Publications by authors named "Graham Brogden"

27 Publications

  • Page 1 of 1

Ketogenic Diet: Impact on Cellular Lipids in Hippocampal Murine Neurons.

Nutrients 2020 Dec 18;12(12). Epub 2020 Dec 18.

Clinic for Pediatric Kidney, Liver, and Metabolic Diseases, Hannover Medical School, Carl-Neuberg Str. 1, 30625 Hannover, Germany.

Background: The mechanism of action of the ketogenic diet (KD), an effective treatment for pharmacotherapy refractory epilepsy, is not fully elucidated. The present study examined the effects of two metabolites accumulating under KD-beta-hydroxybutyrate (ßHB) and decanoic acid (C10) in hippocampal murine (HT22) neurons.

Methods: A mouse HT22 hippocampal neuronal cell line was used in the present study. Cellular lipids were analyzed in cell cultures incubated with high (standard) versus low glucose supplemented with ßHB or C10. Cellular cholesterol was analyzed using HPLC, while phospholipids and sphingomyelin (SM) were analyzed using HPTLC.

Results: HT22 cells showed higher cholesterol, but lower SM levels in the low glucose group without supplements as compared to the high glucose groups. While cellular cholesterol was reduced in both ßHB- and C10-incubated cells, phospholipids were significantly higher in C10-incubated neurons. Ratios of individual phospholipids to cholesterol were significantly higher in ßHB- and C10-incubated neurons as compared to controls.

Conclusion: Changes in the ratios of individual phospholipids to cholesterol in HT22 neurons suggest a possible alteration in the composition of the plasma membrane and organelle membranes, which may provide insight into the working mechanism of KD metabolites ßHB and C10.
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http://dx.doi.org/10.3390/nu12123870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766526PMC
December 2020

Mesenchymal to epithelial transition driven by canine distemper virus infection of canine histiocytic sarcoma cells contributes to a reduced cell motility in vitro.

J Cell Mol Med 2020 08 6;24(16):9332-9348. Epub 2020 Jul 6.

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

Sarcomas especially of histiocytic origin often possess a poor prognosis and response to conventional therapies. Interestingly, tumours undergoing mesenchymal to epithelial transition (MET) are often associated with a favourable clinical outcome. This process is characterized by an increased expression of epithelial markers leading to a decreased invasion and metastatic rate. Based on the failure of conventional therapies, viral oncolysis might represent a promising alternative with canine distemper virus (CDV) as a possible candidate. This study hypothesizes that a CDV infection of canine histiocytic sarcoma cells (DH82 cells) triggers the MET process leading to a decreased cellular motility. Immunofluorescence and immunoblotting were used to investigate the expression of epithelial and mesenchymal markers followed by scratch assay and an invasion assay as functional confirmation. Furthermore, microarray data were analysed for genes associated with the MET process, invasion and angiogenesis. CDV-infected cells exhibited an increased expression of epithelial markers such as E-cadherin and cytokeratin 8 compared to controls, indicating a MET process. This was accompanied by a reduced cell motility and invasiveness. Summarized, these results suggest that CDV infection of DH82 cells triggers the MET process by an increased expression of epithelial markers resulting in a decreased cell motility in vitro.
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http://dx.doi.org/10.1111/jcmm.15585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7417708PMC
August 2020

Axonopathy and Reduction of Membrane Resistance: Key Features in a New Murine Model of Human G-Gangliosidosis.

J Clin Med 2020 Apr 2;9(4). Epub 2020 Apr 2.

Department of Pathology, University of Veterinary Medicine Hannover, D-30559 Hannover, Germany.

G-gangliosidosis is caused by a reduced activity of β-galactosidase (), resulting in intralysosomal accumulations of G. The aim of this study was to reveal the pathogenic mechanisms of G-gangliosidosis in a new knockout mouse model. mice were analyzed clinically, histologically, immunohistochemically, electrophysiologically and biochemically. Morphological lesions in the central nervous system were already observed in two-month-old mice, whereas functional deficits, including ataxia and tremor, did not start before 3.5-months of age. This was most likely due to a reduced membrane resistance as a compensatory mechanism. Swollen neurons exhibited intralysosomal storage of lipids extending into axons and amyloid precursor protein positive spheroids. Additionally, axons showed a higher kinesin and lower dynein immunoreactivity compared to wildtype controls. mice also demonstrated loss of phosphorylated neurofilament positive axons and a mild increase in non-phosphorylated neurofilament positive axons. Moreover, marked astrogliosis and microgliosis were found, but no demyelination. In addition to the main storage material G, G, sphingomyelin, phosphatidylcholine and phosphatidylserine were elevated in the brain. In summary, the current mice exhibit a so far undescribed axonopathy and a reduced membrane resistance to compensate the functional effects of structural changes. They can be used for detailed examinations of axon-glial interactions and therapy trials of lysosomal storage diseases.
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http://dx.doi.org/10.3390/jcm9041004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7230899PMC
April 2020

Different Trafficking Phenotypes of Niemann-Pick C1 Gene Mutations Correlate with Various Alterations in Lipid Storage, Membrane Composition and Miglustat Amenability.

Int J Mol Sci 2020 Mar 19;21(6). Epub 2020 Mar 19.

Department of Natural Sciences, Lebanese American University, Beirut 1102-2801, Lebanon.

