Polysorbate-80 coating enhances uptake of polybutylcyanoacrylate (PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells.

Certain drugs such as dalargin, loperamide or tubocurarine are not transported across the blood-brain barrier (BBB) and therefore exhibit no effects on the central nervous system. However, effects on the central nervous system can be observed when these drugs are loaded onto polybutylcyanoacrylate (PBCA)-nanoparticles and coated with polysorbate 80. The mechanism by which these complexed nanoparticles cross the BBB and exhibit their effects has not been elucidated. Cultured microvessel brain endothelial cells of human and bovine origin were used as an in vitro model for the BBB to gain further insight into the mechanism of uptake of nanoparticles. With cells from these species we were able to show that polysorbate 80-coated nanoparticles were taken up by brain endothelial cells much more rapidly and in significantly higher amounts (20-fold) than uncoated nanoparticles. The process of uptake was followed by fluorescence and confocal laser scanning microscopy. The results demonstrate that the nanoparticles are taken up by cells and that this uptake occurs via an endocytotic mechanism.

How to overcome the disturbing effects of human anti-mouse antibodies (HAMA) on in vitro assays.

The development of human anti-mouse antibodies (HAMA) is a common immune response in patients with ovarian carcinoma after repeated injections of murine anti-CA 125 monoclonal antibodies for immunoscintigraphy. As a tumor marker with significant diagnostic value CA 125 is routinely measured in the follow-up of tumor patients by immunoradiometric assays (IRMA) based on murine anti-CA 125 monoclonal antibodies. HAMA may cause false-positive findings in a CA 125-IRMA. In this report our group demonstrates a simple way of eliminating HAMA by protein A/G affinity chromatography allowing the reliable detection of the tumor marker CA 125 in the serum of patients with ovarian carcinoma.

Generation of human monoclonal anti-idiotypic antibodies with specificity to the murine monoclonal anti-CA 125 antibody B43.13.

Two human monoclonal antibodies, HID-7E7 and ROB-6F2, were produced by EBV transformation of peripheral blood lymphocytes (PBL). PBL were obtained from a patient with ovarian cancer who had been exposed several times to a Tc-99m labeled murine monoclonal anti-CA 125 antibody (B43.13, Biomira, Edmonton) for immunoscintigraphy. The HID-7E7 and ROB-6F2 producing B-cells were cloned with a limiting dilution technique and have shown stable immunoglobulin secretion within a period of three years. The human monoclonal antibodies HID-7E7 and ROB-6F2 are of the IgG isotype, and bind with significant affinity to the murine monoclonal antibody B43.13, which was used for immunoscintigraphy. Binding affinity of ROB-6F2 to other murine antibodies could not be detected. Cross reactivity of HID-7E7 to a murine anti-CEA monoclonal antibody was observed. In order to verify the anti-idiotypic character of the generated human antibodies, the ability of HID-7E7 and ROB-6F2, respectively, to inhibit the formation of the CA125/B43.13 complex is demonstrated via an enzyme-linked immunosorbent assay. These human anti-idiotypic antibodies are possible candidates for immunotherapy of ovarian cancer in patients with a small tumor burden following surgery and/or chemotherapy.

Immunological profile of patients with ovarian-cancer under immunostimulation with murine monoclonal-antibodies.

The longer survival of ovarian cancer patients after immunostimulation has been connected with the induction of an anti-tumor activity triggered by cellular and humoral immune responses. Our interest was to study the long-term influence on the immune system in relation to the various levels of the HAMA response and the disease stage. The immunological profile was evaluated by regularly performing phenotyping and functional analysis of peripheral blood lymphocytes (PBL). We report the statistical analysis of immunological data obtained in a study with 77 ovarian cancer patients examined over a period of up to 28 months. The results demonstrate that these immunological data are important for monitoring cancer patients under immunotherapy whereas they provide no prognostic significance.

