Isolation and purification of vascular endothelial growth factor (VEGF) from ascitic fluid of ovarian cancer patients

Vascular Endothelial Growth Factor (VEGF) or Vascular Permeability Factor (VPF) is an angiogenic cytokine expressed by many human and animal tumors. Because of the importance of VEGF in animal tumors, we purified VEGF/VPF from ascitic fluid of ovarian cancer patients with heparin sepharose column. The purified protein gave protein bands of 37 and 26 kD, respectively in 12% SDS PAGE. The specificity of the purified protein was determined with dot blot, trans-immunoblot and ELISA using polyclonal goat anti-VEGF antibody (Santa Cruz Biotechnology). The vasodilatatory effect of the purified protein was confirmed by a vascular permeability assay on mouse. A polyclonal mouse antibody was raised against the purified protein, which recognized the same protein by ELISA, transimmunoblot and dot-blot analysis. It has been also found that the raised polyclonal antibody in mouse- and the commercial VEGF polyclonal antibody (Santa Cruz Biotechnology) both inhibited in vitrocell proliferation of human MCF-7 cell line. This study shows for the first time an effort to purify VEGF from human source.(Pathology Oncology Research Vol 10, No 2, 104–108)     

Murine erythropoietin in ascitic fluid

Administration of 0.2 ml of a 1.25% solution of phenylhydrazine (PHZ) and 500 rads of X-irradiation to BALB/c mice produced a prompt and severe reduction to hematocrit with a resultant increase in plasma levels of erythropoietin (Ep). By also giving mice treated with PHZ and irradiation an ascites from of a transplantable sarcoma, the amount of recoverable material rich in Ep could be greatly increased because ascitic fluid contained levels of Ep comparable to plasma.     

Angiogenic effects of macrophages isolated from ascitic fluid aspirated from women with advanced ovarian cancer.

The cellular components of ascitic fluid aspirated from the peritoneal cavity of women with advanced ovarian cancer were separated on a Ficoll gradient. Isolated macrophages, which were further purified, elaborated a growth factor which was mitogenic for human endothelial cells isolated from umbilical veins, arteries and the omental microvasculature in vitro, and was angiogenic in vivo. It is postulated that the macrophage-derived factor enhances tumor neovascularization of the widespread ovarian-derived peritoneal malignant lesions appearing in these patients, thus contributing to their rapid growth and metastasis, and the poor prognosis of the disease.     

Immunosuppressive activity of ascitic fluid from patients with cancer metastatic to the peritoneum.

We have examined the immunosuppressive effects of ascitic fluids from patients with advanced cancer metastatic to the peritoneum and compared them with noncancerous abdominal or pleural effusions, serum from cancer patients, or the proteins human serum albumin and bovine gamma-globulin. The ascitic fluids from cancer patients produce a nontoxic, dose-dependent suppression of DNA and protein synthesis of phytohemagglutinin-stimulated human peripheral blood lymphocytes. This suppression reached 100% with an ascitic protein concentration of 4 to 6 mg/ml, whereas control effusions, serum from cancer patients, or added extraneous proteins were not suppressive. Ascitic proteins also suppress the mixed lymphocyte reaction and the response of human peripheral blood lymphocytes to the antigen keyhold limpet hemocyanin, in vivo, the primary plaque-forming response to sheep red blood cells in mice could be suppressed, and this suppression was not due to antigenic competition.     

Biological characterization of a tumour-growth-promoting factor from ascitic fluid of tumour-bearing mice

Tumour-growth-promoting activity has been observed in ascitic fluids of mice with progressively growing SV40- and chemically induced tumours. Administration of ascitic fluid to animals together with a normally non-tumorigenic dose of SV40-transformed tissue culture cells (TU-5) will cause these cells to grow progressively and eventually kill the animals. Also, the mKSA-specific, immunologically non-responsive state (the eclipse phase) of mice bearing large mKSA tumours can be maintained by administration of ascitic fluid or serum from tumour-bearing animals after the tumours have been surgically removed. Normally, complete immunity to a secondary tumour cell challenge is re-established approximately 10 days after removal of the primary tumour, but daily injections of ascitic fluid or serum will maintain eclipse so that a secondary challenge of tumour cells will grow progressively. The active component apparently acts non-specifically, since ascitic fluid from an unrelated tumour also is able to maintain the mKSA-specific eclipse phase. The ascitic fluid has no apparent immunosuppressive activity in vivo.     

Case report. Isolation of Cladosporium cladosporioides from cerebrospinal fluid

Cladosporium cladosporioides was isolated from three subsequent cerebrospinal fluid specimens and from a brain biopsy specimen of a human patient. Susceptibility testing of the isolate was performed against seven antifungal agents.     