Niemann-Pick Type C (NPC) is an autosomal recessive lysosomal storage disease leading to progressive neurodegeneration. Mutations in the gene, which accounts for 95% of the cases, lead to a defect in intra-lysosomal trafficking of cholesterol and an accumulation of storage material including cholesterol and sphingolipids in the endo-lysosomal system. Symptoms are progressive neurological and visceral deterioration, with variable onset and severity of the disease. This study investigates the influence of two different NPC1 mutations on the biochemical phenotype in fibroblasts isolated from NPC patients in comparison to healthy, wild type (WT) cells. Skin derived fibroblasts were cultured from one patient compound-heterozygous for D874V/D948Y mutations, which presented wild-type like intracellular trafficking of NPC1, and a second patient compound- heterozygous for I1061T/P887L mutations, which exhibited a more severe biochemical phenotype as revealed in the delayed trafficking of NPC1. Biochemical analysis using HPLC and TLC indicated that lipid accumulations were mutation-dependent and correlated with the trafficking pattern of NPC1: higher levels of cholesterol and glycolipids were associated with the mutations that exhibited delayed intracellular trafficking, as compared to their WT-like trafficked or wild type (WT) counterparts. Furthermore, variations in membrane structure was confirmed in these cell lines based on alteration in lipid rafts composition as revealed by the shift in flotillin-2 (FLOT2) distribution, a typical lipid rafts marker, which again showed marked alterations only in the NPC1 mutant showing major trafficking delay. Finally, treatment with N-butyldeoxynojirimycin (NB-DNJ, Miglustat) led to a reduction of stored lipids in cells from both patients to various extents, however, no normalisation in lipid raft structure was achieved. The data presented in this study help in understanding the varying biochemical phenotypes observed in patients harbouring different mutations, which explain why the effectiveness of NB-DNJ treatment is patient specific.
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http://dx.doi.org/10.3390/ijms21062101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139583PMC
March 2020

Extracellular Traps: An Ancient Weapon of Multiple Kingdoms.

Biology (Basel) 2020 Feb 18;9(2). Epub 2020 Feb 18.

Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany.

The discovery, in 2004, of extracellular traps released by neutrophils has extended our understanding of the mode of action of various innate immune cells. This fascinating discovery demonstrated the extracellular trapping and killing of various pathogens by neutrophils. During the last decade, evidence has accumulated showing that extracellular traps play a crucial role in the defence mechanisms of various cell types present in vertebrates, invertebrates, and plants. The aim of this review is to summarise the relevant literature on the evolutionary history of extracellular traps used as a weapon in various kingdoms of life.
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http://dx.doi.org/10.3390/biology9020034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168307PMC
February 2020

Antimicrobial and Immunomodulatory Effect of Gum Arabic on Human and Bovine Granulocytes Against and .

Front Immunol 2019 31;10:3119. Epub 2020 Jan 31.

Department of Physiological Chemistry, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Hanover, Germany.

Gum arabic (GA) is a traditional herbal medicine from (L.) Willdenow trees, which consist of a complex mixture of polysaccharides and glycoproteins. It is used in daily applications for several diseases and is considered to protect against bacterial infections. The detailed mechanisms behind these observations are still unclear. In this study, we investigated the direct antibacterial activity of GA water and ethanol extracts against (.) or (.) and the immunomodulating properties of those extracts on granulocytes as a first line of defense against bacteria. Firstly, the direct antimicrobial effect of GA was tested on three different strains and two strains. The growth of bacteria was analyzed in the presence of different GA concentrations over time. GA water as well as ethanol extracts showed a significant growth inhibition in a concentration-dependent manner in the case of Newman, Rd5, and 25922, but not in the case of . USA300 and K1. Transmission electron microscopic analysis confirmed an antibacterial effect of GA on the bacteria. Secondly, the immunomodulatory effect of GA on the antimicrobial activity of bovine or human blood-derived granulocytes was evaluated. Interestingly, water and ethanol extracts enhanced antimicrobial activity of granulocytes by the induction of intracellular ROS production. In line with these data, GA increased the phagocytosis rate of . No effect was seen on neutrophil extracellular trap (NET) formation that mediates killing of extracellular bacteria such as . In conclusion, we show that GA exhibits a direct antibacterial effect against some and strains. Furthermore, GA boosts the antimicrobial activities of granulocytes and increases intracellular ROS production, which may lead to more phagocytosis and intracellular killing. These data might explain the described putative antimicrobial activity of GA used in traditional medicine.
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http://dx.doi.org/10.3389/fimmu.2019.03119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005937PMC
November 2020

Oxidative Stress in Canine Histiocytic Sarcoma Cells Induced by an Infection with Canine Distemper Virus Led to a Dysregulation of HIF-1α Downstream Pathway Resulting in a Reduced Expression of VEGF-B in vitro.

Viruses 2020 02 11;12(2). Epub 2020 Feb 11.

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

Histiocytic sarcomas represent malignant tumors which require new treatment strategies. Canine distemper virus (CDV) is a promising candidate due to its oncolytic features reported in a canine histiocytic sarcoma cell line (DH82 cells). Interestingly, the underlying mechanism might include a dysregulation of angiogenesis. Based on these findings, the aim of the present study was to investigate the impact of a persistent CDV-infection on oxidative stress mediated changes in the expression of hypoxia-inducible factor (HIF)-1α and its angiogenic downstream pathway in DH82 cells in vitro. Microarray data analysis, immunofluorescence for 8-hydroxyguanosine, superoxide dismutase 2 and catalase, and flow cytometry for oxidative burst displayed an increased oxidative stress in persistently CDV-infected DH82 cells (DH82Ond pi) compared to controls. The HIF-1α expression in DH82Ond pi increased, as demonstrated by Western blot, and showed an unexpected, often sub-membranous distribution, as shown by immunofluorescence and immunoelectron microscopy. Furthermore, microarray data analysis and immunofluorescence confirmed a reduced expression of VEGF-B in DH82Ond pi compared to controls. In summary, these results suggest a reduced activation of the HIF-1α angiogenic downstream pathway in DH82Ond pi cells in vitro, most likely due to an excessive, unusually localized, and non-functional expression of HIF-1α triggered by a CDV-induced increased oxidative stress.
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http://dx.doi.org/10.3390/v12020200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077254PMC
February 2020

Membrane Binding, Cellular Cholesterol Content and Resealing Capacity Contribute to Epithelial Cell Damage Induced by Suilysin of .

