the saline group(P=0.023); while the tumor weights were not significantly different between the 6B11-OCIK group with CIK and the PBMNC group(P=0.540; P=0.285). CONCLUSIONS: The application of 6B11-OCIK in vivo has reached the safety standard. 6B11-OCIK has the inhibitory effect on the growth of ovarian cancer cells.

[Identification of T cell epitopes from ovarian cancer associated anti-idiotype antibody].

OBJECTIVE: To identify the T cell epitopes from ovarian cancer associated anti-idiotypic antibody 6B11 in order to explore the molecular basis of 6B11 induced cellular immune responses against ovarian cancer. METHODS: Potential human leukocyte antigen (HLA) A0201 ligands were predicted by using SYFPEITHI algorithm and tested by the T2 binding assay for screening of HLA-A2 binding peptides from 6B11 complimentary determining region (CDR). Cytotoxic T lymphocytes (CTL) to 6B11 or peptides were generated by 3 rounds of in vitro stimulation with 6B11 or peptide-pulsed dendritic cells (DC), and then tested by (51)Cr-release assay to ascertain the CTL epitope of 6B11. Cell proliferation assay was performed by using 6B11 specific CTL as responder cells and peptide-pulsed DC as stimulators to ascertain the helper T lymphocyte (Th) epitope of 6B11. Cytokine assay and interferon-gamma ELISPOT assay were performed to verify the CTL and Th epitope of 6B11 further. RESULTS: Light chain CDR3 peptide (VL CDR3) of 6B11 induced specific CTL to kill HLA-A2 and target antigen positive ovarian cancer cells, which could be blocked by anti human major histocompatibility complex (MHC) I antibody. Heavy chain CDR3 peptide (VH CDR3) of 6B11 stimulated the proliferation of 6B11-primed CTL, which could be blocked mainly by anti human MHC-II antibody, and further experiments showed that part of the VH CDR3 peptide-primed CTL killed K562 cells. Peptides in VL CDR3 and VH CDR3 were the CTL and Th epitope mimicking the original antigen of 6B11 respectively. Collaboration of 6B11 CTL and Th epitope, or 6B11 CTL epitope and keyhole limpet hemocyanin (KLH), the former was more powerful in inducing specific cellular immune responses against ovarian cancer. 6B11 or corresponding CTL and Th epitope specific CTL secreted high levels of interleukin-2 (1630, 1503 ng/L) and interferon-gamma (5620, 5421 ng/L), while basal level of interleukin-4 was detected (253, 274 ng/L). ELISPOT assay confirmed the existence of specific interferon-gamma secreting cells in 6B11 or epitopes specific CTL (196/1 x 10(6) T cell, 184/1 x 10(6) T cell). CONCLUSIONS: VL CDR3 and VH CDR3 peptides of 6B11 are the CTL and Th epitopes mimicking original antigen which could duplicate the activity of intact 6B11 to induce the cellular immune responses against ovarian cancer. The results have significant theoretical and practical value in application of anti-idiotypic antibody as anti tumor vaccine. The acquired CTL and Th epitopes as constituents of future multiple peptides against ovarian cancer could be used in peptide vaccine based ovarian cancer therapy.

[Preparation and characterization of monoclonal anti-anti-idiotypic antibody (3D12) of epithelial ovarian carcinoma].

OBJECTIVE: To generate and characterize the anti-anti-idiotypic monoclonal antibody (MAb) of epithelial ovarian carcinoma (EOC)ìwith the aim of further investigating the mechanism of active immunotherapy of tumor vaccine. METHODS: Using anti-idiotypic MAb (6B11)of EOC as immunogen, the anti-anti-idiotypic MAb 3D12 was generated by hybridoma technology. To characterize the 3D12, ELISA, immunohistochemistry, flow cytometry and western blot were used to define its specific reactivity with both 6B11 and the original EOC antigen OC166-9. RESULTS: One hybridoma cell secreting 3D12 was obtained. The 3D12 showed the ability of specific binding to 6B11 and OC166-9, and of inhibiting the binding of COC166-9 (the MAb against OC166-9) to OC166-9. A positive staining of serous papillary EOC and the specific binding of SKOV3 cell line with 3D12 supernatant were also demonstrated. Western blot showed 3D12 and COC166-9 could immunoprecipitate the same protein. The 3D12 was IgM, with the affinity Kaff approximately 8.34x10(7) L/mol. CONCLUSION: The anti-anti-idiotypic MAb 3D12 is confirmed as Ab1-like Ab3, which indirectly proves that 6B11 bears the internal image of OC166-9.