Pepstatin, an inhibitor of leukokinin formation and ascitic fluid accumulation.

Ascites fluid accumulation accompanying a mastocytoma or L1210 murine tumor is significantly retarded following the i.p. or s.c. injection of moderate quantities of pepstatin, a known acid protease inhibitor. No effect on cell count was noted by pepstatin treatment. The probable mechanism by which pepstatin acts is by inhigiting the enzymatic formation of chemical mediators known as leukokinins. These are pharmoacologically active peptiedes having potent permeability characteristics previously described by this laboratory. Leukokinins are formed by cathepsin D-like enzymes present in the invading cells and in the ascites fluid acting on a protein substrate, leukokininogen. present in the ascites fluid. Pestatin inhibits the action of these leukokinin-forming enzymes invitro but has no effect on kallikreins (bradykinin-forming enzymes) in vitro. Human ascites fluid from a patient with ovarian carcioma was found to have a paepstatin-inhibited, leukokinin-generating system, as does the mouse. A 'chemical mediator' theory is proposed for ascites fromation which broadens the previously held theory of lymphatic blockage (Holm-Nielsen) and may explain the recent findings of Hirabayashi and Graham of increased plasma-ascites exchange in peritoneal carcionmatosis. Pepstatin inhibition of chemical mediator formation may represent a new therapeutic approach to ascites fluid accumulation in neoplastic disease.     

Coagulative and fibrinolytic properties of ascitic fluid associated with ovarian tumors.

Ascitic fluid samples from 19 patients with ovarian carcinoma, 3 with a benign ovarian tumor, and 5 with cirrhosis of the liver were examined for their content of coagulation factors and components of the fibrinolytic system. The concentration of trypsin inhibitors in the ascitic fluid was significantly higher in the presence of carcinoma. Large amounts of FDP were found in the ascitic fluid in all patients with malignant tumors, but not in the other two groups. Determination of FDP may therefore make it possible to differentiate between malignant and nonmalignant ascitic fluid.     

Isolation and characterization of 5T4, a tumour-associated antigen

The monoclonal antibody (MAb) 5T4 defines a human trophoblast antigen marker with a restricted pattern of expression in normal adult tissues but this antigen is expressed on a variety of carcinomas. The purification of 5T4 antigenic molecules is described from term syncytiotrophoblast by a combination of lectin- and immunoaffinity chromatography and gel filtration giving up to 10,000-fold purification with 70% yield. The antigen is carried by non-associated glycoprotein molecules with an apparent molecular weight of 72 kDa on SDS-PAGE and a neutral pl. Removal of N-linked sugars by N-glycanase reveals a core protein of 42 kDa. Treatment with enzymes that cleave O-linked sugars does not substantially alter the molecular size. The native 5T4 molecules are very resistant to proteolysis until the N-linked sugars are removed or the glycoprotein is denatured and reduced. Glycopeptides generated by these approaches will be suitable for amino acid sequencing.     

Isolation and characterization of an immunosuppressive acidic protein from ascitic fluids of cancer patients

Analytical isoelectric focusing in polyacrylamide gel at pH 2.5 to pH 5 was used successfully to detect an acidic protein which can be found in large quantities in the sera of cancer patients but in only small amounts in the sera of healthy persons. The main peak of this acidic protein when obtained from cancer patients revealed a pI of 3.0, while the pI of that obtained from normal subjects was 3.1. The results obtained also demonstrated that qualitative and quantitative differences of such serum acidic proteins discriminate between sera of normal persons and cancer patients. Purification of this acidic protein (pI 3.0) was attempted with cancer ascitic fluids by successive ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative isoelectric focusing. When the purified acidic protein was characterized for physicochemical properties, it was found to have an isoelectric point of 3.0, to have a molecular weight of 50,000, and to contain 31.5% carbohydrate. Its behavior in immunodiffusion and immunoelectrophoresis was indistinguishable from that of normal human alpha 1-acid glycoprotein. However, in gel isoelectric focusing, in molecular weight, and also in carbohydrate content, the acidic protein isolated from cancer ascitic fluid differed from normal alpha 1-acid glycoprotein. Furthermore, this acidic protein was found to suppress both phytohemagglutinin-induced lymphocyte blast formation and mixed-lymphocytes reaction in vitro. The acidic protein, which we called "immunosuppressive acidic protein," and its biological activities were discussed regarding its possible role in the impairment of the immunological surveillance mechanism in the tumor-bearing host.     

The phenotype of ascitic fluid lymphocytes in patients with ovarian carcinoma and other primaries.