Pathogens 2019 Dec 30;9(1). Epub 2019 Dec 30.

Institute for Microbiology, University of Veterinary Medicine Hannover, 30173 Hannover, Germany.

() is a major cause of economic losses in the pig industry worldwide and is an emerging zoonotic pathogen. One important virulence-associated factor is suilysin (SLY), a toxin that belongs to the family of cholesterol-dependent pore-forming cytolysins (CDC). However, the precise role of SLY in host-pathogen interactions is still unclear. Here, we investigated the susceptibility of different respiratory epithelial cells to SLY, including immortalized cell lines (HEp-2 and NPTr cells), which are frequently used in in vitro studies on virulence mechanisms, as well as primary porcine respiratory cells, which represent the first line of barrier during infections. SLY-induced cell damage was determined by measuring the release of lactate dehydrogenase after infection with a virulent serotype 2 strain, its isogenic SLY-deficient mutant strain, or treatment with the recombinant protein. HEp-2 cells were most susceptible, whereas primary epithelial cells were hardly affected by the toxin. This prompted us to study possible explanations for these differences. We first investigated the binding capacity of SLY using flow cytometry analysis. Since binding and pore-formation of CDC is dependent on the membrane composition, we also determined the cellular cholesterol content of the different cell types using TLC and HPLC. Finally, we examined the ability of those cells to reseal SLY-induced pores using flow cytometry analysis. Our results indicated that the amount of membrane-bound SLY, the cholesterol content of the cells, as well as their resealing capacity all affect the susceptibility of the different cells regarding the effects of SLY. These findings underline the differences of in vitro pathogenicity models and may further help to dissect the biological role of SLY during infections.
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http://dx.doi.org/10.3390/pathogens9010033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7168673PMC
December 2019

Influence of Oxygen on Function and Cholesterol Composition of Murine Bone Marrow-Derived Neutrophils.

Methods Mol Biol 2020 ;2087:223-233

Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany.

During inflammation and infection, invading pathogens as well as infiltrating neutrophils locally consume oxygen and reduce the present oxygen level. Since oxygen is an elementary component of the microenvironment required for cell activity and alters multiple cellular functions, it is important to study neutrophil functionality and phenotype at characteristic pathophysiological oxygen levels that reflect the hypoxic phenotype during infection and inflammation. Here, we describe methods to study murine neutrophils under hypoxic compared to normoxic conditions, including analysis of cholesterol content as a key lipid involved in biological functions.
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http://dx.doi.org/10.1007/978-1-0716-0154-9_17DOI Listing
January 2021

Isolation and Quantification of Sphingosine and Sphinganine from Rat Serum Revealed Gender Differences.

Biomolecules 2019 09 7;9(9). Epub 2019 Sep 7.

Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany.

Sphingolipids are an important group of lipids that play crucial roles in living cells, facilitating cell recognition, signal transduction and endocytosis. The concentration of sphingosine and some of its derivatives like sphinganine may serve as a biomarker for the diagnosis of sphingolipidoses or be used for further research into similar diseases. In this study, a sphingolipid extraction and a high resolution detection method specific for sphingosine and sphinganine was adapted and tested. Lipids were extracted from rats' serum, coupled to -phthalaldehyde and detected with a fluorescence detector after running through a silica gel column in a high performance liquid chromatography system. With this method, we analysed 20 male and 20 female rat serum samples and compared the concentrations of sphingosine and sphinganine. The results showed a significant difference between the sphingosine concentrations in the male and female rats. The sphingosine concentration in female rats was 805 ng/mL (standard deviation, SD ± 549), while that in males was significantly lower at (75 ng/mL (SD ± 40)). Furthermore, the sphingosine:sphinganine ratio was almost 15-fold higher in the females' samples. The method presented here facilitates the accurate quantification of sphingosine and sphinganine concentrations down to 2.6 ng and 3.0 ng, respectively, and their ratio in small amounts of rat serum samples to study the sphingolipid metabolism and its potential modulation due to gene mutations or the effect of prevalent toxins.
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http://dx.doi.org/10.3390/biom9090459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770419PMC
September 2019

Hypoxia Modulates the Response of Mast Cells to Infection.

Front Immunol 2017 11;8:541. Epub 2017 May 11.

Department of Physiological Chemistry, University for Veterinary Medicine Hannover, Hanover, Germany.

