Publications by authors named "Dirk Schaudien"

34 Publications

Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL.

Mol Ther Methods Clin Dev 2021 Jun 9;21:621-641. Epub 2021 Apr 9.

Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, 30625 Hannover, Germany.

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent "induced DCs" (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14 monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34 cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.
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http://dx.doi.org/10.1016/j.omtm.2021.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142053PMC
June 2021

Final results from a 90-day quantitative inhalation toxicology study evaluating the dose-response and fate in the lung and pleura of chrysotile-containing brake dust compared to TiO, chrysotile, crocidolite or amosite asbestos: Histopathological examination, confocal microscopy and collagen quantification of the lung and pleural cavity.

Toxicol Appl Pharmacol 2021 May 30;424:115598. Epub 2021 May 30.

Fraunhofer Institute for Toxicology and Experimental Medicine, 1 Nikolai-Fuchs-Strasse, D-30625 Hannover, Germany. Electronic address:

The final results from this multi-dose, 90-day inhalation toxicology study in the rat with life-time post-exposure observation have shown a significant fundamental difference in pathological response and tumorgenicity between brake dust generated from brake pads manufactured with chrysotile or from chrysotile alone in comparison to the amphiboles, crocidolite and amosite asbestos. The groups exposed to brake dust showed no significant pathological or tumorigenic response in the respiratory track compared to the air control group at exposure concentrations and deposited doses well above those at which humans have been exposed. Slight alveolar/interstitial macrophage accumulation of particles was noted. Wagner grades were 1-2 (1 = control group), similar to the TiO particle control group. Chrysotile was not biopersistent, exhibiting in the lung a deterioration of its matrix which results in breakage into particles and short fibers which can be cleared by alveolar macrophages and which can continue to dissolve. Particle-laden macrophage accumulation was observed, leading to a very-slight interstitial inflammatory response (Wagner grade 1-3). There was no peribronchiolar inflammation, occasional very-slight interstitial fibrosis (Wagner grade 4), and no exposure-related tumorigenic response. The pathological response of crocidolite and amosite compared to the brake dust and chrysotile was clearly differentiated by the histopathology and the confocal analysis. Crocidolite and amosite induced persistent inflammation, microgranulomas, persistent fibrosis (Wagner grades 4), and a dose-related lung tumor response. Confocal microscopy quantified extensive inflammatory response and collagen development in the lung, visceral and parietal pleura as well as pleural adhesions. These results provide a clear foundation for differentiating the innocuous effects of brake dust exposure from the adverse effects following amphibole asbestos exposure.
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http://dx.doi.org/10.1016/j.taap.2021.115598DOI Listing
May 2021

Intratumoral Canine Distemper Virus Infection Inhibits Tumor Growth by Modulation of the Tumor Microenvironment in a Murine Xenograft Model of Canine Histiocytic Sarcoma.

Int J Mol Sci 2021 Mar 30;22(7). Epub 2021 Mar 30.

Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany.

Histiocytic sarcomas refer to highly aggressive tumors with a poor prognosis that respond poorly to conventional treatment approaches. Oncolytic viruses, which have gained significant traction as a cancer therapy in recent decades, represent a promising option for treating histiocytic sarcomas through their replication and/or by modulating the tumor microenvironment. The live attenuated canine distemper virus (CDV) vaccine strain Onderstepoort represents an attractive candidate for oncolytic viral therapy. In the present study, oncolytic virotherapy with CDV was used to investigate the impact of this virus infection on tumor cell growth through direct oncolytic effects or by virus-mediated modulation of the tumor microenvironment with special emphasis on angiogenesis, expression of selected MMPs and TIMP-1 and tumor-associated macrophages in a murine xenograft model of canine histiocytic sarcoma. Treatment of mice with xenotransplanted canine histiocytic sarcomas using CDV induced overt retardation in tumor progression accompanied by necrosis of neoplastic cells, increased numbers of intratumoral macrophages, reduced angiogenesis and modulation of the expression of MMPs and TIMP-1. The present data suggest that CDV inhibits tumor growth in a multifactorial way, including direct cell lysis and reduction of angiogenesis and modulation of MMPs and their inhibitor TIMP-1, providing further support for the concept of its role in oncolytic therapies.
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http://dx.doi.org/10.3390/ijms22073578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037597PMC
March 2021

Prenatal developmental toxicity studies on fumes from oxidised asphalt (OA) in the rat.

Reprod Toxicol 2021 Jun 26;102:67-79. Epub 2021 Mar 26.

Nynas, P.O. Box 10 700, SE-121 29 Stockholm, Sweden; Toxicology Group in CONCAWE, Brussels, Belgium. Electronic address:

The prenatal developmental toxicity of the fumes of oxidised asphalt (OA) was tested by nose-only inhalation in the rat. The test material was generated by collecting fumes from the headspace of storage tanks filled with OA. The composition of these fumes was matched to fumes sampled at a workplace where the same OA was applied in a pour-and-roll operation, representing occupational exposure with high concentrations of fumes to not underestimate the possible hazard. In the main study, dams were exposed to 0, 53, 158 and 536 mg/m of fume (as total organic mass), for 6 h/day for 19 days p.c. The maternal NOAEC was 53 mg/m³ (lowest dose tested). In the high-dose group treatment-related effects on body weight gain were seen. In the mid- and high-dose groups treatment-related effects on food consumption, lung weights, and histopathological changes in lungs and the upper respiratory tract were observed. The NOAEC for prenatal developmental toxicity was 536 mg/m³ since no exposure-related effects were found in any of the exposure groups for any of the investigated reproductive endpoints. Furthermore, nose-only exposure to OA fumes in concentrations up to 536 mg/m³ from days 1-19 p.c. did not induce any significant fetal abnormalities.
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http://dx.doi.org/10.1016/j.reprotox.2021.03.003DOI Listing
June 2021

Conditionally immortalised leukaemia initiating cells co-expressing Hoxa9/Meis1 demonstrate microenvironmental adaptation properties ex vivo while maintaining myelomonocytic memory.

Sci Rep 2021 Mar 5;11(1):5294. Epub 2021 Mar 5.

Institute of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany.

