Eberhard Schlücker

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.

Ex vivo– and in vivo–induced dead tumor cells as modulators of antitumor responses

Joint application of standard tumor therapies like radiotherapy and/or chemotherapy with immune therapy has long been considered not to fit. However, it has become accepted that immune responses may contribute to the elimination of cancer cells. We present how in vivo–induced tumor cell death by irradiation, chemotherapeutic agents, or hyperthermia can be rendered more immunogenic. High hydrostatic pressure is introduced as an innovative inactivation method for tumor cells used as vaccines. Annexin A5, being a natural occurring ligand for phosphatidylserine that is exposed by dying tumor cells, renders apoptotic tumor cells immunogenic and induces tumor regression. Combinations of irradiation with hyperthermia may also foster antitumor responses. For preparation of autologous tumor cell vaccines, high hydrostatic pressure is suitable to induce immunogenic cancer cell death. Future work will be aimed toward evaluating which combination and chronological sequence of radiotherapy, chemotherapy, hyperthermia, annexin A5, and/or autologous tumor cell vaccines will induce specific and long‐lasting antitumor immunity.

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.

Ammonothermal Synthesis of Novel Nitrides: Case Study on CaGaSiN3

The first gallium-containing nitridosilicate CaGaSiN3 was synthesized in newly developed high-pressure autoclaves using supercritical ammonia as solvent and nitriding agent. The reaction was conducted in an ammonobasic environment starting from intermetallic CaGaSi with NaN3 as a mineralizer. At 770 K, intermediate compounds were obtained, which were subsequently converted to the crystalline nitride at temperatures up to 1070 K (70-150 MPa). The impact of other mineralizers (e.g., LiN3 , KN3 , and CsN3 ) on the product formation was investigated as well. The crystal structure of CaGaSiN3 was analyzed by powder X-ray diffraction and refined by the Rietveld method. The structural results were further corroborated by transmission electron microscopy, 29 Si MAS-NMR, and first-principle DFT calculations. CaGaSiN3 crystallizes in the orthorhombic space group Cmc21 (no. 36) with lattice parameters a=9.8855(11), b=5.6595(1), c=5.0810(1) Å, (Z=4, Rwp =0.0326), and is isostructural with CaAlSiN3 (CASN). Eu2+ doped samples exhibit red luminescence with an emission maximum of 620 nm and FWHM of 90 nm. Thus, CaGaSiN3 :Eu2+ also represents an interesting candidate as a red-emitting material in phosphor-converted light-emitting diodes (pc-LEDs). In addition to the already known substitution of alkaline-earth metals in (Ca,Sr)AlSiN3 :Eu2+ , inclusion of Ga is a further and promising perspective for luminescence tuning of widely used red-emitting CASN type materials.

Influence of Vortical Flow Structures on the Glottal Jet Location in the Supraglottal Region

Within the fully coupled multiphysics phonation process, the fluid flow plays an important role for sound production. This study addresses phenomena in the flow downstream of synthetic self-oscillating vocal folds. An experimental setup consisting of devices for producing and conditioning the flow including the main test channel was applied. The supraglottal channel was designed to prevent an acoustic coupling to the vocal folds. Hence, the oscillations were aerodynamically driven. The cross-section of the supraglottal channel was systematically varied by increasing the distance between the lateral channel walls. The vocal folds consisted of silicone rubber of homogenous material distribution generating self-sustained oscillations. The airflow was visualized in the immediate supraglottal region using a laser-sheet technique and a digital high-speed camera. Furthermore, the flow was studied by measuring the static pressure distributions on both lateral supraglottal channel walls. The results clearly showed different flow characteristics depending on the supraglottal configuration. In all cases with supraglottal channel, the jet was located asymmetrical and bent in medial-lateral direction. Furthermore, the side to which the jet was deflected changed in between the consecutive cycles showing a bifurcational behavior. Previously, this phenomenon was explained by the Coanda effect. However, the present data suggest that the deflection of the jet was mainly caused by large air vortices in the supraglottal channel produced by the flow field of previous oscillations. In contrast, for the case without supraglottal channel, the air jet was found totally symmetrical stabilized by the constant pressure in the ambient region. The emitted sound signal showed additional subharmonic tonal peaks for the asymmetric flow cases, which are characteristics for diplophonia.