To study the antimicrobial function of immune cells , cells are commonly cultivated under atmospheric oxygen concentrations (20-21%; normoxia), although the physiological oxygen conditions are significantly lower in most tissues. Especially during an acute infection, oxygen concentration locally decreases to hypoxic levels around or below 1%. The goal of this study was to investigate the effect of hypoxia on the activity of mast cells (MCs). MCs were cultivated for 3 or 24 h at 1% O in a hypoxia glove box and co-incubated with heat-inactivated . When incubating the cells for 24 h under hypoxia, the transcriptional regulator hypoxia-inducible factor 1α (HIF-1α) was stabilized and resulted in increased extracellular trap formation and decreased phagocytosis. Interestingly, while phagocytosis of fluorescent bioparticles as well as the release of extracellular traps remained unaffected at 3 h hypoxia, the secretion of the prestored mediator histamine was increased under hypoxia alone. In contrast, the release of TNF-α was generally reduced at 3 h hypoxia. Microarray transcriptome analysis revealed 13 genes that were significantly downregulated in MCs comparing 3 h hypoxia versus normoxia. One interesting candidate is , a member of the pre-budding complex of coat protein complex II (COPII), which is responsible for the anterograde transport of proteins from the ER to the Golgi apparatus. These data lead to the suggestion that synthesized proteins including crucial factors, which are involved in the response to an acute infection like TNF-α, may eventually be retained in the ER under hypoxia. Importantly, the expression of HIF-1α was not altered at 3 h. Thus, our data exhibit a HIF-1α-independent reaction of MCs to short-term hypoxia. We hypothesize that MCs respond to short-term low oxygen levels in a HIF-1α-independent manner by downregulating the release of proinflammatory cytokines like TNF-α, thereby avoiding uncontrolled degranulation, which could lead to excessive inflammation and severe tissue damage.
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http://dx.doi.org/10.3389/fimmu.2017.00541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425595PMC
May 2017

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps.

J Vis Exp 2017 03 29(121). Epub 2017 Mar 29.

Department of Physiological Chemistry, University of Veterinary Medicine Hannover; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover;

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-β-cyclodextrin (MβCD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that MβCD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, MβCD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.
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http://dx.doi.org/10.3791/54667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5564427PMC
March 2017

Case study on the pathophysiology of Fabry disease: abnormalities of cellular membranes can be reversed by substrate reduction .

Biosci Rep 2017 04 28;37(2). Epub 2017 Apr 28.

Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany

It is still not entirely clear how α-galactosidase A (GAA) deficiency translates into clinical symptoms of Fabry disease (FD). The present communication investigates the effects of the mutation N215S in FD on the trafficking and processing of lysosomal GAA and their potential association with alterations in the membrane lipid composition. Abnormalities in lipid rafts (LRs) were observed in fibroblasts isolated from a male patient with FD bearing the mutation N215S. Interestingly, LR analysis revealed that the distribution of cholesterol and flotillin-2 are distinctly altered in the Fabry fibroblasts when compared with that of the wild-type cells. Furthermore, increased levels of glycolipid globotriaosylceramide 3 (Gb3) and sphingomyelin (SM) were observed in non-raft membrane fractions of Fabry cells. Substrate reduction with -butyldeoxynojirimycin (NB-DNJ) was capable of reversing these abnormalities in this patient. These data led to the hypothesis that alterations of LRs may contribute to the pathophysiology of Morbus Fabry. Furthermore, it may be suggested that substrate reduction therapy with NB-DNJ might be a promising approach for the treatment of GAA deficiency at least for the selected patients.
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http://dx.doi.org/10.1042/BSR20160402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5408660PMC
April 2017

Formation of Neutrophil Extracellular Traps under Low Oxygen Level.

Front Immunol 2016 25;7:518. Epub 2016 Nov 25.

Department of Physiological Chemistry, University for Veterinary Medicine Hannover, Hanover, Germany; Research Center for Emerging Infections and Zoonoses (RIZ), University for Veterinary Medicine Hannover, Hanover, Germany.

Since their discovery, neutrophil extracellular traps (NETs) have been characterized as a fundamental host innate immune defense mechanism. Conversely, excessive NET-release may have a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during an infectious challenge. Our own recently published data revealed that stabilization of hypoxia-inducible factor 1α (HIF-1α) by the iron chelating HIF-1α-agonist desferoxamine or AKB-4924 enhanced the release of phagocyte extracellular traps. Since HIF-1α is a global regulator of the cellular response to low oxygen, we hypothesized that NET formation may be similarly increased under low oxygen conditions. Hypoxia occurs in tissues during infection or inflammation, mostly due to overconsumption of oxygen by pathogens and recruited immune cells. Therefore, experiments were performed to characterize the formation of NETs under hypoxic oxygen conditions compared to normoxia. Human blood-derived neutrophils were isolated and incubated under normoxic (21%) oxygen level and compared to hypoxic (1%) conditions. Dissolved oxygen levels were monitored in the primary cell culture using a Fibox4-PSt3 measurement system. The formation of NETs was quantified by fluorescence microscopy in response to the known NET-inducer phorbol 12-myristate 13-acetate (PMA) or wild-type and a nuclease-deficient mutant. In contrast to our hypothesis, spontaneous NET formation of neutrophils incubated under hypoxia was distinctly reduced compared to control neutrophils incubated under normoxia. Furthermore, neutrophils incubated under hypoxia showed significantly reduced formation of NETs in response to PMA. Gene expression analysis revealed that mRNA level of α as well as α target genes was not altered. However, in good correlation to the decreased NET formation under hypoxia, the cholesterol content of the neutrophils was significantly increased under hypoxia. Interestingly, NET formation in response to viable wild-type or nuclease-deficient strain was retained under hypoxia. Our results lead to the conclusion that hypoxia is not the ideal tool to analyze HIF-1α in neutrophils. However, the data clearly suggest that neutrophils react differently under hypoxia compared to normoxia and thereby highlight the importance of the usage of physiological relevant oxygen level when studying neutrophil functions.
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http://dx.doi.org/10.3389/fimmu.2016.00518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122589PMC
November 2016

Alterations in membrane trafficking and pathophysiological implications in lysosomal storage disorders.

