High hydrostatic pressure treatment generates inactivated mammalian tumor cells with immunogeneic features.

Most of the classical therapies for solid tumors have limitations in achieving long-lasting anti-tumor responses. Therefore, treatment of cancer requires additional and multimodal therapeutic strategies. One option is based on the vaccination of cancer patients with autologous inactivated intact tumor cells. The master requirements of cell-based therapeutic tumor vaccines are the: (a) complete inactivation of the tumor cells; (b) preservation of their immunogenicity; and (c) need to remain in accordance with statutory provisions. Physical treatments like freeze-thawing and chemotherapeutics are currently used to inactivate tumor cells for vaccination purposes, but these techniques have methodological, therapeutic, or legal restrictions. For this reason, we have proposed the use of a high hydrostatic pressure (HHP) treatment (p >or= 100 MPa) as an alternative method for the inactivation of tumor cells. HHP is a technique that has been known for more than 100 years to successfully inactivate micro-organisms and to alter biomolecules. In the studies here, we show that the treatment of MCF7, B16-F10, and CT26 tumor cells with HHP >or= 300 MPa results in mainly necrotic tumor cell death forms displaying degraded DNA. Only CT26 cells yielded a notable amount of apoptotic cells after the application of HHP. All tumor cells treated with >or= 200 MPa lost their ability to form colonies in vitro. Furthermore, the pressure-inactivated cells retained their immunogenicity, as tested in a xenogeneic as well as syngeneic mouse models. We conclude that the complete tumor cell inactivation, the degradation of the cell's nuclei, and the retention of the immunogeneic potential of these dead tumor cells induced by HHP favor the use of this technique as a powerful and low-cost technique for the inactivation of tumor cells to be used as a vaccine.

Microbiological stability of biodiesel-diesel-mixtures.

The biodegradation of rapeseed oil methyl ester (RME) in pure and in mixtures with diesel fuel was investigated. Higher ratio of diesel fuel in the mixture resulted in higher count of bacteria. Fungal growth was advanced by higher RME contents. The growth of microorganisms gained from soil was strongest in B 20 (20 vol.% biodiesel and 80 vol.% diesel fuel) mixtures followed by B 5 (5 vol.% biodiesel and 95 vol.% diesel fuel) mixtures and pure RME. The formation of free fatty acids (FFA) in the RME sample was measured according to DIN EN 14214. The content of FFA in inoculated RME samples rose from 0.08 mass% to 0.344 mass% at the beginning. The oxidation stability of inoculated samples of B 20, B 5 and pure RME decreased faster than the oxidation stability of blank samples. An optical evaluation showed the formation of turbidity. Partly, the formation of sediment was observed, especially in B 20 and B 5 samples.

Hydrostatic pressure induced death of mammalian cells engages pathways related to apoptosis or necrosis.

We investigated the response to high hydrostatic pressure (HHP) of mammalian cells, since HHP is proposed to be suitable to inactivate mammalian cells in biopharmaceutics and patient's material. We observed that cells were not restricted in their viability by pressures up to 100 MPa. Mammalian cells die when treated with pressures of 200 MPa or more. But the effects of 200, 300 or 400 MPa do not follow the same pattem. At 200 MPa, cells die in a way that is related to apoptosis. Some apoptotic characteristics like phosphatidylserine (PS) exposure and morphological alterations appear very fast. Other features like a higher exposure of intracellular NPn ligands and pronounced degradation of DNA and lectin ligands are unique features of HHP induced apoptosis. Cells treated with 300 and 400 MPa die immediately following a unique necrotic pathway, since treated cells harbour high DNA and glycoprotein degrading activities.

High hydrostatic pressure inactivated human tumour cells preserve their immunogenicity.

High hydrostatic pressure (HHP) is an established method to inactivate biomolecules and microoganisms. It is routinely used for the sterilization of foodstuff. Recently, new applications as inactivation of microorganisms and tumour cells for bone transplants or for cancer vaccines have emerged. Characterization of the HHP-induced cellular responses are a prerequisite for its clinical use. To this end, we investigated the fate of human cells after HHP by cytofluorometry. We observed that the induction by HHP of cell death is time- and pressure-dependent. Surprisingly, an HHP-treatment of 100 MPa did not reduce viability at any time point. Pressures from 150 to 250 MPa-induced programmed cell death in most cells. However, survivors were observed in long term culture experiments under these conditions. Pressures above 300 MPa immediately induced cell death by necrosis and completely inactivated the cells. In contrast to inactivation by other necrosis inducing treatments like heat, freeze/thaw, or chemical agents, HHP avoids generation of Maillard products and disintegration and lysis of the cells. Instead HHP generates a gelatinised mixture of antigens captured in a distinct and robust particle and maintains their humoral immunogenicity. The high viscosity of the internal matrix of a pressurised cell is reflected by the slow penetration of the low molecular compound propidium iodide and limits the bleeding of antigen before uptake by antigen presenting cells. Taken together, HHP is an alternative method for the inactivation of mammalian cells in clinical settings.