Regulation of haematopoietic stem cell fate through conditional gene expression could improve understanding of healthy haematopoietic and leukaemia initiating cell (LIC) biology. We established conditionally immortalised myeloid progenitor cell lines co-expressing constitutive Hoxa9.EGFP and inducible Meis1.dTomato (H9M-ciMP) to study growth behaviour, immunophenotype and morphology under different cytokine/microenvironmental conditions ex vivo upon doxycycline (DOX) induction or removal. The vector design and drug-dependent selection approach identified new retroviral insertion (RVI) sites that potentially collaborate with Meis1/Hoxa9 and define H9M-ciMP fate. For most cell lines, myelomonocytic conditions supported reversible H9M-ciMP differentiation into neutrophils and macrophages with DOX-dependent modulation of Hoxa9/Meis1 and CD11b/Gr-1 expression. Here, up-regulation of Meis1/Hoxa9 promoted reconstitution of exponential expansion of immature H9M-ciMPs after DOX reapplication. Stem cell maintaining conditions supported selective H9M-ciMP exponential growth. H9M-ciMPs that had Ninj2 RVI and were cultured under myelomonocytic or stem cell maintaining conditions revealed the development of DOX-dependent acute myeloid leukaemia in a murine transplantation model. Transcriptional dysregulation of Ninj2 and distal genes surrounding RVI (Rad52, Kdm5a) was detected. All studied H9M-ciMPs demonstrated adaptation to T-lymphoid microenvironmental conditions while maintaining immature myelomonocytic features. Thus, the established system is relevant to leukaemia and stem cell biology.
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http://dx.doi.org/10.1038/s41598-021-84468-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935976PMC
March 2021

Effects of the Gestagen Levonorgestrel in a Life Cycle Test with Zebrafish (Danio rerio).

Environ Toxicol Chem 2021 Feb 4. Epub 2021 Feb 4.

Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany.

The amount of pharmaceuticals transferred to the aquatic environment via municipal and hospital waste water is steadily increasing. The progress in medical research has resulted in the manufacture of active substances of increased stability, specificity, and potency, which can trigger adverse effects in aquatic organisms. Moreover, advanced analytical methods allow the detection of pharmaceuticals in environmental matrices at very low concentrations, which increases the number of substances to be assessed. Levonorgestrel is a synthetic gestagen commonly used in medicinal products for contraception. Because progestogenic compounds could have an impact on fish maturation processes, a life cycle test was performed to assess the effects of levonorgestrel exposure of the embryonic to the adult stages of zebrafish (Danio rerio) at mean measured concentrations of 0.06, 0.16, 0.47, 1.64, and 5.45 ng/L. Apical endpoints were survival, growth, reproduction, and sex ratio. Determination of endocrine modulation was completed by measurement of vitellogenin and 11-keto testosterone in blood plasma, as well as by histopathological analysis of gonads. For all parameters, control values were within the recommended quality range. The most prominent levonorgestrel effect was a shift toward an increased number of male fish at 1.64 and especially 5.45 ng/L, at which point all fish were histologically determined to be males and no spawning occurred; 11-keto testosterone was significantly decreased. A no-observed-effect concentration (NOEC) of 0.47 ng levonorgestrel/L was confirmed by the fertilization capability of adult fish, the male maturation stages, and female gonad histopathology. Whereas hatch and juvenile growth were not affected, posthatch survival was significantly impeded at ≥0.47 ng levonorgestrel/L, although it was not clearly related to the test concentration. For male length and weight, the same NOEC of 0.16 ng/L was obtained at study termination. Environ Toxicol Chem 2021;00:1-12. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
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http://dx.doi.org/10.1002/etc.5008DOI Listing
February 2021

International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Nonproliferative and Proliferative Lesions of the Minipig.

Toxicol Pathol 2021 01;49(1):110-228

Toxicology & Pathology Consulting, LLC, Ann Arbor, MI, USA.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/inhand.asp) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in most tissues and organs from the minipig used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions as well as lesions induced by exposure to test materials. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.
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http://dx.doi.org/10.1177/0192623320975373DOI Listing
January 2021

Prenatal developmental toxicity studies on fumes from bitumen in the rat.

Reprod Toxicol 2021 01 26;99:15-26. Epub 2020 Nov 26.

Nynas AB, P.O. Box 10 700, Stockholm, SE-121 29, Sweden; Toxicology Group in CONCAWE, Belgium. Electronic address:

The prenatal developmental toxicity of bitumen fume was tested by nose-only inhalation in the rat. The fumes for exposure were collected from the headspace of a storage tank filled with a bitumen corresponding in composition to an anticipated worst-case occupational exposure. The composition of these fumes was compared to actual paving site fumes to ensure its representativeness for workplace exposures. In a dose-range-finding study male and female rats were exposed to 0, 103, 480 or 1043 mg/m of fume (as total organic mass), for 6 h/day during 20 days post conception (p.c.). Dose-related effects on body weight and lungs were observed in the mid- and high-dose groups. In the main study, dams were exposed to 0, 52, 151 and 482 mg/m of fume, for 6 h/day during 19 days p.c. The maternal NOAEL was 52 mg/m³. In the high-dose group treatment-related effects on body weight (gain), food consumption, lung weights, and histopathological changes in lungs and larynx were observed. In the mid-dose group only histopathological changes in the larynx and lungs were found. The NOAEL for prenatal developmental toxicity was 151 mg/m³ based on reduced fetal weight in the high-dose group (482 mg/m³). However, these changes are most likely a consequence of the maternal toxicity, in particular the reduction of maternal body weight gain by 26 % as compared to control. Nose-only exposure to bitumen fumes in concentrations up to 482 mg/m³ from days 1-19 p.c. did not induce any significant fetal anomalies.
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http://dx.doi.org/10.1016/j.reprotox.2020.11.009DOI Listing
January 2021

Hepatotoxic pyrrolizidine alkaloids induce DNA damage response in rat liver in a 28-day feeding study.

Arch Toxicol 2020 05 17;94(5):1739-1751. Epub 2020 May 17.

German Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589, Berlin, Germany.