Biochimie 2016 Nov 21;130:152-162. Epub 2016 Sep 21.

Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany.

Lysosomal storage disorders are a heterogeneous group of more than 50 distinct inborn metabolic diseases affecting about 1 in 5000 to 7000 live births. The diseases often result from mutations followed by functional deficiencies of enzymes or transporters within the acidic environment of the lysosome, which mediate the degradation of a wide subset of substrates, including glycosphingolipids, glycosaminoglycans, cholesterol, glycogen, oligosaccharides, peptides and glycoproteins, or the export of the respective degradation products from the lysosomes. The progressive accumulation of uncleaved substrates occurs in multiple organs and finally causes a broad spectrum of different pathologies including visceral, neurological, skeletal and hematologic manifestations. Besides deficient lysosomal enzymes and transporters other defects may lead to lysosomal storage disorders, including activator defects, membrane defects or defects in modifier proteins. In this review we concentrate on four different lysosomal storage disorders: Niemann-Pick type C, Fabry disease, Gaucher disease and Pompe disease. While the last three are caused by defective lysosomal hydrolases, Niemann-Pick type C is caused by the inability to export LDL-derived cholesterol out of the lysosome. We want to emphasise potential implications of membrane trafficking defects on the pathology of these diseases, as many mutations interfere with correct lysosomal protein trafficking and alter cellular lipid homeostasis. Current therapeutic strategies are summarised, including substrate reduction therapy as well as pharmacological chaperone therapy which directly aim to improve folding and lysosomal transport of misfolded mutant proteins.
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http://dx.doi.org/10.1016/j.biochi.2016.09.011DOI Listing
November 2016

Cholesterol-rich lipid rafts play an important role in the Cyprinid herpesvirus 3 replication cycle.

Vet Microbiol 2015 Sep 31;179(3-4):204-12. Epub 2015 May 31.

Fish Disease Research Unit, University of Veterinary Medicine Hanover, Germany. Electronic address:

The Cyprinus herpesvirus 3 (CyHV-3) is a member of the new Alloherpesviridae virus family in the Herpesvirales order. CyHV-3 has been implicated in a large number of disease outbreaks in carp populations causing up to 100% mortality. The aim of this study was to investigate the requirement of cholesterol-rich lipid rafts in CyHV-3 entry and replication in carp cells. Plasma membrane cholesterol was depleted from common carp brain (CCB) cells with methyl-β-cyclodextrin (MβCD). Treated and non-treated cells were infected with CyHV-3 and virus binding and infection parameters were assessed using RT-qPCR, immunocytochemistry and virus titration. The effect of cholesterol reduction severely stunted virus entry in vitro, however after cholesterol replenishment virus entry and subsequent replication rates were similar to the control infection. Furthermore, cholesterol depletion did not significantly influence virus binding and the subsequent post-entry replication stage, however had an impact on virus egress. Comparative analysis of the lipid compositions of CyHV-3 and CCB membrane fractions revealed strong similarities between the lipid composition of the CyHV-3 and CCB lipid rafts. The results presented here show that cholesterol-rich lipid rafts are important for the CyHV-3 replication cycle especially during entry and egress.
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http://dx.doi.org/10.1016/j.vetmic.2015.05.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7117466PMC
September 2015

Central Nervous System Demyelination and Remyelination is Independent from Systemic Cholesterol Level in Theiler's Murine Encephalomyelitis.

Brain Pathol 2016 Jan 10;26(1):102-19. Epub 2015 Jun 10.

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

High dietary fat and/or cholesterol intake is a risk factor for multiple diseases and has been debated for multiple sclerosis. However, cholesterol biosynthesis is a key pathway during myelination and disturbances are described in demyelinating diseases. To address the possible interaction of dyslipidemia and demyelination, cholesterol biosynthesis gene expression, composition of the body's major lipid repositories and Paigen diet-induced, systemic hypercholesterolemia were examined in Theiler's murine encephalomyelitis (TME) using histology, immunohistochemistry, serum clinical chemistry, microarrays and high-performance thin layer chromatography. TME-virus (TMEV)-infected mice showed progressive loss of motor performance and demyelinating leukomyelitis. Gene expression associated with cholesterol biosynthesis was overall down-regulated in the spinal cord of TMEV-infected animals. Spinal cord levels of galactocerebroside and sphingomyelin were reduced on day 196 post TMEV infection. Paigen diet induced serum hypercholesterolemia and hepatic lipidosis. However, high dietary fat and cholesterol intake led to no significant differences in clinical course, inflammatory response, astrocytosis, and the amount of demyelination and remyelination in the spinal cord of TMEV-infected animals. The results suggest that down-regulation of cholesterol biosynthesis is a transcriptional marker for demyelination, quantitative loss of myelin-specific lipids, but not cholesterol occurs late in chronic demyelination, and serum hypercholesterolemia exhibited no significant effect on TMEV infection.
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http://dx.doi.org/10.1111/bpa.12266DOI Listing
January 2016

Interaction between type I interferon and Cyprinid herpesvirus 3 in two genetic lines of common carp Cyprinus carpio.

Dis Aquat Organ 2014 Sep;111(2):107-18

Fish Disease Research Unit, Centre of Infectious Diseases, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany.