[The values of serum human epididymis secretory protein 4 and CA(125) assay in the diagnosis of ovarian malignancy].

OBJECTIVE: To evaluate the value of human epididymis secretory protein 4 (HE4) and CA(125) in the diagnosis of ovarian malignancy. METHODS: HE4 and CA(125) in the serum specimens of malignant ovarian tumor group (30 cases), benign ovarian diseases (110 cases; 45 benign ovarian tumor, 57 endometriotic diseases and 8 pelvic inflammation were included) and healthy women group (137 cases) were assayed double blindly. The levels and the diagnosis efficiency of the HE4 and CA(125) were analyzed. RESULTS: (1) The median levels of HE4 and CA(125) were significantly higher in malignant ovarian tumor group (244 pmol/L and 601 kU/L respectively) than those of the benign ovarian diseases group (32 pmol/L and 22 kU/L respectively) and healthy women group (32 pmol/L and 11 kU/L respectively) (P = 0.000 - 0.029). The median levels of CA(125) were also higher in endometriotic diseases and pelvic inflammation groups (53 and 41 kU/L respectively) than those of benign ovarian tumor group and healthy women group (12 and 11 kU/L respectively; P = 0.000 - 0.031). (2) The positive rate of HE4 was lower than that of CA(125) in malignant ovarian tumor group (P = 0.036). HE4 was negative in benign diseases and healthy women groups. But the positive rates of CA(125) were 56.1% and 5/8 respectively in endometriotic diseases and pelvic inflammation groups and there were significant differences compared with HE4 (P = 0.000). (3) The HE4 assay had advantage over the CA(125) assay in receiver operating characteristic-area under the curve (ROC-AUC) and sensitivity with a specificity of 100% when ovarian malignancy was compared with controls having benign diseases and healthy women, benign tumor or benign diseases groups respectively. The CA(125) assay had advantage over the HE4 assay in ROC-AUC and sensitivity with the same specificity when ovarian cancers were compared with controls having healthy women group. (4) Combined assay of HE4 and CA(125) was better than CA(125) alone when ovarian malignancy was compared with controls having any group. (5) Combined assay was better than HE4 alone in ROC-AUC and sensitivity with the same specificity when ovarian cancers were compared with controls having benign diseases and healthy women or healthy women groups. And combined assay was lower in the ROC-AUC and the sensitivity with specificity of 100% than HE4 when ovarian cancers were compared with controls having benign tumors or benign diseases groups respectively. (6) The diagnosis efficiency of the HE4 assay at the level 86 pmol/L determined in ROC curve with controls having benign diseases and healthy women group and at the 95% reference level 50 pmol/L of healthy women or 150 pmol/L recommended by the kit respectively was compared. The sensitivity of 50 pmol/L was 73% higher than 150 pmol/L and 86 pmol/L, while the specificity and positive predictive value were lower (P = 0.002, P = 0.000). The specificity, accuracy and positive predictive value of HE4 assay at the set point of 150 pmol/L and 86 pmol/L were 100%, 96% and 96%. The set point of 86 pmol/L had advantage over 150 pmol/L at the sensitivity of diagnosis, 70% and 63% respectively. But the positive predictive value was 95% lower than 150 pmol/L, being 100%. There was no significant difference (P = 0.883, P = 0.883). CONCLUSIONS: The specificity of HE4 assay is higher than CA(125) assay in the diagnosis of ovarian cancer and HE4 combined with CA(125) assay can improve the diagnoses. The set point of 150 pmol/L is advantageous for the accurate diagnosis, while the set point of 86 pmol/L is advantageous for the screening of malignant ovarian cancer.