Pyrrolizidine alkaloids (PA) are secondary plant metabolites that occur as food and feed contaminants. Acute and subacute PA poisoning can lead to severe liver damage in humans and animals, comprising liver pain, hepatomegaly and the development of ascites due to occlusion of the hepatic sinusoids (veno-occlusive disease). Chronic exposure to low levels of PA can induce liver cirrhosis and liver cancer. However, it is not well understood which transcriptional changes are induced by PA and whether all hepatotoxic PA, regardless of their structure, induce similar responses. Therefore, a 28-day subacute rat feeding study was performed with six structurally different PA heliotrine, echimidine, lasiocarpine, senecionine, senkirkine, and platyphylline, administered at not acutely toxic doses from 0.1 to 3.3 mg/kg body weight. This dose range is relevant for humans, since consumption of contaminated tea may result in doses of ~ 8 µg/kg in adults and cases of PA ingestion by contaminated food was reported for infants with doses up to 3 mg/kg body weight. ALT and AST were not increased in all treatment groups. Whole-genome microarray analyses revealed pronounced effects on gene expression in the high-dose treatment groups resulting in a set of 36 commonly regulated genes. However, platyphylline, the only 1,2-saturated and, therefore, presumably non-hepatotoxic PA, did not induce significant expression changes. Biological functions identified to be affected by high-dose treatments (3.3 mg/kg body weight) comprise cell-cycle regulation associated with DNA damage response. These functions were found to be affected by all analyzed 1,2-unsaturated PA.In conclusion, 1,2-unsaturated hepatotoxic PA induced cell cycle regulation processes associated with DNA damage response. Similar effects were observed for all hepatotoxic PA. Effects were observed in a dose range inducing no histopathological alterations and no increase in liver enzymes. Therefore, transcriptomics studies identified changes in expression of genes known to be involved in response to genotoxic compounds at PA doses relevant to humans under worst case exposure scenarios.
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http://dx.doi.org/10.1007/s00204-020-02779-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261731PMC
May 2020

Fate of Barium Sulfate Nanoparticles Deposited in the Lungs of Rats.

Sci Rep 2019 06 3;9(1):8163. Epub 2019 Jun 3.

Molecular and Integrative Physiological Sciences Program, Department of Environmental Health, Harvard T.H. Chan School of Public Health, 665 Huntington Avenue, Boston, MA, 02115, USA.

We have shown that barium [from BaSO nanoparticles (NPs)] was cleared from the lungs faster than other poorly soluble NPs and translocated mostly to bone. We now studied barium biokinetics in rats during Study 1: two-year inhalation exposure to 50 mg/m BaSO NP aerosols, and Study 2: single intratracheal (IT) instillation of increasing doses of BaSO NPs or BaCl. Study 1 showed that lung barium content measured by inductively coupled plasma mass spectrometry increased during 360 days of BaSO NP aerosol exposures. An equilibrium was established from that time until 2 years. Barium concentrations in BaSO-exposed animals were in the order (lungs > lymph nodes > hard bone > bone marrow > liver). In Study 2, there was an increase in lung barium post-IT instillation of BaSO NPs while barium from BaCl was mostly cleared by day 28. Transmission electron microscopy showed intact BaSO NPs in alveolar macrophages and type II epithelial cells, and in tracheobronchial lymph nodes. Using stimulated Raman scattering microscopy, specific BaSO Raman spectra were detected in BaSO NP-instilled lungs and not in other organs. Thus, we posit that barium from BaSO NPs translocates from the lungs mainly after dissolution. Barium ions are then incorporated mostly into the bone and other organs.
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http://dx.doi.org/10.1038/s41598-019-44551-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546789PMC
June 2019

Signatures of T and B Cell Development, Functional Responses and PD-1 Upregulation After HCMV Latent Infections and Reactivations in Nod.Rag.Gamma Mice Humanized With Cord Blood CD34 Cells.

Front Immunol 2018 22;9:2734. Epub 2018 Nov 22.

Clinic of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.

Human cytomegalovirus (HCMV) latency is typically harmless but reactivation can be largely detrimental to immune compromised hosts. We modeled latency and reactivation using a traceable HCMV laboratory strain expressing the luciferase reporter gene (HCMV/GLuc) in order to interrogate the viral modulatory effects on the human adaptive immunity. Humanized mice with long-term (more than 17 weeks) steady human T and B cell immune reconstitutions were infected with HCMV/GLuc and 7 weeks later were further treated with granulocyte-colony stimulating factor (G-CSF) to induce viral reactivations. Whole body bio-luminescence imaging analyses clearly differentiated mice with latent viral infections vs. reactivations. of vigorous viral reactivations were detectable in liver, lymph nodes and salivary glands. The number of viral genome copies in various tissues increased upon reactivations and were detectable in sorted human CD14, CD169, and CD34 cells. Compared with non-infected controls, mice after infections and reactivations showed higher thymopoiesis, systemic expansion of Th, CTL, Treg, and Tfh cells and functional antiviral T cell responses. Latent infections promoted vast development of memory CD4 T cells while reactivations triggered a shift toward effector T cells expressing PD-1. Further, reactivations prompted a marked development of B cells, maturation of IgG plasma cells, and HCMV-specific antibody responses. Multivariate statistical methods were employed using T and B cell immune phenotypic profiles obtained with cells from several tissues of individual mice. The data was used to identify combinations of markers that could predict an HCMV infection vs. reactivation status. In spleen, but not in lymph nodes, higher frequencies of effector CD4 T cells expressing PD-1 were among the factors most suited to distinguish HCMV reactivations from infections. These results suggest a shift from a T cell dominated immune response during latent infections toward an exhausted T cell phenotype and active humoral immune response upon reactivations. In sum, this novel humanized model combined with advanced analyses highlights a dynamic system clearly specifying the immunological spatial signatures of HCMV latency and reactivations. These signatures can be merged as predictive biomarker clusters that can be applied in the clinical translation of new therapies for the control of HCMV reactivation.
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http://dx.doi.org/10.3389/fimmu.2018.02734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6262073PMC
October 2019

Digital Microscopy, Image Analysis, and Virtual Slide Repository.

ILAR J 2018 12;59(1):66-79

Famke Aeffner, DVM PhD DACVP, is a principal pathologist in the Comparative Biology and Safety Sciences Department at Amgen Inc. in South San Francisco, California. Hibret Adissu, DVM PhD DVSc DACVP, is an investigative pathologist in the Laboratory of Cancer Biology and Genetics, Center for Cancer Research, at the National Cancer Institute in Bethesda, Maryland. Michael C. Boyle, DVM PhD DACVP DABT, is a principal pathologist in the Comparative Biology and Safety Sciences at Amgen Inc. in Thousand Oaks, California. Robert D. Cardiff, MD PhD, is a distinguished professor of pathology (emeritus) at the Center for Comparative Medicine at the University of California in Davis, California. Erik Hagendorn is a senior scientist of informatics at AbbVie Bioresearch in Worcester, Massachusetts. Mark J. Hoenerhoff, DVM PhD DACVP, is an associate professor and veterinary pathologist at the In Vivo Animal Core, Unit for Laboratory Animal Medicine, at the University of Michigan in Ann Arbor, Michigan. Robert Klopfleisch, DVM PhD DACVP, is an associate professor at the Institute of Veterinary Pathology of the Freie Universitaet Berlin, in Berlin, Germany. Susan Newbigging, BSc MSc DVM DVSc, is a pathologist and Director of The Pathology Core at the Toronto Center of Phenogenomics in Toronto, Ontario, Canada. Dirk Schaudien, DVM PhD DACVP, is a veterinary pathologist at the Fraunhofer Institute for Toxicology and Experimental Medicine, in Hannover, Germany. Oliver Turner, BSC(Hons), BVSc MRCVS PhD DACVP DABT, is a senior pathologist in the Preclinical Safety department of Novartis Pharmaceuticals in East Hanover, New Jersey. Kristin Wilson, DVM PhD DACVP, is a pathologist at Flagship Biosciences Inc. in Westminster, Colorado.