Cyprinid herpesvirus 3 (CyHV-3) infection in common carp Cyprinus carpio L. and its ornamental koi varieties can induce the severe systemic disease known as koi herpesvirus disease. This disease is characterised by a rapid replication and spreading of the virus through multiple organs and results in a fast onset of mortality (starting on Day 6 post infection) in up to 100% of infected fish. During the first phase of viral infections, type I interferons (IFNs) have generally been proven to be essential in inducing an innate immune response; however, very little is known about the type I IFN response to herpesviruses in fish. The aim of this work was to study the type I IFN responses during CyHV-3 infection in 2 genetically divergent lines of common carp which presented differing survival rates. Our results show that CyHV-3 induced a systemic type I IFN response in carp, and the magnitude of type I IFN expression is correlated with the virus load found in skin and head kidney. In this in vivo experimental setup, the level of type I IFN response cannot be linked with higher survival of carp during CyHV-3 infection.
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http://dx.doi.org/10.3354/dao02773DOI Listing
September 2014

Lipid alterations in human blood-derived neutrophils lead to formation of neutrophil extracellular traps.

Eur J Cell Biol 2014 Aug-Sep;93(8-9):347-54. Epub 2014 Aug 2.

Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany. Electronic address:

The formation of neutrophil extracellular traps (NETs) as a host innate immune defence mechanism has been shown to be the result of a novel cell death process called NETosis. The objective of this study was to investigate the role of cholesterol in the formation of NETs. To this end, primary human neutrophils were treated with different concentrations of methy-β-cyclodetxrin (MβCD) to reduce cholesterol level in the cell. The formation of NETs was studied using immunofluorescence microscopy and Picogreen-quantification of released dsDNA. Neutrophils treated with MβCD showed a significant release of NETs in a process that is independent of NADPH-oxidase. The effect of MβCD on the lipid composition of the cells was determined using high performance thin layer chromatography (HPTLC). The identities of lipids separated by HPTLC were confirmed by mass spectrometry. Treatment of neutrophils with MβCD revealed distinct changes in the lipid composition: The percentage of cholesterol in the cell was significantly reduced; other lipids as sphingomyelin were only slightly affected. Interestingly, neutrophils treated with sphingomyelin-degrading sphingomyelinase also showed significant release of NETs. In conclusion, this study shows that lipid alterations facilitate formation of NETs.
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http://dx.doi.org/10.1016/j.ejcb.2014.07.005DOI Listing
March 2015

The effect of β-glucan on formation and functionality of neutrophil extracellular traps in carp (Cyprinus carpio L.).

Dev Comp Immunol 2014 Jun 14;44(2):280-5. Epub 2014 Jan 14.

Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany. Electronic address:

The formation of neutrophil extracellular traps (NETs) has been characterised as a novel antimicrobial host defence strategy of neutrophils besides phagocytosis and degranulation, which may lead to entrapment and subsequent immobilisation and/or killing of bacterial pathogens. Here we studied the effect of the feed additive β-glucan, namely MacroGard(®), on the formation and functionality of NETs in carp. Therefore, common carp (Cyprinus carpio) head kidney and kidney cells were isolated and treated with or without β-glucan over time. The formation of NETs was analysed by immunofluorescence microscopy and revealed a distinct increase of NET-formation with β-glucan. Furthermore the subsequent entrapment of Aeromonas hydrophila, an important fish pathogen, was increased after stimulating the cells with β-glucan. However, β-glucan did not lead to a stimulation of antimicrobial activity of neutrophils against A. hydrophila. In conclusion, the data underline the fact that the feed additive β-glucan is able to modulate carp neutrophil functions.
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http://dx.doi.org/10.1016/j.dci.2014.01.003DOI Listing
June 2014

Isolation and analysis of membrane lipids and lipid rafts in common carp (Cyprinus carpio L.).

Comp Biochem Physiol B Biochem Mol Biol 2014 Mar 8;169:9-15. Epub 2013 Dec 8.

Fish Disease Research Unit, Institute of Parasitology, University of Veterinary Medicine, Bünteweg 17, 30599 Hannover, Germany. Electronic address:

Cell membranes act as an interface between the interior of the cell and the exterior environment and facilitate a range of essential functions including cell signalling, cell structure, nutrient uptake and protection. It is composed of a lipid bilayer with integrated proteins, and the inner leaflet of the lipid bilayer comprises of liquid ordered (Lo) and liquid disordered (Ld) domains. Lo microdomains, also named as lipid rafts are enriched in cholesterol, sphingomyelin and certain types of proteins, which facilitate cell signalling and nutrient uptake. Lipid rafts have been extensively researched in mammals and the presence of functional lipid rafts was recently demonstrated in goldfish, but there is currently very little knowledge about their composition and function in fish. Therefore a protocol was established for the analysis of lipid rafts and membranous lipids in common carp (Cyprinus carpio L.) tissues. Twelve lipids were identified and analysed in the Ld domain of the membrane with the most predominant lipids found in all tissues being; triglycerides, cholesterol, phosphoethanolamine and phosphatidylcholine. Four lipids were identified in lipid rafts in all tissues analysed, triglycerides (33-62%) always found in the highest concentration followed by cholesterol (24-32%), phosphatidylcholine and sphingomyelin. Isolation of lipid rafts was confirmed by identifying the presence of the lipid raft associated protein flotillin, present at higher concentrations in the detergent resistant fraction. The data provided here build a lipid library of important carp tissues as a baseline for further studies into virus entry, protein trafficking or environmental stress analysis.
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http://dx.doi.org/10.1016/j.cbpb.2013.12.001DOI Listing
March 2014

Intestinal barrier of carp (Cyprinus carpio L.) during a cyprinid herpesvirus 3-infection: molecular identification and regulation of the mRNA expression of claudin encoding genes.

