Publications by authors named "Ralf Bergmann"

157 Publications

Two Be or Not Two Be: The Nuclear Autoantigen La/SS-B Is Able to Form Dimers and Oligomers in a Redox Dependent Manner.

Int J Mol Sci 2021 Mar 25;22(7). Epub 2021 Mar 25.

Department of Radioimmunology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), 01328 Dresden, Germany.

According to the literature, the autoantigen La is involved in Cap-independent translation. It was proposed that one prerequisite for this function is the formation of a protein dimer. However, structural analyses argue against La protein dimers. Noteworthy to mention, these structural analyses were performed under reducing conditions. Here we describe that La protein can undergo redox-dependent structural changes. The oxidized form of La protein can form dimers, oligomers and even polymers stabilized by disulfide bridges. The primary sequence of La protein contains three cysteine residues. Only after mutation of all three cysteine residues to alanine La protein becomes insensitive to oxidation, indicating that all three cysteines are involved in redox-dependent structural changes. Biophysical analyses of the secondary structure of La protein support the redox-dependent conformational changes. Moreover, we identified monoclonal anti-La antibodies (anti-La mAbs) that react with either the reduced or oxidized form of La protein. Differential reactivities to the reduced and oxidized form of La protein were also found in anti-La sera of autoimmune patients.
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http://dx.doi.org/10.3390/ijms22073377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036718PMC
March 2021

Development of a ghrelin receptor inverse agonist for positron emission tomography.

Oncotarget 2021 Mar 2;12(5):450-474. Epub 2021 Mar 2.

Institute of Biochemistry, Faculty of Life Sciences, Universität Leipzig, Leipzig, Germany.

Imaging of Ghrelin receptors provides unique potential to gain deeper understanding on Ghrelin and its receptors in health and disease, in particular, in cancer. Ghrelin, an octanoylated 28-mer peptide hormone activates the constitutively active growth hormone secretagogue receptor type 1a (GHS-R1a) with nanomolar activity. We developed novel compounds, derived from the potent inverse agonist K-(D-1-Nal)-FwLL-NH but structurally varied by lysine conjugation with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), palmitic acid and/or diethylene glycol (PEG2) to allow radiolabeling and improve pharmacokinetics, respectively. All compounds were tested for receptor binding, potency and efficacy , for biodistribution and -kinetics in rats and in preclinical prostate cancer models on mice. Radiolabeling with Cu-64 and Ga-68 was successfully achieved. The Cu-64- or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH radiotracer were specifically accumulated by the GHS-R1a in xenotransplanted human prostate tumor models (PC-3, DU-145) in mice. The tumors were clearly delineated by PET. The radiotracer uptake was also partially blocked by K-(D-1-Nal)-FwLL-NH in stomach and thyroid. The presence of the GHS-R1a was also confirmed by immunohistology. In the arterial rat blood plasma, only the original compounds were found. The Cu-64 or Ga-68-NODAGA-NH-K-K-(D-1-NaI)-F-w-L-L-NH radiolabeled inverse agonists turned out to be potent and safe. Due to their easy synthesis, high affinity, medium potency, metabolic stability, and the suitable pharmacokinetic profiles, they are excellent tools for imaging and quantitation of GHS-R1a expression in normal and cancer tissues by PET. These compounds can be used as novel biomarkers of the Ghrelin system in precision medicine.
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http://dx.doi.org/10.18632/oncotarget.27895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939532PMC
March 2021

Modulated Electro-Hyperthermia Facilitates NK-Cell Infiltration and Growth Arrest of Human A2058 Melanoma in a Xenograft Model.

Front Oncol 2021 25;11:590764. Epub 2021 Feb 25.

Institute of Translational Medicine, Semmelweis University, Budapest, Hungary.

Modulated electro-hyperthermia (mEHT), induced by 13.56 MHz radiofrequency, has been demonstrated both in preclinical and clinical studies to efficiently induce tumor damage and complement other treatment modalities. Here, we used a mouse xenograft model of human melanoma (A2058) to test mEHT (~42°C) both alone and combined with NK-cell immunotherapy. A single 30 min shot of mEHT resulted in significant tumor damage due to induced stress, marked by high hsp70 expression followed by significant upregulation of cleaved/activated caspase-3 and p53. When mEHT was combined with either primary human NK cells or the IL-2 independent NK-92MI cell line injected subcutaneously, the accumulation of NK cells was observed at the mEHT pretreated melanoma nodules but not at the untreated controls. mEHT induced the upregulation of the chemoattractant CXCL11 and increased the expression of the matrix metalloproteinase MMP2 which could account for the NK-cell attraction into the treated melanoma. In conclusion, mEHT monotherapy of melanoma xenograft tumors induced irreversible heat and cell stress leading to caspase dependent apoptosis to be driven by p53. mEHT could support the intratumoral attraction of distantly injected NK-cells, contributed by CXCL11 and MMP2 upregulation, resulting in an additive tumor destruction and growth inhibition. Therefore, mEHT may offer itself as a good partner for immunotherapy.
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http://dx.doi.org/10.3389/fonc.2021.590764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959784PMC
February 2021

Suppression of Metastatic Melanoma Growth in Lung by Modulated Electro-Hyperthermia Monitored by a Minimally Invasive Heat Stress Testing Approach in Mice.

Cancers (Basel) 2020 Dec 21;12(12). Epub 2020 Dec 21.

Institute of Translational Medicine, Semmelweis University, 1094 Budapest, Hungary.