Advancements in technology and digitization have ushered in novel ways of enhancing tissue-based research via digital microscopy and image analysis. Whole slide imaging scanners enable digitization of histology slides to be stored in virtual slide repositories and to be viewed via computers instead of microscopes. Easier and faster sharing of histologic images for teaching and consultation, improved storage and preservation of quality of stained slides, and annotation of features of interest in the digital slides are just a few of the advantages of this technology. Combined with the development of software for digital image analysis, digital slides further pave the way for the development of tools that extract quantitative data from tissue-based studies. This review introduces digital microscopy and pathology, and addresses technical and scientific considerations in slide scanning, quantitative image analysis, and slide repositories. It also highlights the current state of the technology and factors that need to be taken into account to insure optimal utility, including preanalytical considerations and the importance of involving a pathologist in all major steps along the digital microscopy and pathology workflow.
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http://dx.doi.org/10.1093/ilar/ily007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927898PMC
December 2018

Matrix metalloproteinases expression in spontaneous canine histiocytic sarcomas and its xenograft model.

Vet Immunol Immunopathol 2018 Apr 14;198:54-64. Epub 2018 Mar 14.

Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, 30559, Hannover, Germany. Electronic address:

Canine histiocytic sarcoma (HS) represents a malignant neoplastic disorder often with a rapid and progressive clinical course. A better understanding of the interaction between tumor cells and the local microenvironment may provide new insights into mechanisms of tumor growth and metastasis. The influence of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) on tumor angiogenesis, invasion and metastasis has been detailed in previous studies. In addition, inflammatory cells infiltrating neoplasms especially tumor associated macrophages (TAM) may contribute significantly to tumor progression. Due to the high variability of spontaneously occurring canine HS, standardized models are highly required to investigate tumor progression and interaction with its microenvironment. Therefore, the present study comparatively characterized the intratumoral macrophage infiltration as well as the expression of MMP-2, MMP-9, MMP-14 and TIMP-1 in spontaneous canine HS and its murine model. In spontaneous canine HS, scattered MAC 387-positive macrophages were randomly found in tumor center and periphery, whereas tumor cells were negative for this marker. Interestingly, quantitative analysis revealed that MMPs and TIMP-1 were mainly expressed at the invasive front while tumor centers exhibited significantly reduced immunoreactivity. Similar findings were obtained in xenotransplanted HS. Interestingly, murine tumor associated macrophages (TAM), characterized by Mac3 expression (CD107b/LAMP2), which was not present in xenotransplanted histiocytic sarcoma cells, strongly express MMPs and TIMP-1. In addition, MMPs are known to regulate angiogenesis and a positive correlation between MMP-14 expression and microvessel density was demonstrated in xenotransplanted histiocytic sarcomas. Summarized similar findings with respect to MMP and TIMP distribution and the role of macrophages in spontaneously-occurring and xenotransplanted HS indicate the high suitability of this murine model to further investigate HS under standardized conditions. Moreover results indicate that MMP expression contributes to tumor progression and invasion and TAMs seem to be major players in the interaction between neoplastic cells, the microenvironment and vessel formation indicating that therapeutic approaches modulating TAM associated molecules might represent promising future treatment options.
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http://dx.doi.org/10.1016/j.vetimm.2018.03.002DOI Listing
April 2018

Cerium oxide and barium sulfate nanoparticle inhalation affects gene expression in alveolar epithelial cells type II.

J Nanobiotechnology 2018 Feb 20;16(1):16. Epub 2018 Feb 20.

Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Nikolai-Fuchs-Straße 1, 30625, Hannover, Germany.

Background: Understanding the molecular mechanisms of nanomaterial interacting with cellular systems is important for appropriate risk assessment. The identification of early biomarkers for potential (sub-)chronic effects of nanoparticles provides a promising approach towards cost-intensive and animal consuming long-term studies. As part of a 90-day inhalation toxicity study with CeO NM-212 and BaSO NM-220 the present investigations on gene expression and immunohistochemistry should reveal details on underlying mechanisms of pulmonary effects. The role of alveolar epithelial cells type II (AEII cells) is focused since its contribution to defense against inhaled particles and potentially resulting adverse effects is assumed. Low dose levels should help to specify particle-related events, including inflammation and oxidative stress.

Results: Rats were exposed to clean air, 0.1, 0.3, 1.0, and 3.0 mg/m CeO NM-212 or 50.0 mg/m BaSO NM-220 and the expression of 391 genes was analyzed in AEII cells after one, 28 and 90 days exposure. A total number of 34 genes was regulated, most of them related to inflammatory mediators. Marked changes in gene expression were measured for Ccl2, Ccl7, Ccl17, Ccl22, Ccl3, Ccl4, Il-1α, Il-1ß, and Il-1rn (inflammation), Lpo and Noxo1 (oxidative stress), and Mmp12 (inflammation/lung cancer). Genes related to genotoxicity and apoptosis did not display marked regulation. Although gene expression was less affected by BaSO compared to CeO the gene pattern showed great overlap. Gene expression was further analyzed in liver and kidney tissue showing inflammatory responses in both organs and marked downregulation of oxidative stress related genes in the kidney. Increases in the amount of Ce were measured in liver but not in kidney tissue. Investigation of selected genes on protein level revealed increased Ccl2 in bronchoalveolar lavage of exposed animals and increased Lpo and Mmp12 in the alveolar epithelia.