Humoral immune responses induced by anti-idiotypic antibody fusion protein of 6B11scFv/hGM-CSF in BALB/c mice.

BACKGROUND: We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B11, which mimics an ovarian carcinoma associated antigen OC166 - 9 and whose corresponding monoclonal antibody is COC166 - 9 (Ab1). In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment (scFv)/human granulocyte macrophage colony-stimulating factor (hGM-CSF) and 6B11scFv in BALB/c mice. METHODS: The fusion protein 6B11scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSF. BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively. RESULTS: The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F (ab) 2' and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6B11ScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11scFv/hGM-CSF compared with the 6B11scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week. CONCLUSION: The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11scFv/hGM-CSF for active immunotherapy of ovarian cancer.

[In vitro study of the antitumor immune responses induced by anti-idiotypic minibody vaccine of ovarian cancer].

OBJECTIVE: To evaluate whether anti-tumor immune response can be induced in vitro with 6B11 anti-idiotypic minibody. and to explore its probability as ovarian cancer vaccine. METHODS: Separated human peripheral blood mononuclear cells (PBMC) were stimulated and cultured by 6B11 minibody. The proliferations of PBMC and cytotoxin were observed by (3)HTdR and (51)Cr release test respectively. ELISA(Enzyme-Linked Immunosorbent Assay) test and Immune Flow Cytometry were used to analyze IFN-gamma in supernatant of the cultured cdlls and the change of T lymphocyte phenotype of PBMC with 6B11 minibody stimulated. RESULTS: 6B11 minibody could stimulate PBMC to proliferate, the best dose was 20 mg/L; it performed cytotoxin function to ovarian carcinoma cell line expressing OC166-9. IFN-gamma maintained at high level after stimulation. It stimulated proliferation of CD3(+) T cell and CD4(+) from PBMC after stimulation respectively. CD8(+) T cell proliferation was not clear. There was significant difference between stimulation and unstimulation in CD4(+)/CD8(+) ratio. CONCLUSION: 6B11 anti-idiotypic minibody can induce both humoral and cellular immunity against ovarian carcinoma in vitro. This paper has provided strong experimental evidence for clinical use of 6B11 minibody as anti-idiotype vaccines against ovarian carcinoma.

[Induction of T cell responses against autologous ovarian cancer by anti-idiotype minibody-pulsed dendritic cells].

BACKGROUND & OBJECTIVE: Immunotherapy of sensitizing dendritic cells (DCs) with antigen,protein,and frozen cancer cell has been widely used in treating various cancers. The 6B11 anti-idiotype-antibody,a fusion protein prepared by our research center,can mimic ovarian cancer-associated antigen OC166-9. This study was to induce T cell cytotoxicity against autologous tumor cells of patients with ovarian cancer by 6B11 anti-idiotype-antibody. METHODS: Peripheral blood samples were collected from 10 patients with epithelial ovarian cancer,Monocytes were isolated and cultured to obtain DCs. Immature DCs were stimulated with 6B11 anti-idiotype-antibody (MINI-DC group); unpulsed DCs (unpulsed-DC group),mouse F(ab) '2 fragments pulsed DCs [F(ab)'2-DC group],and T cells alone (T group) were served as controls. Mature DCs were harvested. (3)H-thymidine ((3)H-TdR) incorporation approach was used to measure effect of DCs on stimulating auto-T cell proliferation. Cytotoxicity of DC-activated T cells against auto-tumor cells was measured with (51)Cr 6-h release test,tumor cell lines,SKOV3,HLE,and K562, were used as controls. RESULTS: In 4 cases,cpm value of (3)H-TdR incorporation,as symbol of auto-T cell proliferation, in MINI-DC group was significantly higher than those in control groups. In 5 cases,specific cytotoxicity effect of T cells on auto-tumor cells was observed in MINI-DC group at effect-target ratio of 20:1,the toxicity effect of T cells in MINI-DC group was 25%-100%,significantly higher than those in F(ab)'2-DC group (18%-40%), unpulsed-DC group (13%-43%),and T group (9%-58%). In 4 cases,the toxicity effect of T cells in MINI-DC group, at effect-target ratio of 20:1,on auto-tumor cells was 25%-100%, higher than those on SKOV3 cells (5%-51%),HLE cells (2%-38%),and K562 cells (2%-25%). Moreover,the toxicity effect of T cells in MINI-DC group on auto-tumor cells can be partially blocked by anti-MHC-I antibody,which indicated that the toxicity was antigen-specific. CONCLUSION: DCs loaded with 6B11 anti-idiotype antibody that mimic ovarian cancer antigen can induce antigen specific T cell cytotoxicity against auto-ovarian tumor cells in vitro.