Modulated electro-hyperthermia (mEHT) is a novel complementary therapy in oncology which is based on the higher conductivity and permittivity of cancerous tissues due to their enhanced glycolytic activity and ionic content compared to healthy normal tissues. We aimed to evaluate the potential of mEHT, inducing local hyperthermia, in the treatment of pulmonary metastatic melanoma. Our primary objective was the optimization of mEHT for targeted lung treatment as well as to identify the mechanism of its potential anti-tumor effect in the B16F10 mouse melanoma pulmonary metastases model while investigating the potential treatment-related side effects of mEHT on normal lung tissue. Repeated treatment of tumor-bearing lungs with mEHT induced significant anti-tumor effects as demonstrated by the lower number of tumor nodules and the downregulation of Ki67 expression in treated tumor cells. mEHT treatment provoked significant DNA double-strand breaks indicated by the increased expression of phosphorylated H2AX protein in treated tumors, although treatment-induced elevation of cleaved/activated caspase-3 expression was insignificant, suggesting the minimal role of apoptosis in this process. The mEHT-related significant increase in p21 positive tumor cells suggested that p21-mediated cell cycle arrest plays an important role in the anti-tumor effect of mEHT on melanoma metastases. Significantly increased CD3+, CD8+ T-lymphocytes, and F4/80+CD11b+ macrophage density in the whole lung and tumor of treated animals emphasizes the mobilizing capability of mEHT on immune cells. In conclusion, mEHT can reduce the growth potential of melanoma, thus offering itself as a complementary therapeutic option to chemo- and/or radiotherapy.
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http://dx.doi.org/10.3390/cancers12123872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767533PMC
December 2020

Fast 3D form measurement using a tunable lens profiler based on imaging with LED illumination.

Opt Express 2021 Jan;29(1):385-399

We present a fast shape measurement of micro-parts based on depth discrimination in imaging with LED illumination. It is based on a 4f-setup with an electrically adjusted tunable lens at the common Fourier plane. Using such a configuration, the opportunity to implement a fast depth scan by means of a tunable lens without the requirement of mechanically moving parts and depth discrimination using the limited spatial coherence of LED illumination is investigated. The technique allows the use of limited spatially partially coherent illumination which can be easily adapted to the test object by selecting the geometrical parameters of the system accordingly. Using this approach, we demonstrate the approach by measuring the 3D form of a tilted optically rough surface and a cold-formed micro-cup. The approach is robust, fast since required images are captured in less than a second, and eye-safe and offers an extended depth of focus in the range of few millimetres. Using a step height standard, we determine a height error of ±1.75 μm (1σ). This value may be further decreased by lowering the spatial coherence length of the illumination or by increasing the numerical aperture of the imaging system.
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http://dx.doi.org/10.1364/OE.413182DOI Listing
January 2021

Radiotherapy enhances uptake and efficacy of Y-cetuximab: A preclinical trial.

Radiother Oncol 2021 02 21;155:285-292. Epub 2020 Nov 21.

German Cancer Consortium (DKTK), Partner Site Dresden, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany; OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, TU Dresden, Helmholtz-Zentrum Dresden - Rossendorf, Germany; Department of Radiotherapy and Radiation Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, TU, Dresden, Germany; Helmholtz-Zentrum Dresden - Rossendorf, Institute of Radiooncology - OncoRay, Dresden, Germany; National Center for Tumor Diseases (NCT), Partner Site Dresden, German Cancer Research Center (DKFZ), Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany, Helmholtz Association / Helmholtz-Zentrum Dresden - Rossendorf (HZDR), Dresden, Germany.

Background And Purpose: Systemic molecular radiotherapy utilizes internal irradiation by radionuclide-labeled tumor-targeting agents with the potential to destroy (micro-)metastases. However, doses that are applicable in solid tumors do not reach the levels nessecary for tumor control. Thus, the combination of molecular and external radiotherapy is a promising treatment strategy, as enhanced tumor doses can be delivered with and without minor overlapping toxicities. Here, we combined a Y-labeled anti-EGFR antibody (Cetuximab) with clinically relevant fractionated radiotherapy in a preclinical trial using head and neck squamous cell carcinoma xenograft tumors.

Materials And Methods: To model Y-Cetuximab uptake for treatment schedule optimization, FaDu-bearing mice were injected with near-infrared-labeled-Cetuximab at different time points during radiotherapy with differing doses. Cetuximab uptake was longitudinally followed by in vivo-optical imaging. Tumor control probability experiments with fractionated radiotherapy (30 fx, 6 weeks, 8 dose groups/ arm) in combination with Y-Cetuximab were performed to test the curative potential.

Results: Imaging of near-infrared-labeled-Cetuximab uptake revealed that low to moderate external beam doses can enhance antibody uptake. Using the optimized schedule, combination of molecular and external radiotherapy using Y-Cetuximab at a dose that did not result in permanent tumor inactivation in previous experiments, led to substantially increased tumor control compared to radiotherapy alone.

Conclusion: Our results indicate that combination of radiolabeled therapeutics with clinically relevant fractionated radiotherapy has a remarkable potential to improve curative treatment outcome. Application of some radiation dose prior to injection may improve drug uptake and enable patient stratification and treatment personalization via a corresponding PET-tracer during therapy.
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http://dx.doi.org/10.1016/j.radonc.2020.11.013DOI Listing
February 2021

Fast form measurements using a digital micro-mirror device in imaging with partially coherent illumination.

Opt Lett 2020 Nov;45(22):6154-6157

We present a new technique for fast form measurement based on imaging with partially coherent illumination. It consists of a 4-imaging system with a digital micro-mirror device (DMD) located in the Fourier plane of its two lenses. The setup benefits from spatially partially coherent illumination that allows for depth discrimination and a DMD that enables a fast depth scan. Evaluating the intensity contrast, the 3D form of an object is reconstructed. We show that the technique additionally offers extended depth of focus imaging in microscopy and short measurement times of less than a second.
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http://dx.doi.org/10.1364/OL.411106DOI Listing
November 2020

Multiple Aperture Shear-Interferometry (MArS): a solution to the aperture problem for the form measurement of aspheric surfaces.

