N. Ebel

Cells under pressure - treatment of eukaryotic cells with high hydrostatic pressure, from physiologic aspects to pressure induced cell death.

The research on high hydrostatic pressure in medicine and life sciences is multifaceted. According to the used pressure head the research has to be divided into two different parts. To study physiological aspects of pressure on eukaryotic cells physiological pressure (pHHP; < 100 MPa) is used. pHHP induces morphological alterations in the cellular organelles and evokes a reversible stress response similar to the well known heat shock response. pHHP induces highly reversible alterations and normally does not affect cellular viability. The treatment of eukaryotic cells with non-physiological pressure (HHP; > or = 100 MPa) reveals different outcomes. Treatment with HHP < 150 MPa does not markedly affect viability of human cells, but induces apoptosis in murine cells. In human cells apoptosis is observed after treatment with > or = 200 MPa. Moreover, HHP treatment with > 300 MPa leads to necrosis. Therefore, HHP plays a role for the sterilisation of human transplants, of food stuff, and pharmaceuticals. Human tumour cells subjected to HHP > 300 MPa display a necrotic phenotype along with a gelificated cytoplasm, preserve their shape, and retain their immunogenicity. These observations favour the use of HHP to produce whole cell based tumour vaccines. Further experiments revealed that the increment of pressure as well as the pressure holding time influences the cell death of tumour cells. We conclude that high hydrostatic pressure offers both, an economic, easy to apply, clean, and fast technique for the generation of vaccines, and a promising tool to study physiological aspects.

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 ≥ 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 ≥ 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 ≥ 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.

Selected anti-tumor vaccines merit a place in multimodal tumor therapies.

Multimodal approaches are nowadays successfully applied in cancer therapy. Primary locally acting therapies such as radiotherapy (RT) and surgery are combined with systemic administration of chemotherapeutics. Nevertheless, the therapy of cancer is still a big challenge in medicine. The treatments often fail to induce long-lasting anti-tumor responses. Tumor recurrences and metastases result. Immunotherapies are therefore ideal adjuncts to standard tumor therapies since they aim to activate the patients immune system against malignant cells even outside the primary treatment areas (abscopal effects). Especially cancer vaccines may have the potential both to train the immune system against cancer cells and to generate an immunological memory, resulting in long-lasting anti-tumor effects. However, despite promising results in phase I and II studies, most of the concepts finally failed. There are some critical aspects in development and application of cancer vaccines that may decide on their efficiency. The time point and frequency of medication, usage of an adequate immune adjuvant, the vaccines immunogenic potential, and the tumor burden of the patient are crucial. Whole tumor cell vaccines have advantages compared to peptide-based ones since a variety of tumor antigens (TAs) are present. The master requirements of cell-based, therapeutic tumor vaccines are the complete inactivation of the tumor cells and the increase of their immunogenicity. Since the latter is highly connected with the cell death modality, the inactivation procedure of the tumor cell material may significantly influence the vaccines efficiency. We therefore also introduce high hydrostatic pressure (HHP) as an innovative inactivation technology for tumor cell-based vaccines and outline that HHP efficiently inactivates tumor cells by enhancing their immunogenicity. Finally studies are presented proving that anti-tumor immune responses can be triggered by combining RT with selected immune therapies.

Characterization of Escherichia coli suspensions using UV/Vis/NIR absorption spectroscopy.

In this paper we demonstrate that conventional absorption spectroscopy in the ultraviolet, visible and near-infrared spectral range facilitates characterization of Escherichia coli (E. coli) suspensions. Two kinds of samples have been studied: (1) Untreated E. coli suspensions with systematically varied cell concentration and (2) E. coli treated by different inactivation procedures. For the purpose of inactivation the bacteria have been treated by either heat at elevated temperature as an established method or by hydrostatic or dynamic high pressure. The results show that at cell concentrations above a certain threshold extinction measurements in the ultraviolet region can yield a quantitative measure of the cell number density with optimal sensitivity and precision. Furthermore, examining suitable spectral regions the absorption spectra reveal characteristic features hence allowing identification of the treatment procedure later on. In conclusion, this study establishes a simple and cost-efficient approach for online and in-situ monitoring of processes for the inactivation of microbiological organisms. Moreover, the method provides a tool for the investigation of the inactivation mechanisms.