Conclusion: AEII cells contribute to CeO nanoparticle caused inflammatory and oxidative stress reactions in the respiratory tract by the release of related mediators. Effects of BaSO exposure are low. However, overlap between both substances were detected and support identification of potential early biomarkers for nanoparticle effects on the respiratory system. Signs for long-term effects need to be further evaluated by comparison to a respective exposure setting.
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http://dx.doi.org/10.1186/s12951-018-0343-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819288PMC
February 2018

Influence of modified atmosphere and vacuum packaging with and without nanosilver-coated films on different quality parameters of pork.

J Food Sci Technol 2017 Sep 8;54(10):3251-3259. Epub 2017 Aug 8.

Institute of Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany.

Pork is often marketed in packages with high oxygen atmosphere (MAP) or vacuum to improve shelf life and appearance. As silver ions have antibacterial effects, food contact films coated with silver might improve the shelf life of meat. In the present study, pork was wrapped in commercially available films, coated with nanosilver particles, and stored in the two packaging variants MAP and vacuum for 12 days. During storage, samples were analyzed on days 1 (before packaging), 4, 8 and 12 for microbiological contamination, meat quality (e.g., pH, color), and for the percentages of the myoglobin (Mb) redox forms. In addition, the effects of the film were examined after inoculation of the meat with high quantities of methicillin-resistant (MRSA) cells before vacuum storage for 8 days. MAP storage resulted in higher lightness (L*) values, lower liquid loss and higher Mb oxidation compared to vacuum. Microbiological spoilage was partly affected by the packaging variants with reducing effects of the MAP. The nanosilver-coating only affects the Mb redox form percentages of the pork cutlets and on day 4 the L* values, whereas microbiological parameters were not influenced. As the nanosilver coating had no influence on the total viable bacteria counts as well as spp., and MRSA counts, an advantage of the nanosilver coating on the shelf life could be excluded.
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http://dx.doi.org/10.1007/s13197-017-2768-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602988PMC
September 2017

Effects from a 90-day inhalation toxicity study with cerium oxide and barium sulfate nanoparticles in rats.

Part Fibre Toxicol 2017 07 12;14(1):23. Epub 2017 Jul 12.

Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Nikolai-Fuchs-Straße 1, 30625, Hannover, Germany.

Background: Nanomaterials like cerium oxide and barium sulfate are frequently processed in industrial and consumer products and exposure of humans and other organisms is likely. Generally less information is given on health effects and toxicity, especially regarding long-term exposure to low nanoparticle doses. Since inhalation is still the major route of uptake the present study focused on pulmonary effects of CeONM-212 (0.1, 0.3, 1.0, 3.0 mg/m) and BaSONM-220 nanoparticles (50.0 mg/m) in a 90-day exposure setup. To define particle-related effects and potential mechanisms of action, observations in histopathology, bronchoalveolar lavage and immunohistochemistry were linked to pulmonary deposition and clearance rates. This further allows evaluation of potential overload related effects.

Results: Lung burden values increased with increasing nanoparticle dose levels and ongoing exposure. At higher doses, cerium clearance was impaired, suggesting lung overload. Barium elimination was extremely rapid and without any signs of overload. Bronchoalveolar lavage fluid analysis and histopathology revealed lung tissue inflammation with increasing severity and post-exposure persistency for CeO. Also, marker levels for genotoxicity and cell proliferation were significantly increased. BaSO showed less inflammation or persistency of effects and particularly affected the nasal cavity.

Conclusion: CeO nanoparticles penetrate the alveolar space and affect the respiratory tract after inhalation mainly in terms of inflammation. Effects at low dose levels and post-exposure persistency suggest potential long-term effects and a notable relevance for human health. The generated data might be useful to improve nanoparticle risk assessment and threshold value generation. Mechanistic investigations at conditions of non-overload and absent inflammation should be further investigated in future studies.
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http://dx.doi.org/10.1186/s12989-017-0204-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508701PMC
July 2017

Translation of Angiotensin-Converting Enzyme 2 upon Liver- and Lung-Targeted Delivery of Optimized Chemically Modified mRNA.

Mol Ther Nucleic Acids 2017 Jun 13;7:350-365. Epub 2017 Apr 13.

Ethris GmbH, 82152 Planegg, Germany. Electronic address:

Changes in lifestyle and environmental conditions give rise to an increasing prevalence of liver and lung fibrosis, and both have a poor prognosis. Promising results have been reported for recombinant angiotensin-converting enzyme 2 (ACE2) protein administration in experimental liver and lung fibrosis. However, the full potential of ACE2 may be achieved by localized translation of a membrane-anchored form. For this purpose, we advanced the latest RNA technology for liver- and lung-targeted ACE2 translation. We demonstrated in vitro that transfection with ACE2 chemically modified messenger RNA (cmRNA) leads to robust translation of fully matured, membrane-anchored ACE2 protein. In a second step, we designed eight modified ACE2 cmRNA sequences and identified a lead sequence for in vivo application. Finally, formulation of this ACE2 cmRNA in tailor-made lipidoid nanoparticles and in lipid nanoparticles led to liver- and lung-targeted translation of significant amounts of ACE2 protein, respectively. In summary, we provide evidence that RNA transcript therapy (RTT) is a promising approach for ACE2-based treatment of liver and lung fibrosis to be tested in fibrotic disease models.
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http://dx.doi.org/10.1016/j.omtn.2017.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423349PMC
June 2017

Assessment of long-term cultivated human precision-cut lung slices as an ex vivo system for evaluation of chronic cytotoxicity and functionality.

J Occup Med Toxicol 2017 26;12:13. Epub 2017 May 26.

Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Centre for Lung Research (DZL), Member of the REBIRTH Cluster of Excellence, Hanover, Germany.

Background: Investigation of basic chronic inflammatory mechanisms and development of new therapeutics targeting the respiratory tract requires appropriate testing systems, including those to monitor long- persistence. Human precision-cut lung slices (PCLS) have been demonstrated to mimic the human respiratory tract and have potential of an alternative, ex-vivo system to replace or augment in-vitro testing and animal models. So far, most research on PCLS has been conducted for short cultivation periods (≤72 h), while analyses of slowly metabolized therapeutics require long-term survival of PCLS in culture. In the present study, we evaluated viability, physiology and structural integrity of PCLS cultured for up to 15 days.

Methods: PCLS were cultured for 15 days and various parameters were assessed at different time points.

Results: Structural integrity and viability of cultured PCLS remained constant for 15 days. Moreover, bronchoconstriction was inducible over the whole period of cultivation, though with decreased sensitivity (EC1d = 4 × 10 M vs. EC15d = 4 × 10 M) and reduced maximum of initial airway area (1d = 0.5% vs. 15d = 18.7%). In contrast, even though still clearly inducible compared to medium control, LPS-induced TNF-α secretion decreased significantly from day 1 to day 15 of culture.