Opt Express 2020 Nov;28(23):34677-34691

Multiple Aperture Shear-Interferometry (MArS) is a shape measurement technique that uses multi-spot illumination to overcome the problem of a limited observation aperture of conventional interferometric techniques and thus considerably simplifies the measurement of optical aspheres and freeform surfaces. Using a shear interferometry setup, MArS measures the coherence function in order to obtain wave vector distributions created from multi-spot LED illumination reflected by the specimen. Based on the wave vectors we reconstruct the surface topography of aspheric lenses using an inverse ray tracing approach and prior knowledge about the individual source locations. We present the topographic measurement of two aspheric lenses with different global curvature radii measured with the same identical reflection setup. In addition, we examine the achievable accuracy of the wave vector measurement using a single light source to find physical limits of MArS.
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http://dx.doi.org/10.1364/OE.408979DOI Listing
November 2020

Versatile chimeric antigen receptor platform for controllable and combinatorial T cell therapy.

Oncoimmunology 2020 07 3;9(1):1785608. Epub 2020 Jul 3.

Department of Radioimmunology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Dresden, Germany.

Chimeric antigen receptor (CAR) T cells show remarkable therapeutic effects in some hematological malignancies. However, CAR T cells can also cause life-threatening side effects. In order to minimize off-target and on-target/off-tumor reactions, improve safety, enable controllability, provide high flexibility, and increase tumor specificity, we established a novel humanized artificial receptor platform termed RevCARs. RevCAR genes encode for small surface receptors lacking any antigen-binding moiety. Steering of RevCAR T cells occurs via bispecific targeting molecules (TMs). The small size of RevCAR-encoding genes allows the construction of polycistronic vectors. Here, we demonstrate that RevCAR T cells efficiently kill tumor cells, can be steered by TMs, flexibly redirected against multiple targets, and used for combinatorial targeting following the "OR" and "AND" gate logic.
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http://dx.doi.org/10.1080/2162402X.2020.1785608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458653PMC
July 2020

Highly Efficient Targeting of EGFR-Expressing Tumor Cells with UniCAR T Cells via Target Modules Based on Cetuximab.

Onco Targets Ther 2020 12;13:5515-5527. Epub 2020 Jun 12.

German Cancer Consortium (DKTK), Dresden, Germany.

Introduction: Since epithelial growth factor receptor (EGFR) overexpression is linked to a variety of malignancies, it is an attractive target for immune therapy including chimeric antigen receptor (CAR)-engineered T cells. Unfortunately, CAR T cell therapy harbors the risk of severe, even life-threatening side effects. Adaptor CAR T cell platforms such as the previously described UniCAR system might be able to overcome these problems. In contrast to conventional CARs, UniCAR T cells are per se inert. Their redirection towards target cells occurs only in the presence of a tumor-specific target molecule (TM). TMs are bifunctional molecules being able to recognize a tumor-associated antigen and to cross-link the CAR T cell via a peptide epitope recognized by the UniCAR domain.

Materials And Methods: Here, we compare αEGFR TMs: a nanobody (nb)-based αEGFR TM derived from the camelid αEGFR antibody 7C12 with a murine and humanized single-chain fragment variable (scFv) based on the clinically used antibody Cetuximab.

Results: In principle, both the nb- and scFv-based TM formats are able to redirect UniCAR T cells to eliminate EGFR-expressing tumor cells in an antigen-specific and TM-dependent manner. However, the scFv-based αEGFR TM was significantly superior to the nb-based TM especially with respect to lysis of tumor cells.

Discussion: Improved efficiency of the scFv-based TM allowed the redirection of UniCAR T cells towards tumor cells expressing high as well as low EGFR levels in comparison to nb-based αEGFR TMs.
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http://dx.doi.org/10.2147/OTT.S245169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297505PMC
June 2020

Development and In Vivo Application of a Water-Soluble Anticancer Copper Ionophore System Using a Temperature-Sensitive Liposome Formulation.

Pharmaceutics 2020 May 20;12(5). Epub 2020 May 20.

Laboratory for Environmental Chemistry and Bioanalytics, Institute of Chemistry, Eötvös Loránd University, H-1117 Budapest, Pázmány Péter Stny. 1/A, Hungary.

Liposomes containing copper and the copper ionophore neocuproine were prepared and characterized for in vitro and in vivo anticancer activity. Thermosensitive PEGylated liposomes were prepared with different molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and hydrogenated soybean phosphatidylcholine (HSPC) in the presence of copper(II) ions. Optimal, temperature dependent drug release was obtained at 70:30 DPPC to HSPC weight ratio. Neocuproine (applied at 0.2 mol to 1 mol phospholipid) was encapsulated through a pH gradient while using unbuffered solution at pH 4.5 inside the liposomes, and 100 mM HEPES buffer pH 7.8 outside the liposomes. Copper ions were present in excess, yielding 0.5 mM copper-(neocuproine) complex and 0.5 mM free copper. Pre-heating to 45 °C increased the toxicity of the heat-sensitive liposomes in short-term in vitro experiments, whereas at 72 h all investigated liposomes exhibited similar in vitro toxicity to the copper(II)-neocuproine complex (1:1 ratio). Thermosensitive liposomes were found to be more effective in reducing tumor growth in BALB/c mice engrafted with C26 cancer cells, regardless of the mild hyperthermic treatment. Copper uptake of the tumor was verified by PET/CT imaging following treatment with [Cu]Cu-neocuproine liposomes. Taken together, our results demonstrate the feasibility of targeting a copper nanotoxin that was encapsulated in thermosensitive liposomes containing an excess of copper.
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http://dx.doi.org/10.3390/pharmaceutics12050466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284829PMC
May 2020

UniCAR T cell immunotherapy enables efficient elimination of radioresistant cancer cells.

Oncoimmunology 2020 5;9(1):1743036. Epub 2020 Apr 5.

Department of Radioimmunology, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Institute of Radiopharmaceutical Cancer Research, Dresden, Germany.