Fish Shellfish Immunol 2013 Jan 26;34(1):305-14. Epub 2012 Nov 26.

Fish Disease Research Unit, Centre of Infectious Diseases, University of Veterinary Medicine Hannover, Hannover, Germany.

As a major part of tight junctions in the intestinal epithelium of vertebrates, claudin proteins are crucial to develop a selective permeable function and to maintain integrity of the barrier. The intestine has been reported as one of the targeted tissue of the cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus (KHV) which causes major disease problems in carp production worldwide. To analyse the impact of the disease on the epithelial barrier of the intestine, carp claudin encoding genes were cloned, their tissue expression was described, and the abundance of gene transcripts in the intestine of carp under CyHV-3 infection was determined. Some of the carp claudin genes such as claudin-7 and -11 were expressed in various tissues, whilst others, like claudin-2 and -23, showed more tissue-specific expression profiles, which may reflect specific functions of these particular claudins. Along the gut axis, a spatial distribution of claudin gene expressions was found, with a lower abundance of gene transcripts in anterior regions of the intestine and increased expression in the distal section of the intestine, which might indicate specific functions of different regions in the intestinal tract of carp. In carp under CyHV-3 infection, an up-regulation in the expression of IFN-a2, IL-1beta and iNOS was observed, together with an elevation of transcriptional levels of claudin-2, -3c, -11, and -23. The data suggest that expression of claudins is involved in the reorganisation of the intestinal epithelium in CyHV-3-infected carp, which may be responsible for changes in the paracellular permeability. An increased expression of the claudins might be a response to the disturbance of the hydromineral balance in carp under CyHV-3 infection.
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http://dx.doi.org/10.1016/j.fsi.2012.11.010DOI Listing
January 2013

Cyprinid herpesvirus 3 infection disrupts the skin barrier of common carp (Cyprinus carpio L.).

Vet Microbiol 2013 Mar 7;162(2-4):456-470. Epub 2012 Nov 7.

Fish Disease Research Unit, Centre of Infectious Diseases, University of Veterinary Medicine Hanover, Bünteweg 17, D-30559 Hanover, Germany.

Cyprinid herpesvirus-3 (CyHV-3) is recognised as a pathogen which causes mass mortality in populations of carp, Cyprinus carpio. One of the characteristic symptoms of the disease associated with CyHV-3 infection is the occurrence of skin lesions, sloughing off the epithelium and a lack of mucus. Furthermore, fish then seem to be more susceptible to secondary infections by bacterial, parasitic or fungal pathogens which may cause further mortality within the population. The observed pathological alterations lead to the assumption that the carp skin barrier is strongly challenged during CyHV-3 associated disease. Therefore we examined mRNA expression of genes encoding inflammatory mediators, type I interferons, and the following skin defence molecules: antimicrobial peptides, claudins, and mucin. In addition, we monitored changes in the bacterial flora of the skin during disease conditions. Our results show that CyHV-3 associated disease in the skin of common carp leads to a reduction in mRNA expression of genes encoding several important components of the mucosal barrier, in particular mucin 5B, beta defensin 1 and 2, and the tight junction proteins claudin 23 and 30. This caused changes in the bacterial flora and the development of secondary bacterial infection among some individual fish. To our knowledge this is the first report showing that under disease conditions associated with virus infection, the mucosal barrier of fish skin is disrupted resulting in a higher susceptibility to secondary infections. The reported clinical signs of CyHV-3 skin infection can now be explained by our results at the molecular level, although the mechanism of a probable virus induced immunomodulation has to be investigated further.
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http://dx.doi.org/10.1016/j.vetmic.2012.10.033DOI Listing
March 2013

β-Glucan protects neutrophil extracellular traps against degradation by Aeromonas hydrophila in carp (Cyprinus carpio).

Fish Shellfish Immunol 2012 Oct 21;33(4):1060-4. Epub 2012 Aug 21.

Fish Disease Research Unit, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany.

A novel host innate immune defence mechanism against invading pathogens, namely the formation of neutrophil extracellular traps (NETs), has recently been discovered. These NETs are described as DNA fibres released by dying neutrophils, which are able to entrap and kill various microbes. Here we studied the effect of the feed additive β-glucan, namely MacroGard(®), on the degradation of NETs by the important fish pathogen Aeromonas hydrophila. Therefore, common carp (Cyprinus carpio) head kidney cells consisting of approximately 45% neutrophils were isolated and treated with or without β-glucan. The degradation of NETs after co-incubation with A. hydrophila was analysed by immunofluorescence microscopy. The data show that A. hydrophila is able to degrade NETs and that treatment of cells with β-glucan significantly protects the NETs against bacterial degradation. Control experiments revealed that β-glucan augments nuclease activity of the bacteria at the same time while protecting the NETs against its degradation. In conclusion the data indicate that β-glucan might affect the composition and stabilisation of NETs and thereby protecting them against degradation by A. hydrophila nuclease.
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http://dx.doi.org/10.1016/j.fsi.2012.08.009DOI Listing
October 2012

Interferon type I responses to virus infections in carp cells: In vitro studies on Cyprinid herpesvirus 3 and Rhabdovirus carpio infections.