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.

Cavitation In Reciprocating Positive Displacement Pumps

Owing to their impressive properties reciprocating positive displacement pumps are used in many applications. Pumping fluids at high delivery pressures and metering are the areas of frequent use. The design of reciprocating pumps requires an exact knowledge of the appearing phenomena such as unacceptable pipeline pulsation and harmful cavitation. But cavitation in reciprocating positive displacement pumps is still an insufficiently understood problem. For a better understanding of the effects of incipient, partial and full cavitation in reciprocating positive displacement pumps high-speed camera measurements were done under real operating conditions using a horizontal single-acting plunger pump. Inspection windows were placed to capture all cavitation phenomena. Exemplarily the cavitation phenomena and their erosive potential are to be described on the basis of high-speed sequences for selected cavitation conditions. Also the mechanism of the incipient cavitation and the opening of the self acting valves could be clearly investigated with this experimental setup. Standards and guidelines were discussed concerning the economical operating of reciprocating positive displacement pumps.

Vibration-induced particle formation during yogurt fermentation — Industrial vibration measurements and development of an experimental setup

The aim of the study was to investigate the effects of vibrations during yogurt fermentation. Machinery such as pumps and switching valves generate vibrations that may disturb the gelation by inducing large particles. Oscillation measurements on an industrial yogurt production line showed that oscillations are transferred from pumps right up to the fermentation tanks. An experimental setup (20L) was developed to study the effect of vibrations systematically. The fermenters were decoupled with air springs to enable reference fermentations under idle conditions. A vibration exciter was used to stimulate the fermenters. Frequency sweeps (25-1005Hz, periodic time 10s) for 20min from pH5.4 induced large particles. The number of visible particles was significantly increased from 35±4 (reference) to 89±9 particles per 100g yogurt. Rheological parameters of the stirred yogurt samples were not influenced by vibrations.

Vibration-induced particle formation during yogurt fermentation—Effect of frequency and amplitude

Machinery such as pumps used for the commercial production of fermented milk products cause vibrations that can spread to the fermentation tanks. During fermentation, such vibrations can disturb the gelation of milk proteins by causing texture defects including lumpiness and syneresis. To study the effect of vibrations on yogurt structure systematically, an experimental setup was developed consisting of a vibration exciter to generate defined vibrational states and accelerometers for monitoring. During the fermentation of skim milk, vibrations (frequency sweep: 25 to 1,005 Hz) were introduced at different pH (5.7 to 5.1, step width 0.1 units) for 200 s. Physical properties of set gels (syneresis, firmness) and resultant stirred yogurts (visible particles, rheology, laser diffraction) were analyzed. Vibrational treatments at pH 5.5 to 5.2 increased syneresis, gel firmness, and the number of large particles (d > 0.9 mm); hence, this period was considered critical. The particle number increased from 34 ± 5 to 242 ± 16 particles per 100 g of yogurt due to vibrations at pH 5.4. In further experiments, yogurts were excited with fixed frequencies (30, 300, and 1,000 Hz). All treatments increased syneresis, firmness, and particle formation. As the strongest effect was observed by applying 30 Hz, the amplitude was set to vibration accelerations of a = 5, 10, 15, 20, and 25 m/s2 in the final experiments. The number of large particles was increased due to each treatment and a positive correlation with the amplitude was found. We concluded that vibrations during gelation increase the collision probability of aggregating milk proteins, resulting in a compressed set gel with syneresis. Resultant stirred yogurts exhibit large particles with a compact structure leading to a reduced water-holding capacity and product viscosity.