Induction or selection of radioresistant cancer (stem) cells following standard radiotherapy is presumably one of the major causes for recurrence of metastatic disease. One possibility to prevent tumor relapse is the application of targeted immunotherapies including, e.g., chimeric antigen receptor (CAR) T cells. In light of long-term remissions, it is highly relevant to clarify whether radioresistant cancer cells are susceptible to CAR T cell-mediated killing. To answer this question, we evaluated the anti-tumor activity of the switchable universal chimeric antigen receptor (UniCAR) system against highly radioresistant head and neck squamous cell carcinoma cells both and . Following specific UniCAR T cell engagement via EGFR or CD98 target modules, T cell effector mechanisms were induced including secretion of pro-inflammatory cytokines, up-regulation of granzyme B and perforin, as well as T cell proliferation. CD98- or EGFR-redirected UniCAR T cells further possess the capability to efficiently lyse radioresistant tumor cells. Observed anti-tumor effects were comparable to those against the radiosensitive parental cell lines. Finally, redirected UniCAR T cells significantly inhibited the growth of radioresistant cancer cells in immunodeficient mice. Taken together, our obtained data underline that the UniCAR system is able to overcome radioresistance. Thus, it represents an attractive technology for the development of combined radioimmunotherapeutic approaches that might improve the outcome of patients with metastatic radioresistant tumor diseases.
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http://dx.doi.org/10.1080/2162402X.2020.1743036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7219270PMC
April 2020

Extended half-life target module for sustainable UniCAR T-cell treatment of STn-expressing cancers.

J Exp Clin Cancer Res 2020 May 5;39(1):77. Epub 2020 May 5.

Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Institute of Radiopharmaceutical Cancer Research, Bautzner Landstrasse 400, 01328, Dresden, Germany.

Background: Adapter chimeric antigen receptor (CAR) approaches have emerged has promising strategies to increase clinical safety of CAR T-cell therapy. In the UniCAR system, the safety switch is controlled via a target module (TM) which is characterized by a small-size and short half-life. The rapid clearance of these TMs from the blood allows a quick steering and self-limiting safety switch of UniCAR T-cells by TM dosing. This is mainly important during onset of therapy when tumor burden and the risk for severe side effects are high. For long-term UniCAR therapy, the continuous infusion of TMs may not be an optimal setting for the patients. Thus, in later stages of treatment, single infusions of TMs with an increased half-life might play an important role in long-term surveillance and eradication of residual tumor cells. Given this, we aimed to develop and characterize a novel TM with extended half-life targeting the tumor-associated carbohydrate sialyl-Tn (STn).

Methods: The extended half-life TM is composed of the STn-specific single-chain variable fragment (scFv) and the UniCAR epitope, fused to the hinge region and Fc domain of a human immunoglobulin 4 (IgG4) antibody. Specific binding and functionality of the αSTn-IgG4 TM as well as pharmacokinetic features were assessed using in vitro and in vivo assays and compared to the already established small-sized αSTn TM.

Results: The novel αSTn-IgG4 TM efficiently activates and redirects UniCAR T-cells to STn-expressing tumors in a target-specific and TM-dependent manner, thereby promoting the secretion of proinflammatory cytokines and tumor cell lysis in vitro and in experimental mice. Moreover, PET-imaging results demonstrate the specific enrichment of the αSTn-IgG4 TM at the tumor site, while presenting a prolonged serum half-life compared to the short-lived αSTn TM.

Conclusion: In a clinical setting, the combination of TMs with different formats and pharmacokinetics may represent a promising strategy for retargeting of UniCAR T-cells in a flexible, individualized and safe manner at particular stages of therapy. Furthermore, as these molecules can be used for in vivo imaging, they pose as attractive candidates for theranostic approaches.
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http://dx.doi.org/10.1186/s13046-020-01572-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201957PMC
May 2020

Ultrasmall silicon nanoparticles as a promising platform for multimodal imaging.

Faraday Discuss 2020 06;222(0):362-383

Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstraße 400, Dresden, D-01328, Germany.

Bimodal systems for nuclear and optical imaging are currently being intensively investigated due to their comparable detection sensitivity and the complementary information they provide. In this perspective, we have implemented both modalities on biocompatible ultrasmall silicon nanoparticles (Si NPs). Such nanoparticles are particularly interesting since they are highly biocompatible, have covalent surface functionalization and demonstrate very fast body clearance. We prepared monodisperse citrate-stabilized Si NPs (2.4 ± 0.5 nm) with more than 40 accessible terminal amino groups per particle and, for the first time, simultaneously, a near-infrared dye (IR800-CW) and a radiolabel (64Cu-NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid) have been covalently linked to the surface of such Si NPs. The obtained nanomaterials have been fully characterized using HR-TEM, XPS, UV-Vis and FT-IR spectroscopy. These dual-labelled particles do not exhibit any cytotoxicity in vitro. In vivo studies employing both positron emission tomography (PET) and optical imaging (OI) techniques revealed rapid renal clearance of dual-labelled Si NPs from mice.
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http://dx.doi.org/10.1039/c9fd00091gDOI Listing
June 2020

Synthesis, Labeling and Preclinical Evaluation of a Squaric Acid Containing PSMA Inhibitor Labeled with Ga: A Comparison with PSMA-11 and PSMA-617.

ChemMedChem 2020 04 19;15(8):695-704. Epub 2020 Mar 19.

Institute of Nuclear Chemistry, Johannes Gutenberg University, Fritz-Strassmann-Weg 2, 55128, Mainz, Germany.

The L-lysine urea-L-glutamate (KuE) represents a key motif in recent diagnostic and therapeutic radiopharmaceuticals targeting the prostate specific membrane antigen (PSMA). Using a squaric acid moiety for coupling of KuE with a radioactive label, the squaric acid as a linker in the PSMA ligand seems to mimic the aromatic structure of the naphthylalanine unit on PSMA-617. In this work, we investigate the influence of squaric acid moiety on the biological activity of the compound carrying a KuE motif and three typical chelates. The derivatives TRAM.SA.KuE, DOTAGA.SA.KuE and NODAGA.SA.KuE were all synthesized in straightforward organic reactions and purified by HPLC afterward. Different amounts of tracer were labeled at different temperatures with Ga. PET examinations were performed on NMRInu/nu nude mice with an LNCaP tumor on the right hind leg including ex vivo investigations of the organs. For comparison, Ga-derivatives of PSMA-11 and PSMA-617, the derivatives most commonly used in clinics, were investigated in the same animal model.
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http://dx.doi.org/10.1002/cmdc.201900559DOI Listing
April 2020

"UniCAR"-modified off-the-shelf NK-92 cells for targeting of GD2-expressing tumour cells.