Conclusions: Overall, though long-term cultivation of PCLS need further investigation for cytokine secretion, possibly on a cellular level, PCLS are feasible for bronchoconstriction studies and toxicity assays.
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http://dx.doi.org/10.1186/s12995-017-0158-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446749PMC
May 2017

Maintenance of high quality rat precision cut liver slices during culture to study hepatotoxic responses: Acetaminophen as a model compound.

Toxicol In Vitro 2017 Aug 2;42:200-213. Epub 2017 May 2.

Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Member of the German Center for Lung Research (DZL), Biomedical Research in End Stage and Obstructive Lung Disease (BREATH) Research Network, Member of the Cluster of Excellence Regenerative Biology to Reconstructive Therapy (REBIRTH), Nikolai-Fuchs-Straße 1, 30625 Hannover, Germany. Electronic address:

Precision cut liver slices (PCLiS) represent a promising tool in reflecting hepatotoxic responses. However, the culture of PCLiS varies considerably between laboratories, which can affect the performance of the liver slices and thus the experimental outcome. In this study, we describe an easily accessible culture method, which ensures optimal slice viability and functionality, in order to set the basis for reproducible and comparable PCLiS studies. The quality of the incubated rat PCLiS was assessed during a 24h culture period using ten readouts, which covered viability (lactate dehydrogenase-, aspartate transaminase- and glutamate dehydrogenase-leakage, ATP content) and functionality parameters (urea, albumin production) as well as histomorphology and other descriptive characteristics (protein content, wet weight, slice thickness). The present culture method resulted in high quality liver slices for 24h. Finally, PCLiS were exposed to increasing concentrations of acetaminophen to assess the suitability of the model for the detection of hepatotoxic responses. Six out of ten readouts revealed a toxic effect and showed an excellent mutual correlation. ATP, albumin and histomorphology measurements were identified as the most sensitive readouts. In conclusion, our results indicate that rat PCLiS are a valuable liver model for hepatotoxicity studies, particularly if they are cultured under optimal standardized conditions.
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http://dx.doi.org/10.1016/j.tiv.2017.05.001DOI Listing
August 2017

Human Effector Memory T Helper Cells Engage with Mouse Macrophages and Cause Graft-versus-Host-Like Pathology in Skin of Humanized Mice Used in a Nonclinical Immunization Study.

Am J Pathol 2017 Jun 20;187(6):1380-1398. Epub 2017 Apr 20.

Regenerative Biology to Reconstructive Therapies (REBIRTH), Laboratory of Regenerative Immune Therapies Applied, Hannover Medical School, Hannover, Germany; Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany. Electronic address:

Humanized mice engrafted with human hematopoietic stem cells and developing functional human T-cell adaptive responses are in critical demand to test human-specific therapeutics. We previously showed that humanized mice immunized with long-lived induced-dendritic cells loaded with the pp65 viral antigen (iDCpp65) exhibited a faster development and maturation of T cells. Herein, we evaluated these effects in a long-term (36 weeks) nonclinical model using two stem cell donors to assess efficacy and safety. Relative to baseline, iDCpp65 immunization boosted the output of effector memory CD4 T cells in peripheral blood and lymph nodes. No weight loss, human malignancies, or systemic graft-versus-host (GVH) disease were observed. However, for one reconstitution cohort, some mice immunized with iDCpp65 showed GVH-like signs on the skin. Histopathology analyses of the inflamed skin revealed intrafollicular and perifollicular human CD4 cells near F4/80 mouse macrophages around hair follicles. In spleen, CD4 cells formed large clusters surrounded by mouse macrophages. In plasma, high levels of human T helper 2-type inflammatory cytokines were detectable, which activated in vitro the STAT5 pathway of murine macrophages. Despite this inflammatory pattern, human CD8 T cells from mice with GVH reacted against the pp65 antigen in vitro. These results uncover a dynamic cross-species interaction between human memory T cells and mouse macrophages in the skin and lymphatic tissues of humanized mice.
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http://dx.doi.org/10.1016/j.ajpath.2017.02.015DOI Listing
June 2017

Viral Infection of the Central Nervous System Exacerbates Interleukin-10 Receptor Deficiency-Mediated Colitis in SJL Mice.

PLoS One 2016 9;11(9):e0161883. Epub 2016 Sep 9.

Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany.

Theiler´s murine encephalomyelitis virus (TMEV)-infection is a widely used animal model for studying demyelinating disorders, including multiple sclerosis (MS). The immunosuppressive cytokine Interleukin (IL)-10 counteracts hyperactive immune responses and critically controls immune homeostasis in infectious and autoimmune disorders. In order to investigate the effect of signaling via Interleukin-10 receptor (IL-10R) in infectious neurological diseases, TMEV-infected SJL mice were treated with IL-10R blocking antibody (Ab) in the acute and chronic phase of the disease. The findings demonstrate that (i) Ab-mediated IL-10 neutralization leads to progressive colitis with a reduction in Foxp3+ regulatory T cells and increased numbers of CD8+CD44+ memory T cells as well as activated CD4+CD69+ and CD8+CD69+ T cells in uninfected mice. (ii) Concurrent acute TMEV-infection worsened enteric disease-mediated by IL-10R neutralization. Virus-triggered effects were associated with an enhanced activation of CD4+ T helper cells and CD8+ cytotoxic T lymphocytes and augmented cytokine expression. By contrast, (iii) IL-10R neutralization during chronic TMEV-infection was not associated with enhanced peripheral immunopathology but an increased CD3+ T cell influx in the spinal cord. IL-10R neutralization causes a breakdown in peripheral immune tolerance in genetically predisposed mice, which leads to immune-mediated colitis, resembling inflammatory bowel disease. Hyperactive immune state following IL-10R blockade is enhanced by central nervous system-restricted viral infection in a disease phase-dependent manner.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161883PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5017624PMC
August 2017

Translocation and biokinetic behavior of nanoscaled europium oxide particles within 5 days following an acute inhalation in rats.

J Appl Toxicol 2016 Mar 20;36(3):474-8. Epub 2015 Nov 20.

Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany.