Fish Shellfish Immunol 2012 Sep 7;33(3):482-93. Epub 2012 Jun 7.

Fish Disease Research Unit, Centre of Infectious Diseases, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany.

Interferons (IFNs) are secreted mediators that play a fundamental role in the innate immune response against viruses among all vertebrate classes. Common carp is a host for two highly contagious viruses: spring viraemia of carp virus (Rhabdovirus carpio, SVCV) and the Cyprinid herpesvirus 3 (CyHV-3), which belong to Rhabdoviridae and Alloherpesviridae families, respectively. Both viruses are responsible for significant losses in carp aquaculture. In this paper we studied the mRNA expression profiles of genes encoding for proteins promoting various functions during the interferon pathway, from pattern recognition receptors to antiviral genes, during in vitro viral infection. Furthermore, we investigated the impact of the interferon pathway (stimulated with poly I:C) on CyHV-3 replication and the speed of virus spreading in cell culture. The results showed that two carp viruses, CyHV-3 and SVCV induced fundamentally different type I IFN responses in CCB cells. SVCV induced a high response in all studied genes, whereas CyHV-3 seems to induce no response in CCB cells, but it induces a response in head kidney leukocytes. The lack of an IFN type I response to CyHV-3 could be an indicator of anti-IFN actions of the virus, however the nature of this mechanism has to be evaluated in future studies. Our results also suggest that an activation of type I IFN in CyHV-3 infected cells can limit the spread of the virus in cell culture. This would open the opportunity to treat the disease associated with CyHV-3 by an application of poly I:C in certain cases.
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http://dx.doi.org/10.1016/j.fsi.2012.05.031DOI Listing
September 2012

Gene expression analysis of common carp (Cyprinus carpio L.) lines during Cyprinid herpesvirus 3 infection yields insights into differential immune responses.

Dev Comp Immunol 2012 May 24;37(1):65-76. Epub 2011 Dec 24.

Polish Academy of Sciences, Institute of Ichthyobiology & Aquaculture in Gołysz, Kalinowa 2, 43-520 Chybie, Poland.

Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is the etiological agent of a virulent and lethal disease in common and koi carp. This study aimed to determine the genetic basis underlying the common carp immune response to the CyHV-3 virus. Two common carp lines (R3 and K) were infected with CyHV-3 by immersion. The R3 line presented a 20% higher survival rate compared to the K line and significantly lower viral loads as measured at day 3 post infection (p.i.). Microarray analysis using a common carp slides containing a number of 10,822 60-mer probes, revealed that 581 genes in line K (330 up-regulated, 251 down-regulated) and 107 genes in line R3 (77 up-regulated, 30 down-regulated), showed at least a 2-fold difference in expression at day 3 p.i. compared to day 0. Genes which showed at least a 4-fold difference in expression in both lines were selected as potential markers of a CyHV-3 infection in common carp. Additionally, 76 genes showed at least 2-fold differentially expression between K and R3 lines at day 3 p.i. Significantly higher expression of several immune-related genes including number of those which are involve in pathogen recognition, complement activation, MHC class I-restricted antigen presentation and development of adaptive mucosal immunity was noted in more resistant R3 line. Further real-time PCR based analysis provided evidence for higher activation of CD8(+) T cells in R3 line. This study uncovered wide array of immune-related genes involved into antiviral response of common carp toward CyHV-3. It is also demonstrated that the outcome of this severe disease in large extent could be controlled by genetic factors of the host.
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http://dx.doi.org/10.1016/j.dci.2011.12.006DOI Listing
May 2012

A modified lipid composition in Fabry disease leads to an intracellular block of the detergent-resistant membrane-associated dipeptidyl peptidase IV.

J Inherit Metab Dis 2010 Aug 22;33(4):445-9. Epub 2010 May 22.

Department of Physiological Chemistry, University of Veterinary Medicine, Buenteweg 17, 30559, Hannover, Germany.

Fabry disease is an X-linked lysosomal storage disorder that leads to abnormal accumulation of glycosphingolipids due to a deficiency of alpha-galactosidase A (AGAL). The consequences of these alterations on the targeting of membrane proteins are poorly understood. Glycosphingolipids are enriched in Triton-X-100- resistant lipid rafts [detergent-resistant membranes (DRMs)] and play an important role in the transport of several membrane-associated proteins. Here, we show that In fibroblasts of patients suffering from Fabry disease, the colocalization of AGAL with the lysosomal marker LAMP2 is decreased compared with wild-type fibroblasts concomitant with a reduced transport of AGAL to lysosomes. Furthermore, overall composition of membrane lipids in the patients' fibroblasts as well as in DRMs reveals a substantial increase in the concentration of glycolipids and a slight reduction of phosphatidylethanolamine (PE). The altered glycolipid composition in Fabry fibroblasts is associated with an intracellular accumulation and impaired trafficking of the Triton-X-100 DRM-associated membrane glycoprotein dipeptidyl peptidase IV (DPPIV) in transfected Fabry cells, whereas no effect could be observed on the targeting of aminopeptidase N (ApN) that is not associated with this type of DRM. We propose that changes in the lipid composition of cell membranes in Fabry disease disturb the ordered Triton X-100 DRMs and have implications on the trafficking and sorting of DRM-associated proteins and the overall protein-lipid interaction at the cell membrane. Possible consequences could be altered signalling at the cell surface triggered by DRM-associated proteins, with implications on gene regulation and subsequent protein expression.
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http://dx.doi.org/10.1007/s10545-010-9114-6DOI Listing
August 2010