Sci Rep 2020 02 7;10(1):2141. Epub 2020 Feb 7.

Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Institute of Radiopharmaceutical Cancer Research, Dresden, Germany.

Antigen-specific redirection of immune effector cells with chimeric antigen receptors (CARs) demonstrated high therapeutic potential for targeting cancers of different origins. Beside CAR-T cells, natural killer (NK) cells represent promising alternative effectors that can be combined with CAR technology. Unlike T cells, primary NK cells and the NK cell line NK-92 can be applied as allogeneic off-the-shelf products with a reduced risk of toxicities. We previously established a modular universal CAR (UniCAR) platform which consists of UniCAR-expressing immune cells that cannot recognize target antigens directly but are redirected by a tumour-specific target module (TM). The TM contains an antigen-binding moiety fused to a peptide epitope which is recognized by the UniCAR molecule, thereby allowing an on/off switch of CAR activity, and facilitating flexible targeting of various tumour antigens depending on the presence and specificity of the TM. Here, we provide proof of concept that it is feasible to generate a universal off-the-shelf cellular therapeutic based on UniCAR NK-92 cells targeted to tumours expressing the disialoganglioside GD2 by GD2-specific TMs that are either based on an antibody-derived single-chain fragment variable (scFv) or an IgG4 backbone. Redirected UniCAR NK-92 cells induced specific killing of GD2-expressing cells in vitro and in vivo, associated with enhanced production of interferon-γ. Analysis of radiolabelled proteins demonstrated that the IgG4-based format increased the in vivo half-life of the TM markedly in comparison to the scFv-based molecule. In summary, UniCAR NK-92 cells represent a universal off-the-shelf platform that is highly effective and flexible, allowing the use of different TM formats for specific tumour targeting.
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http://dx.doi.org/10.1038/s41598-020-59082-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005792PMC
February 2020

Radiometal-Containing Aryl Diazonium Salts for Chemoselective Bioconjugation of Tyrosine Residues.

ACS Omega 2019 Dec 10;4(26):22101-22107. Epub 2019 Dec 10.

Department of Oncology, Faculty of Pharmacy and Pharmaceutical Sciences, Department of Chemistry, and Cancer Research Institute of Northern Alberta, University of Alberta, Edmonton AB T6G 2R3, Canada.

Tyrosine is an attractive target for chemo- and site-selective protein modification. The particular chemical nature of tyrosine residues allows bioconjugation chemistry with reactive aryl diazonium salts via electrophilic aromatic substitution to produce diazo compounds. In this work, we describe the preparation of Cu- and Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-diazonium salts as building blocks for azo coupling chemistry with tyrosine and tyrosine-containing peptides and proteins under mild conditions. 2--(4-aminobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (-NH-Bn-NOTA) was used to form the corresponding Cu- and Ga-labeled complexes, followed by diazotization with NaNO in the presence of HCl. Cu- and Ga-labeled NOTA complexes were prepared in high radiochemical yields >80% starting from 20 μg of -NH-Bn-NOTA. Conversion of -NH-Bn-NOTA complexes into diazonium salts followed by azo coupling with l-tyrosine afforded Cu- and Ga-labeled tyrosine in radiochemical yields of 80 and 56%, respectively. Azo coupling with tyrosine-containing hexapeptide neurotensin NT(8-13) afforded Cu- and Ga-labeled NT(8-13) in radiochemical yields of 45 and 11%, respectively. Azo coupling of Cu-labeled NOTA-diazonium salt with human serum albumin (HSA) gave Cu-labeled HSA in radiochemical yields of 20%. The described azo coupling chemistry represents an innovative and versatile bioconjugation strategy for selective targeting of tyrosine residues in peptides and proteins.
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http://dx.doi.org/10.1021/acsomega.9b03248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933782PMC
December 2019

Versatile Bispidine-Based Bifunctional Chelators for Cu -Labelling of Biomolecules.

Chemistry 2020 Feb 9;26(9):1989-2001. Epub 2020 Jan 9.

Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Bautzner Landstrasse 400, 01328, Dresden, Germany.

Bifunctional chelators as parts of modular metal-based radiopharmaceuticals are responsible for stable complexation of the radiometal ion and for covalent linkage between the complex and the targeting vector. To avoid loss of complex stability, the bioconjugation strategy should not interfere with the radiometal chelation by occupying coordinating groups. The C9 position of the very stable Cu chelator 3,7-diazabicyclo[3.3.1]nonane (bispidine) is virtually predestined to introduce functional groups for facile bioconjugation as this functionalisation does not disturb the metal binding centre. We describe the preparation and characterisation of a set of novel bispidine derivatives equipped with suitable functional groups for diverse bioconjugation reactions, including common amine coupling strategies (bispidine-isothiocyanate) and the Cu-free strain-promoted alkyne-azide cycloaddition. We demonstrate their functionality and versatility in an exemplary way by conjugation to an antibody-based biomolecule and validate the obtained conjugate in vitro and in vivo.
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http://dx.doi.org/10.1002/chem.201904654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028042PMC
February 2020

A theranostic PSMA ligand for PET imaging and retargeting of T cells expressing the universal chimeric antigen receptor UniCAR.

Oncoimmunology 2019;8(11):1659095. Epub 2019 Sep 7.

Department of Radioimmunology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Dresden, Germany.