Nanoscaled europium oxide (Eu2O3) particles were inhaled by rats after acute exposure and the potential translocation of particles followed by chemical analysis and transmission electron microscopy (TEM) was investigated. An aqueous dispersion (phosphate buffer/bovine serum albumin) of a commercially available Eu2O3 particle fraction consisting partially of nanoscaled particles was aerosolized with pressurized air. After rapid evaporation, rats inhaled the dry aerosol for 6 h in a single exposure resulting in an alveolar calculated dose of approximately 39.5 μg Eu2O3. Using chemical analysis, 36.8 μg Eu2O3 was detected 1 h after lung inhalation. The amount declined slightly to 34.5 μg after 1 day and 35.0 μg after 5 days. The liver showed an increase of Eu2O3 from 32.3 ng 1 h up to 294 ng 5 days after inhalation. Additionally, lung-associated lymph nodes, thymus, kidneys, heart and testis exhibited an increase of europium over the period investigated. In the blood, the highest amount of europium was found 1 h after treatment whereas feces, urine and mesenteric lymph nodes revealed the highest amount 1 day after treatment. Using TEM analysis, particles could be detected only in lungs, and in the liver, no particles were detectable. In conclusion, the translocation of Eu2O3 within 5 days following inhalation could be determined very precisely by chemical analysis. A translocation of Eu2O3 particulate matter to liver was not detectable by TEM analysis; thus, the overproportional level of 0.8% of the lung load observed in the liver after 5 days suggests a filtering effect of dissolved europium with accumulation.
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http://dx.doi.org/10.1002/jat.3259DOI Listing
March 2016

The carcinogenic effect of various multi-walled carbon nanotubes (MWCNTs) after intraperitoneal injection in rats.

Part Fibre Toxicol 2014 Nov 20;11:59. Epub 2014 Nov 20.

Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Nikolai-Fuchs-Str. 1, 30625, Hannover, Germany.

Background: Biological effects of tailor-made multi-walled carbon nanotubes (MWCNTs) without functionalization were investigated in vivo in a two-year carcinogenicity study. In the past, intraperitoneal carcinogenicity studies in rats using biopersistent granular dusts had always been negative, whereas a number of such studies with different asbestos fibers had shown tumor induction. The aim of this study was to identify possible carcinogenic effects of MWCNTs. We compared induced tumors with asbestos-induced mesotheliomas and evaluated their relevance for humans by immunohistochemical methods.

Methods: A total of 500 male Wistar rats (50 per group) were treated once by intraperitoneal injection with 10⁹ or 5 × 10⁹ WHO carbon nanotubes of one of four different MWCNTs suspended in artificial lung medium, which was also used as negative control. Amosite asbestos (10⁸ WHO fibers) served as positive control. Morbid rats were sacrificed and necropsy comprising all organs was performed. Histopathological classification of tumors and, additionally, immunohistochemistry were conducted for podoplanin, pan-cytokeratin, and vimentin to compare induced tumors with malignant mesotheliomas occurring in humans.

Results: Treatments induced tumors in all dose groups, but incidences and times to tumor differed between groups. Most tumors were histologically and immunohistochemically classified as malignant mesotheliomas, revealing a predominantly superficial spread on the serosal surface of the abdominal cavity. Furthermore, most tumors showed invasion of peritoneal organs, especially the diaphragm. All tested MWCNT types caused mesotheliomas. We observed highest frequencies and earliest appearances after treatment with the rather straight MWCNT types A and B. In the MWCNT C groups, first appearances of morbid mesothelioma-bearing rats were only slightly later. Later during the two-year study, we found mesotheliomas also in rats treated with MWCNT D - the most curved type of nanotubes. Malignant mesotheliomas induced by intraperitoneal injection of different MWCNTs and of asbestos were histopathologically and immunohistochemically similar, also compared with mesotheliomas in man, suggesting similar pathogenesis.

Conclusion: We showed a carcinogenic effect for all tested MWCNTs. Besides aspect ratio, curvature seems to be an important parameter influencing the carcinogenicity of MWCNTs.
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http://dx.doi.org/10.1186/s12989-014-0059-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243371PMC
November 2014

Pathogen reduction by ultraviolet C light effectively inactivates human white blood cells in platelet products.

Transfusion 2015 Feb 19;55(2):337-47. Epub 2014 Aug 19.

German Red Cross Blood Service NSTOB, Institute Springe, Springe, Germany.

Background: Residual white blood cells (WBCs) in cellular blood components induce a variety of adverse immune events, including nonhemolytic febrile transfusion reactions, alloimmunization to HLA antigens, and transfusion-associated graft-versus-host disease (TA-GVHD). Pathogen reduction (PR) methods such as the ultraviolet C (UVC) light-based THERAFLEX UV-Platelets system were developed to reduce the risk of transfusion-transmitted infection. As UVC light targets nucleic acids, it interferes with the replication of both pathogens and WBCs. This preclinical study aimed to evaluate the ability of UVC light to inactivate contaminating WBCs in platelet concentrates (PCs).

Study Design And Methods: The in vitro and in vivo function of WBCs from UVC-treated PCs was compared to that of WBCs from gamma-irradiated and untreated PCs by measuring cell viability, proliferation, cytokine secretion, antigen presentation in vitro, and xenogeneic GVHD responses in a humanized mouse model.

Results: UVC light was at least as effective as gamma irradiation in preventing GVHD in the mouse model. It was more effective in suppressing T-cell proliferation (>5-log reduction in the limiting dilution assay), cytokine secretion, and antigen presentation than gamma irradiation.

Conclusions: The THERAFLEX UV-Platelets (MacoPharma) PR system can substitute gamma irradiation for TA-GVHD prophylaxis in platelet (PLT) transfusion. Moreover, UVC treatment achieves suppression of antigen presentation and inhibition of cytokine accumulation during storage of PCs, which has potential benefits for transfusion recipients.
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http://dx.doi.org/10.1111/trf.12836DOI Listing
February 2015

Nanoparticles and pop-off technique for electron microscopy: a known technique for a new purpose.

Toxicol Pathol 2014 Aug 6;42(6):1041-6. Epub 2014 May 6.

Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany

Because of the size of the nanoparticles, their detection and exact anatomical localization in tissue samples are very difficult. Consequently, suitable methods are needed to prove their presence, especially co-localized to histological lesions. Therefore, the aim of this study was to investigate whether nanoparticles were detectable in specimens after reprocessing samples from glass slides using the pop-off technique. Tissue slides containing agglomerates of titanium dioxide nanoparticles already visible on a light microscopic level and amorphous silicium dioxide (SiO2) particles not observable in tissue slides were reprocessed. Furthermore, cytospots of bronchoalveolar lavage acquired from rats that previously inhaled carbon nanotubes were used. After reprocessing the samples, they were investigated using transmission electron microscopy. In all the reprocessed samples, the respective nanoparticles were detectable. Even the light microscopically invisible amorphous SiO2 particles were observed as electron dense structures. Titanium and silicium were additionally confirmed in the respective nanoparticles by energy-dispersive X-ray spectroscopy (EDX). In summary, the pop-off technique represents a fast and easy way to detect nanoparticles in histological sections. This enables further characterization of these particles by additional techniques such as EDX, and their direct correlation with light microscopic lesions at exactly the same location is investigated.
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http://dx.doi.org/10.1177/0192623313509906DOI Listing
August 2014

Change in agglomeration status and toxicokinetic fate of various nanoparticles in vivo following lung exposure in rats.

Inhal Toxicol 2012 Oct;24(12):821-30

Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany.

The deposition characteristics in lungs following inhalation, the potential toxic effects induced and the toxicokinetic fate including a possible translocation to other sites of the body are predominantly determined by the agglomeration status of nanoscaled primary particles. Systemic particle effects, i.e. effects on remote organs besides the respiratory tract are considered to be of relevant impact only for de-agglomerated particles with a nanoscaled aspect. Rats were exposed to various types of nanoscaled particles, i.e. titanium dioxide, carbon black and constantan. These were dispersed in physiologically compatible media, e.g. phosphate buffer, sometimes including auxiliaries. Rats were treated with aqueous nanoparticle dispersions by intratracheal instillation or were exposed to well-characterized nanoparticle aerosols. Subsequently, alterations in the particle size distribution were studied using transmission electron microscopy (TEM) as well as the bronchoalveolar lavage (BAL) technique. Based on the results in various approaches, a tendency of nanoscaled particles to form larger size agglomerates following deposition and interaction with cells or the respiratory tract is predominant. The contrary trend, i.e. the increase of particle number due to a disintegration of agglomerates seems not to be of high relevance.
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http://dx.doi.org/10.3109/08958378.2012.721097DOI Listing
October 2012

TNF-overexpression in Borna disease virus-infected mouse brains triggers inflammatory reaction and epileptic seizures.

PLoS One 2012 25;7(7):e41476. Epub 2012 Jul 25.

Department of Pathology, University of Veterinary Medicine, Hannover, Germany.

Proinflammatory state of the brain increases the risk for seizure development. Neonatal Borna disease virus (BDV)-infection of mice with neuronal overexpression of tumor necrosis factor-α (TNF) was used to investigate the complex relationship between enhanced cytokine levels, neurotropic virus infection and reaction pattern of brain cells focusing on its role for seizure induction. Viral antigen and glial markers were visualized by immunohistochemistry. Different levels of TNF in the CNS were provided by the use of heterozygous and homozygous TNF overexpressing mice. Transgenic TNF, total TNF (native and transgenic), TNF-receptor (TNFR1, TNFR2), IL-1 and N-methyl-D-aspartate (NMDA)-receptor subunit 2B (NR2B) mRNA values were measured by real time RT-PCR. BDV-infection of TNF-transgenic mice resulted in non-purulent meningoencephalitis accompanied by epileptic seizures with a higher frequency in homozygous animals. This correlated with lower weight gain, stronger degree and progression of encephalitis and early, strong microglia activation in the TNF-transgenic mice, most obviously in homozygous animals. Activation of astroglia could be more intense and associated with an unusual hypertrophy in the transgenic mice. BDV-antigen distribution and infectivity in the CNS was comparable in TNF-transgenic and wild-type animals. Transgenic TNF mRNA-expression was restricted to forebrain regions as the transgene construct comprised the promoter of NMDA-receptor subunit2B and induced up-regulation of native TNF mRNA. Total TNF mRNA levels did not increase significantly after BDV-infection in the brain of transgenic mice but TNFR1, TNFR2 and IL-1 mRNA values, mainly in the TNF overexpressing brain areas. NR2B mRNA levels were not influenced by transgene expression or BDV-infection. Neuronal TNF-overexpression combined with BDV-infection leads to cytokine up-regulation, CNS inflammation and glial cell activation and confirmed the presensitizing effect of elevated cytokine levels for the development of spontaneous epileptic seizures when exposed to additional infectious noxi.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041476PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405098PMC
March 2013

Impacts after inhalation of nano- and fine-sized titanium dioxide particles: morphological changes, translocation within the rat lung, and evaluation of particle deposition using the relative deposition index.

Inhal Toxicol 2012 Aug 19;24(9):557-69. Epub 2012 Jul 19.

Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Germany.

A 3-week inhalation study with nano- and fine-sized titanium dioxide (TiO(2)) with 3, 28, and 90 days recovery time was performed in female Wistar rats. Lung volume measurements, histology, electron microscopy, hematology, and bronchoalveolar lavage (BAL) fluid analyses were conducted and the relative deposition index (RDI) was calculated. Minimal inflammatory changes in the lungs, leucopenia, and a decrease in β-glucuronidase were observed. Particles were mainly deposited in alveolar macrophages and, to a lesser extent, in type-I pneumocytes, and this was quantified using the RDI. Rarely, particle-laden cells were observed inside capillaries. Therefore, minimal translocation of particles into the bloodstream has to be considered. Significant changes, e.g. in elicited effects or translocation behavior, between nano- and fine-particle-treated groups were not observed.
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http://dx.doi.org/10.3109/08958378.2012.697494DOI Listing
August 2012

Interleukin-10 expression during the acute phase is a putative prerequisite for delayed viral elimination in a murine model for multiple sclerosis.

J Neuroimmunol 2012 Aug 14;249(1-2):27-39. Epub 2012 May 14.

Department of Pathology, University of Veterinary Medicine Hannover, Bünteweg 17, Hannover, Germany.

Reduced protective immunity leads to viral persistence and demyelination in Theiler's murine encephalomyelitis. The aim of the present study was to compare the phenotype of brain-infiltrating leukocytes and cytokine expression in susceptible SJL and resistant C57BL/6 mice during Theilervirus-induced acute polioencephalitis. In contrast to C57/BL6 mice, SJL mice show an increased number of Foxp3(+) regulatory T cells and CD45R(+) B cells associated with delayed viral elimination and elevated IL-10 mRNA transcripts in the brain. Results substantiate the hypothesis that an imbalanced cytokine milieu during the early infection phase contributes to ineffective antiviral immunity in animals with a susceptible genetic background.
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http://dx.doi.org/10.1016/j.jneuroim.2012.04.010DOI Listing
August 2012