[Human pituitary tumor transforming gene 1 expression in ovarian carcinoma and its clinical significance].

BACKGROUND & OBJECTIVE: Human pituitary tumor transforming gene 1 (hPTTG1) is a newly identified oncogene. We performed a large-scale screening survey to identify related genes expressed in advanced and poorly differentiated serous ovarian carcinoma using cDNA microarray analysis, and found that hPTTG1 is one of highly up-regulated genes in ovarian carcinoma. This study was to examine hPTTG1 mRNA and protein expression in various epithelial ovarian carcinoma,analyze the relationship of its expression level with FIGO stage and histological grade, and explore the functional role of hPTTG1 in ovarian carcinoma pathogenesis. METHODS: HPTTG1 mRNA expression in 27 cases of epithelial human ovarian carcinoma, and 4 cases of normal ovary was assessed with non-competitive reverse transcription polymerase chain reaction (RT-PCR), and hPTTG1 protein expression in these 27 cases of human ovarian carcinoma, and 18 cases of normal ovary was examined by immunohistochemistry. RESULTS: HPTTG1 mRNA expression level in normal ovary was low, while in ovarian carcinoma was significantly higher than that in normal counterparts (P < 0.01). Fold increase of ovarian cancer tissues to normal ovary tissues is 1.1-4.8 (median 2.4).HPTTG1 mRNA high expression level was related to poor tumor differentiation (r = 0.686, P < 0.05), but the correlation with FIGO stage was not detected. HPTTG1 protein was expressed in all cases of ovarian carcinoma, but not in normal ovaries. CONCLUSIONS: Increased hPTTG1 expression may be an important factor involved in early tumorogenesis, and may be associated with poor tumor differentiation. HPTTG1 could be a potential molecular marker of epithelial ovarian carcinoma.

[In vivo study of antitumor immune responses induced by anti-idiotypic minibody vaccine of ovarian cancer].

BACKGROUND & OBJECTIVE: 6B11 anti-idiotypic minibody can be constructed by fusion of anti-idiotypic single chain antibody (6B11scFv) to human IgG(1) hinge and CH3, which can simulate the function of ovarian carcinoma antigen. It has immune activity of both 6B11 and human immunoglobulin IgG(1) Fc. This study was designed to evaluate whether anti-tumor immune response can be induced in BALB/c mice immunized with 6B11anti-idiotypic minibody and explore its probability as ovarian cancer vaccine. METHODS: BALB/c mice were immunized repeatedly by 6B11 anti-idiotypic minibody. Competition inhibition test, indirect ELISA test, and immune flow cytometry were used for analyzing the serum characterization of anti-anti-idiotypic antibody (Ab3) and the changes of T lymphocyte phenotype of spleen. RESULTS: The specific antitumor immune response could be induced in BALB/c mice after immunized with anti-idiotypic minibody without carrier proteins and adjuvant. The Ab3 maintained at a high level till 30 day after the last immunization. It stimulated proliferation of CD4+ T cells and CD8+ T cells from the spleen of BALB/c mice on 4th day, 14th day, 24th day, and 30th day after the last immunity respectively. CONCLUSIONS: 6B11 anti-idiotypic minibody can induce both humoral and cellular immunity against ovarian carcinoma in vivo, which provided experimental evidence for clinical use of fusion protein 6B11 minibody as anti-idiotype vaccines against ovarian carcinoma.