Chimeric antigen receptor (CAR) T cells have shown impressive therapeutic potential. Due to the lack of direct control mechanisms, therapy-related adverse reactions including cytokine release- and tumor lysis syndrome can even become life-threatening. In case of target antigen expression on non-malignant cells, CAR T cells can also attack healthy tissues. To overcome such side effects, we have established a modular CAR platform termed UniCAR: UniCAR T cells per se are inert as they recognize a peptide epitope (UniCAR epitope) that is not accessible on the surface of living cells. Bifunctional adapter molecules termed target modules (TM) can cross-link UniCAR T cells with target cells. In the absence of TMs, UniCAR T cells automatically turn off. Until now, all UniCAR TMs were constructed by fusion of the UniCAR epitope to an antibody domain. To open up the wide field of low-molecular-weight compounds for retargeting of UniCAR T cells to tumor cells, and to follow in parallel the progress of UniCAR T cell therapy by PET imaging we challenged the idea to convert a PET tracer into a UniCAR-TM. For proof of concept, we selected the clinically used PET tracer PSMA-11, which binds to the prostate-specific membrane antigen overexpressed in prostate carcinoma. Here we show that fusion of the UniCAR epitope to PSMA-11 results in a low-molecular-weight theranostic compound that can be used for both retargeting of UniCAR T cells to tumor cells, and for non-invasive PET imaging and thus represents a member of a novel class of theranostics.
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http://dx.doi.org/10.1080/2162402X.2019.1659095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791425PMC
September 2019

Conventional CARs versus modular CARs.

Cancer Immunol Immunother 2019 Oct 21;68(10):1713-1719. Epub 2019 Sep 21.

Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Institute of Radiopharmaceutical Cancer Research, Bautzner Landstraße 400, 01328, Dresden, Germany.

The clinical application of immune effector cells genetically modified to express chimeric antigen receptors (CARs) has shown impressive results including complete remissions of certain malignant hematological diseases. However, their application can also cause severe side effects such as cytokine release syndrome (CRS) or tumor lysis syndrome (TLS). One limitation of currently applied CAR T cells is their lack of regulation. Especially, an emergency shutdown of CAR T cells in case of life-threatening side effects is missing. Moreover, targeting of tumor-associated antigens (TAAs) that are not only expressed on tumor cells but also on vital tissues requires the possibility of a switch allowing to repeatedly turn the activity of CAR T cells on and off. Here we summarize the development of a modular CAR variant termed universal CAR (UniCAR) system that promises to overcome these limitations of conventional CARs.
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http://dx.doi.org/10.1007/s00262-019-02399-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805801PMC
October 2019

T cells engrafted with a UniCAR 28/z outperform UniCAR BB/z-transduced T cells in the face of regulatory T cell-mediated immunosuppression.

Oncoimmunology 2019;8(9):e1621676. Epub 2019 Jun 7.

Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Dresden, Germany.

Adoptive transfer of chimeric antigen receptor (CAR)-equipped T cells have demonstrated astonishing clinical efficacy in hematological malignancies recently culminating in the approval of two CAR T cell products. Despite this tremendous success, CAR T cell approaches have still achieved only moderate efficacy against solid tumors. As a major obstacle, engineered conventional T cells (Tconvs) face an anti-inflammatory, hostile tumor microenvironment often infiltrated by highly suppressive regulatory T cells (Tregs). Thus, potent CAR T cell treatment of solid tumors requires efficient activation of Tconvs via their engrafted CAR to overcome Treg-mediated immunosuppression. In that regard, selecting an optimal intracellular signaling domain might represent a crucial step to achieve best clinical efficiency. To shed light on this issue and to investigate responsiveness to Treg inhibition, we engrafted Tconvs with switchable universal CARs (UniCARs) harboring intracellularly the CD3ζ domain alone or in combination with costimulatory CD28 or 4-1BB. Our studies reveal that UniCAR ζ-, and UniCAR BB/ζ-engineered Tconvs are strongly impaired by activated Tregs, whereas UniCARs providing CD28 costimulation overcome Treg-mediated suppression both and . Compared to UniCAR ζ- and UniCAR BB/ζ-modified cells, UniCAR 28/ζ-armed Tconvs secrete significantly higher amounts of Th1-related cytokines and, furthermore, levels of these cytokines are elevated even upon exposure to Tregs. Thus, in contrast to 4-1BB costimulation, CD28 signaling in UniCAR-transduced Tconvs seems to foster a pro-inflammatory milieu, which contributes to enhanced resistance to Treg suppression. Overall, our results may have significant implications for CAR T cell-based immunotherapies of solid tumors strongly invaded by Tregs.
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http://dx.doi.org/10.1080/2162402X.2019.1621676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685520PMC
February 2021

Anti-CAR-engineered T cells for epitope-based elimination of autologous CAR T cells.

Cancer Immunol Immunother 2019 Sep 14;68(9):1401-1415. Epub 2019 Aug 14.

Department of Radioimmunology, Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Bautzner Landstraße 400, 01328, Dresden, Germany.

Although CAR T-cell therapy has demonstrated tremendous clinical efficacy especially in hematological malignancies, severe treatment-associated toxicities still compromise the widespread application of this innovative technology. Therefore, developing novel approaches to abrogate CAR T-cell-mediated side effects is of great relevance. Several promising strategies pursue the selective antibody-based depletion of adoptively transferred T cells via elimination markers. However, given the limited half-life and tissue penetration, dependence on the patients' immune system and on-target/off-side effects of proposed monoclonal antibodies, we sought to exploit αCAR-engineered T cells to efficiently eliminate CAR T cells. For comprehensive and specific recognition, a small peptide epitope (E-tag) was incorporated into the extracellular spacer region of CAR constructs. We provide first proof-of-concept for targeting this epitope by αE-tag CAR T cells, allowing an effective killing of autologous E-tagged CAR T cells both in vitro and in vivo whilst sparing cells lacking the E-tag. In addition to CAR T-cell cytotoxicity, the αE-tag-specific T cells can be empowered with cancer-fighting ability in case of relapse, hence, have versatile utility. Our proposed methodology can most probably be implemented in CAR T-cell therapies regardless of the targeted tumor antigen aiding in improving overall safety and survival control of highly potent gene-modified cells.
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http://dx.doi.org/10.1007/s00262-019-02376-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6768917PMC
September 2019

An oligo-His-tag of a targeting module does not influence its biodistribution and the retargeting capabilities of UniCAR T cells.

Sci Rep 2019 07 22;9(1):10547. Epub 2019 Jul 22.

Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Dresden, Germany.