A preliminary study on the functional analysis of peripheral blood lymphocytes from ovarian cancer patients developing HAMA after immunoscintigraphy.

In the follow-up of ovarian cancer patients, rising levels of the tumor-associated antigen CA 125 are an indication for immunoscintigraphy. Human anti-mouse antibodies (HAMA) are frequently found after immunoscintigraphy with murine MAb directed against CA 125. Since we observed that patients developing high HAMA-levels in serum remained free of tumor or had stable disease, we examined the cytotoxic activity of peripheral blood lymphocytes (PBL) by a fluorescence-based assay. Our results demonstrate that PBLs of patients with high anti-idiotypic antibodies show an increased cytotoxic activity (by a factor of 4) compared to those of patients with low HAMA levels. The clinical course of the patients after the first injection of murine monoclonal antibody was observed over a period of 1 to 3 years. Improvement or deterioration of patients' clinical condition corresponded with the results obtained by functional analysis. Further investigations concerning the course of cytotoxic activity in HAMA-positive patients will have to clarify HAMA's role in the immune response.

Interactions between tumor cells and tumor infiltrating lymphocytes in human ovarian carcinoma.

The aim of our study was to investigate the lymphocytic infiltration rate of ovarian tumors and the possible use of tumor-infiltrating lymphocytes (TIL) as a therapeutic tool in gynecologic oncology. Twelve tumors were treated with digesting enzymes in order to isolate tumor-infiltrating lymphocytes as well as tumor cells, TIL were expanded by culture in the presence of human interleukin-2 (IL-2). Freshly prepared tumor cells were allowed to grow in culture medium for several days before the first investigations were performed. TIL could only be isolated from 50% of the investigated tumors. In contrast to this the isolated tumor cells could be largely expanded in 73% of the cases. The expression of the CA125 antigen in the culture supernatants served as control and could still be evaluated up to three months after isolation. In parallel the antigen expression on the cellular surface was estimated by immunocytochemistry. Evaluating the phenotypes of TIL showed predominantly CD3+, their expansion rate was only poor. Tumor cells were isolated and expanded in order to test the tumor-directed cytotoxic efficacy of TIL and for further use in transplantation to nude mice.

Relevance of immunological parameters in progression of colorectal-carcinoma.

The long-term influence on the immunological stage from surgery and/or adjuvant or palliative therapy of 23 patients with metastatic colorectal carcinoma was investigated by performing regular phenotyping and functional analysis of peripheral blood lymphocytes (PBL). The following groups were chosen: A (n=6); patients after resection of primary tumor and liver-metastases without chemotherapy. B (n=3); patients with catheter implantation after resection of primary tumor and liver metastases receiving an adjuvant arterial chemotherapy with 5-fluorouracil (5-FU) and folinic acid (5-formyltetrahydrofolic acid, FA). C (n=7); patients with non-resectable liver-metastases, receiving arterial or systemic chemotherapy after catheter implantation. D (n=7); patients with extrahepatic filiae receiving systemic palliative chemotherapy. Lymphocytes of 10 healthy volunteers served as controls. Furthermore, we were able to show effects of 5-FU and FA on the immune system.

Functional analysis of peripheral blood lymphocytes from healthy volunteers.

Functional analysis of peripheral blood lymphocytes (PBL) from 46 healthy donors is described. For quantitation of cytotoxic activity a fluorescence-based assay is performed with commercially available human tumor cell lines as targets. For evaluation of natural-killer cell activity, the cell line K562 (NK-cell sensitive) and for T-cell activity Raji cells (NK-cell resistant) are used as targets. Since we are not using autologous cells as targets we are measuring the function of non MHC-restricted cytotoxic T-lymphocytes. The results show a median NK-cell activity of 77.4% (given in % lysis) and a median T-cell activity of 21.4% in healthy individuals at an effector-/target-cell ratio (E/T ratio) of 40:1. For correlating immunological parameters such as functionality of PBL from tumor patients with the progression of tumor growth, normal values are of great importance.