[Establishment and comparison of two intraperitoneally transplanted human ovarian carcinoma models with immune reconstitution in severe combined immunodeficient mice].

BACKGROUND & OBJECTIVE: Ovarian carcinoma is leading cause of death in gynecologic malignancies. The survival rate cannot be improved after routine surgery, chemotherapy, and radiotherapy. Therefore biotherapy becomes the fourth treatment pattern for ovarian carcinoma. Adequate experimental models for the development of biologic therapeutic strategies are needed. Our purpose was to establish and compare two intraperitoneally transplanted human ovarian carcinoma models with human immune reconstitution in severe combined immunodeficient (SCID) mice. METHODS: Six and ten C.B17/SCID mice were intraperitoneally injected with human ovarian adenocarcinoma SKOV3 and SKOV3.ip1 cells, respectively. Their biological, histological, and immunological features were compared. Ascites of the mice in SKOV3.ip1 group was injected into other six mice. All the 22 SCID mice were intraperitoneally injected with human peripheral blood lymphocytes (PBL) to establish immune reconstituted model. RESULTS: The taken rates of the SKOV3 and SKOV3.ip1 groups were both 100%. The latent periods of tumor growth were 20-41 days and 22-30 days, respectively (P >0.05). While the mean survival time were 50-78 days and 32-43 days, respectively (P< 0.0001). 83.3% (5/6) of the mice injected with ascites of the mice in SKOV3.ip1 group successfully formed new tumor mass and ascites. Autopsy showed the tumors of the two models were widespread in pelvic cavity. The SKOV3.ip1 group also had 0.35-5.60 ml bloody ascites that was similar to the clinical behavior of most patients, while only 0.2 ml in one mice of SKOV3 group. Histological results showed the tumors of the two groups remained the characteristics of serous papillary adenocarcinoma of human ovary, and immunohistochemistry staining showed the ovarian associated antigen OC166-9 were both positive. Human IgG were detected in 72.7% (16/22) of the mice, and human CD4(+) and CD8(+) T cells were positive in 54.5% (12/22) of the mice. CONCLUSION: The two intraperitoneally transplanted human ovarian carcinoma models had been established in human PBL reconstituted SCID mice. The SKOV3.ip1 model may be an ideal animal model for biotherapy research of ovarian carcinoma as it simulates the intraperitoneally disseminating behavior of human ovarian carcinoma in the patients with immune function, and it took relatively shorter time to be established.

[Establishment and biological characteristics of an immortalized human ovarian sarcomatoid carcinoma cell line BUPH:OVSC-2].

BACKGROUND & OBJECTIVE: Cell immortalization technique has been an important method to establish cell lines with expected phenotype. However, there was few report about the establishment of immortalized human ovarian carcinoma cell lines. The purpose of this study was to establish an immortalized human ovarian sarcomatoid carcinoma cell line, and to investigate its biological characteristics. METHODS: A specimen derived from metastatic tumor of a human ovarian sarcomatoid carcinoma in abdominal wall was obtained and cultured in vitro. The 10th passage was transfected with SV40 T gene, colonies were isolated by G418 selection and expanded to immortalized cell lines. The morphology of the cells was observed by microscope and electromicroscope, Growth curve, karyotype analysis, culturing in soft agar, nude mice transplantation, immunohistochemistry, etc. were used to investigate its biologic characteristics. The biologic characteristics of BUPH: OVSC-2 were compared with its original cells. RESULTS: After transfection and selection, an immortalized ovarian carcinoma cell line BUPH: OVSC-2 was established. The cell line has been maintained for over 90 passages. The H&E stain finding of transplanted tumor showed that the cells were morphologically sarcomatoid. However, the transmission electron microscopic observation exposed its epithelial origin. The cells grew exuberantly and had high malignant characteristics. Comparative studies revealed that the biological properties of BUPH: OVSC-2 and its original cells were identical. CONCLUSIONS: BUPH: OVSC-2 was demonstrated as an immortalized human ovarian sarcomatoid carcinoma cell line with high malignancy. It retains similar properties of their parental cells and could be a useful model for the study of human ovarian sarcomatoid carcinoma.