Recently, we established the controllable modular UniCAR platform technology to advance the efficacy and safety of CAR T cell therapy. The UniCAR system is composed of (i) target modules (TMs) and (ii) UniCAR armed T cells. TMs are bispecific molecules that are able to bind to the tumor cell surface and simultaneously to UniCAR T cells. For interaction with UniCAR T cells, TMs contain a peptide epitope sequence which is recognised by UniCAR T cells. So far, a series of TMs against a variety of tumor targets including against the prostate stem cell antigen (PSCA) were constructed and functionally characterised. In order to facilitate their purification all these TMs are expressed as recombinant proteins equipped with an oligo-His-tag. The aim of the here presented manuscript was to learn whether or not the oligo-His-tag of the TM influences the UniCAR system. For this purpose, we constructed TMs against PSCA equipped with or lacking an oligo-His-tag. Both TMs were compared side by side including for functionality and biodistribution. According to our data, an oligo-His-tag of a UniCAR TM has only little if any effect on its binding affinity, in vitro and in vivo killing capability and in vivo biodistribution.
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http://dx.doi.org/10.1038/s41598-019-47044-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6646371PMC
July 2019

Instant kit preparation of Ga-radiopharmaceuticals via the hybrid chelator DATA: clinical translation of [Ga]Ga-DATA-TOC.

EJNMMI Res 2019 May 23;9(1):48. Epub 2019 May 23.

Institute of Nuclear Chemistry, Johannes Gutenberg-University Mainz, Mainz, Germany.

Purpose: The widespread use of Ga for positron emission tomography (PET) relies on the development of radiopharmaceutical precursors that can be radiolabelled and dispensed in a simple, quick, and convenient manner. The DATA (6-amino-1,4-diazapine-triacetate) scaffold represents a novel hybrid chelator architecture possessing both cyclic and acyclic character that may allow for facile access to Ga-labelled tracers in the clinic. We report the first bifunctional DATA chelator conjugated to [Tyr]octreotide (TOC), a somatostatin subtype 2 receptor (SST)-targeting vector for imaging and functional characterisation of SSTR expressing tumours.

Methods: The radiopharmaceutical precursor, DATA-TOC, was synthesised as previously described and used to complex Ga(III) and Ga(III). Competition binding assays of [Ga]Ga-DATA-TOC or [Ga]Ga-DOTA-TOC against [I-Tyr]LTT-SS28 were conducted in membranes of HEK293 cells transfected to stably express one of the hSST receptor subtypes (HEK293-hSST cells). First in vivo studies were performed in female NMRI-nude mice bearing SST-positive mouse phaeochromocytoma mCherry (MPC-mCherry) tumours to compare the in vivo SST-specific tumour-targeting of [Ga]Ga-DATA-TOC and its overall pharmacokinetics versus the [Ga]Ga-DOTA-TOC reference. A direct comparison of [Ga]Ga-DATA-TOC with the well-established PET radiotracer [Ga]Ga-DOTA-TOC was additionally performed in a 46-year-old male patient with a well-differentiated NET (neuroendocrine tumour), representing the first in human administration of [Ga]Ga-DATA-TOC.

Results: DATA-TOC was labelled with Ga with a radiolabelling efficiency of > 95% in less than 10 min at ambient temperature. A molar activity up to 35 MBq/nmol was achieved. The hSST-affinities of [Ga]Ga-DATA-TOC and [Ga]Ga-DOTA-TOC were found similar with only sub-nanomolar differences in the respective IC values. In mice, [Ga]Ga-DATA-TOC was able to visualise the tumour lesions, showing standardised uptake values (SUVs) similar to [Ga]Ga-DOTA-TOC. Direct comparison of the two PET tracers in a NET patient revealed very similar tumour uptake for the two Ga-radiotracers, but with a higher tumour-to-liver contrast for [Ga]Ga-DATA-TOC.

Conclusion: [Ga]Ga-DATA-TOC was prepared, to a quality appropriate for in vivo use, following a highly efficient kit type process. Furthermore, the novel radiopharmaceutical was comparable or better than [Ga]Ga-DOTA-TOC in all preclinical tests, achieving a higher tumour-to-liver contrast in a NET-patient. The results illustrate the potential of the DATA-chelator to facilitate the access to and preparation of Ga-radiotracers in a routine clinical radiopharmacy setting.
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http://dx.doi.org/10.1186/s13550-019-0516-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533321PMC
May 2019

Radiochemical and radiopharmacological characterization of a Cu-labeled α-MSH analog conjugated with different chelators.

J Labelled Comp Radiopharm 2019 06 17;62(8):495-509. Epub 2019 Jun 17.

Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.

Radiolabeled α-melanocyte-stimulating hormone (α-MSH) derivatives have a high potential for diagnosis and treatment of melanoma, because of high specificity and binding affinity to the melanocortin-1 receptor (MC1R). Hence, the α-MSH-derived peptide NAP-NS1 with a β-Ala linker (ε-Ahx-β-Ala-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH ) was conjugated to different chelators: either to NOTA (p-SCN-Bn-1,4,7-triazacyclononane-1,4,7-triacetic acid), to a hexadentate bispidine carbonate derivative (dimethyl-9-(((4-nitrophenoxy)carbonyl)oxy)-2,4-di(pyridin-2-yl)-3,7-bis(pyridin-2-ylmethyl)-3,7-diazabicyclo[3.3.1]nonane-1,5-dicarboxylate), or to DMPTACN (p-SCN-Ph-bis(2-pyridyl-methyl)-1,4,7-triaza-cyclononane), labeled with Cu, and investigated in terms of radiochemical and radiopharmacological properties. For the three Cu-labeled conjugates negligible transchelation, suitable buffer and serum stability, as well as appropriate water solubility, was determined. The three conjugates exhibited high binding affinity (low nanomolar range) in murine B16F10, human MeWo, and human TXM13 cells. The B values of [ Cu]Cu-bispidine-NAP-NS1 ([ Cu]Cu-2) and [ Cu]Cu-DMPTACN-NAP-NS1 ([ Cu]Cu-3) were higher than those of [ Cu]Cu-NOTA-NAP-NS1 ([ Cu]Cu-1), implying that different charged chelate units might have an impact on binding capacity. Preliminary in vivo biodistribution studies suggested the main excretion pathway of [ Cu]Cu-1 and [ Cu]Cu-3 to be renal, while that of [ Cu]Cu-2 seemed to be both renal and hepatobiliary. An initial moderate uptake in the kidney decreased clearly after 60 minutes. All three Cu-labeled conjugates should be considered for further in vivo investigations using a suitable xenograft mouse model.
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http://dx.doi.org/10.1002/jlcr.3728DOI Listing
June 2019

Theranostic CAR T cell targeting: A brief review.