BACKGROUND: Ascitic tumor-infiltrating lymphocytes (TIL) are a potential source of effectors for adoptive immunotherapy. PATIENTS AND METHODS: The TIL phenotype was examined by two-color flow cytometry in malignancy-related ascites of 49 patients with different primaries. Interleukin-10 (IL-10) and neopterin were determined in ascitic fluid by enzyme-linked immunoassay. RESULTS: Malignant melanoma patients had significantly higher CD3(+), CD3(+)CD8(+) and CD3(+)CD95(+), and lower CD3(+)CD4(+) lymphocyte numbers than patients with other primaries. Ovarian cancer patients had higher CD3(+)CD45RO(+), CD8(+)CD28(+), CD19(+)CD86(+), CD19(+) and CD19(+)CD86(+) lymphocyte numbers, and lower NK cell numbers than patients with gastrointestinal and pancreatic primaries. Pretreated patients had significantly lower concentrations of IL-10, lower CD8(+)CD28(+), CD3(+)CD45RA(+), and higher CD3(+)CD80(+) numbers than chemotherapy-na?ve patients. Patients with hepatic metastases had lower CD3(+), CD3(+)CD4(+) and CD3(+)CD45RO(+), and higher CD3(+)CD25(+) and NK cell numbers than patients without liver metastases. A substantial number of cells exhibited dendritic cell phenotype. Significant correlations were observed between neopterin and IL-10 concentrations, and numbers of CD8(+)CD28(+) and CD3(+)CD80(+) lymphocytes. CONCLUSIONS: Some parameters of TIL phenotype differ depending on primary, previous treatment, or the presence of liver metastases. A negative correlation was observed between IL-10 and neopterin, and an opposing effect of local concentrations of IL-10 and neopterin on the numbers of CD8(+)CD28(+) and CD3(+)CD80(+) was noted.     

Monoclonal antibody FW6 generated against a mucin-carbohydrate of human amniotic fluid recognises a colonic tumour-associated epitope.

Mucus glycoproteins of human amniotic fluid were used to generate a monoclonal antibody (MAb) FW6, which stained the intestine of a 24 week stage fetus. In adults, 76% of colonic adenocarcinomas (13/17) showed strong expression of the FW6 epitope, but it was not detected in the histologically normal mucosae adjacent to the tumours or in normal left colon mucosa. In addition, MAb FW6 stained large cell carcinomas of the lung (2/3), gastric carcinomas (5/11), and ovary adenocarcinomas (3/4). The expression in carcinomas can also be called ectopic for testing normal tissues. MAb FW6 was also reactive with pyloric mucus glands, Brunner's glands of the duodenum, Paneth cells of the ileum, pancreatic ducts, absorptive cells of the right colon, bronchiolar glands, kidney urothelia, and with a restricted number of normal mucinous tubuli of salivary gland. It was demonstrated to be under the control of the secretion gene only in intestinal Paneth cells and absorptive cells of the right colon. Comparative histochemical analysis comprising a panel of MAbs suggests that the corresponding epitope of the MAb FW6 is a type II chain related carbohydrate structure belonging to the Lex/Ley-antigen family, but is different from short chain Lex and Ley.     

Isolation of a bone-resorptive factor from human cancer ascites fluid

A protein fraction that induces the resorption of bone explants in organ culture was isolated from the ascitic fluid of patients with advanced cancer metastatic to the peritoneal cavity. Partial purification was achieved by means of gel filtration, affinity chromatography, and ion-exchange chromatography. The isolated fraction, the components of which have an apparent molecular weight of 60,000, was found to be heterogeneous by disc gel electrophoresis and to be composed primarily of proteins with relatively acidic electrophoretic properties. The specific bone-resorptive activity of this protein fraction was greatly increased over that of the unfractionated starting material, and the activity could be completely destroyed upon incubation with pronase and on heating. As determined by immunoassay and extraction procedures with various solvents, the bone-resorptive action of the isolated fraction was not attributable to the presence of parathyroid hormone, prostaglandin E2 or vitamin D-like sterols. In parallel experiments the supernatants of phytohemagglutinin-stimulated normal human peripheral leukocytes were subjected to identical chromatographic techniques, and a proten fraction with a molecular weight of 60,000, which resembled the resorptive fraction isolated from cancer ascites fluid and which contained significant bone-resorptive activity, was also partially purified.     

Microheterogeneity of transthyretin in serum and ascitic fluid of ovarian cancer patients

Background  

Transthyretin (TTR), a traditional biomarker for nutritional and inflammatory status exists in different molecular variants of yet unknown importance. A truncated form of TTR has recently been described to be part of a set of biomarkers for the diagnosis of ovarian cancer. The main aim of the study was therefore to characterize differences in microheterogeneity between ascitic fluid and plasma of women affected with ovarian cancer and to evaluate the tumor site as the possible source of TTR.