J Labelled Comp Radiopharm 2019 Jun 6;62(8):533-540. Epub 2019 Jun 6.

Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Institute of Radiopharmaceutical Cancer Research, Dresden, Germany.

More than hundred years ago, Paul Ehrlich postulated that our immune system should be able to recognize tumor cells. Just recently, the development of check point inhibitors, bispecific antibodies, and T cells genetically modified to express chimeric antigen receptors (CARs) underlines the true power of our immune system. T cells genetically modified with CARs can lead to complete remission of malignant hematologic diseases. However, they can also cause life-threatening side effects. In case of cytokine release syndrome, tumor lysis syndrome, or deadly side effects on the central nervous system, an emergency shut down of CAR T cells is needed. Targeting of tumor-associated antigens that are also expressed on vital tissues require a possibility to repeatedly switch the activity of CAR T cells on and off on demand and to follow the treatment by imaging. Theranostic, modular CARs such as the UniCAR system may help to overcome these problems.
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http://dx.doi.org/10.1002/jlcr.3727DOI Listing
June 2019

Multimodal PET/MRI Imaging Results Enable Monitoring the Side Effects of Radiation Therapy.

Contrast Media Mol Imaging 2018 1;2018:5906471. Epub 2018 Nov 1.

CROmed Translational Research Centers, H-1047 Budapest, Hungary.

Radiotherapy is one of the most frequently applied treatments in oncology. Tissue-absorbed ionizing radiation damages not only targeted cells but the surrounding cells too. The consequent long-term induced oxidative stress, irreversible tissue damage, or second malignancies draw attention to the urgent need of a follow-up medical method by which personalized treatment could be attained and the actually dose-limiting organ could be monitored in the clinical practice. We worked out a special hemisphere irradiation technique for mice which mimics the radiation exposure during radiotherapy. We followed up the changes of possible brain imaging biomarkers of side effects, such as cerebral blood flow, vascular endothelial function, and cellular metabolic processes for 60 days. BALB/c mice were divided into two groups (=6 per group) based on the irradiation doses (5 and 20 Gy). After the irradiation procedure arterial spin labeling (ASL), diffusion-weighted imaging (DWI) in magnetic resonance modality and [F]fluoro-deoxy-D-glucose positron emission tomography (FDG-PET) scans of the brain were obtained at several time points (3, 7, 30, and 60 days after the irradiation). Significant physiological changes were registered in the brain of animals following the irradiation by both applied doses. Elevated standard uptake values were detected all over the brain by FDG-PET studies 2 months after the irradiation. The apparent diffusion coefficients from DWI scans significantly decreased one month after the irradiation procedure, while ASL studies did not show any significant perfusion changes in the brain. Altogether, our sensitive multimodal imaging protocol seems to be an appropriate method for follow-up of the health status after radiation therapy. The presented approach makes possible parallel screening of healthy tissues and the effectiveness of tumor therapy without any additional radiation exposure.
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http://dx.doi.org/10.1155/2018/5906471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6236670PMC
May 2019

Spatial multiplexing and autofocus in holographic contouring for inspection of micro-parts.

Opt Express 2018 Oct;26(22):28576-28588

We present a method for fast geometrical inspection of micro deep drawing parts. It is based on single-shot two-wavelength contouring digital holographic microscopy (DHM). Within the capturing process, spatial multiplexing is utilized in order to record the two required holograms in a single-shot. For fast evaluation, determining the locations where the object is in focus and stitching all focus object's areas together is achieved digitally without the need for any external intervention using an autofocus algorithm. Thus, the limited depth of focus of the microscope objective is improved. The autofocus algorithm is based on minimizing the total variation (TV) of phase difference residuals of the two-wavelength measurements. In contrast to standard DHM, an object side telecentric microscope objective is used for overcoming the image scaling distortions caused by a conventional microscope objective. The method is used to reconstruct the 3D geometrical shape of a cold drawing micro cup. Experimental results verify the improvement of DHM's depth of focus.
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http://dx.doi.org/10.1364/OE.26.028576DOI Listing
October 2018

Form determination of optical surfaces by measuring the spatial coherence function using shearing interferometry.

Opt Express 2018 Oct;26(21):27991-28001

We present a new method for the form measurement of optical surfaces using the spatial coherence function, which enables a shearing interferometer in combination with an LED multispot illumination to function as a measurement device. A new evaluation approach connects the measured data with the surface form by inverse raytracing. First measurement results with the inverse evaluation procedures are shown. We present the whole measurement in combination with the evaluation procedure. In addition, the convergence and stability of the implemented optimization task is investigated.
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http://dx.doi.org/10.1364/OE.26.027991DOI Listing
October 2018

Isolation of Proteins from Polyacrylamide Gels.

Methods Mol Biol 2019 ;1855:461-465

Institute of Radiopharmaceutical Cancer Research, Department of Radio-/Tumorimmunology, Helmholtz-Zentrum Dresden Rossendorf (HZDR), Dresden, Germany.

Minute amounts of proteins are required for immunization of mice for the development of antibodies including monoclonal antibodies. Here we describe a rapid procedure for the isolation of proteins from polyacrylamide gels after sodium dodecyl sulfate polyacrylamide gel electrophoresis in sufficient amounts for immunization of animals.
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http://dx.doi.org/10.1007/978-1-4939-8793-1_38DOI Listing
June 2019