Identification and functional characterization of nonmammalian Toll-like receptor 20
Pietretti, D. ; Scheer, M.H. ; Fink, I.R. ; Taverne, N. ; Savelkoul, H.F.J. ; Spaink, H.P. ; Forlenza, M. ; Wiegertjes, G. - \ 2014
Immunogenetics 66 (2014)2. - ISSN 0093-7711 - p. 123 - 141.
carp cyprinus-carpio - leucine-rich repeats - common carp - ictalurus-punctatus - monoclonal-antibodies - pathogen recognition - accessory molecules - expression analysis - sequence-analysis - channel catfish
Like other vertebrate Toll-like receptors (TLRs), the TLRs of teleost fish can be subdivided into six major families, each of which recognize a general class of molecular patterns. However, there also are a number of Tlrs with unknown function, the presence of which seems unique to the bony fish, among which is Tlr20. We identified full-length complementary DNA (cDNA) sequences for tlr20 of zebrafish and common carp, two closely related fish species. Zebrafish have six copies of tlr20, whereas carp express only a single copy. Both zebrafish Tlr20 (at least Tlr20a–d) and carp Tlr20 have 26 leucine-rich repeats (LRRs). Three-dimensional modeling indicates a best fit to the crystal structure of TLR8. Phylogenetic analyses place Tlr20 in the TLR11 family closest to Tlr11 and Tlr12, which sense ligands from protozoan parasites in the mouse. Conservation of genes on zebrafish chromosome 9, which carries tlr20, with genes on mouse chromosome 14, which carries tlr11, indicates Tlr11 could be a possible ortholog of Tlr20. Confocal microscopy suggests a subcellular localization of Tlr20 at the endoplasmatic reticulum. Although in vitro reporter assays could not identify a ligand unique to Tlr20, in vivo infection experiments indicate a role for Tlr20 in the immune response of carp to protozoan parasites (Trypanoplasma borreli). Carp tlr20 is mainly expressed in peripheral blood leukocytes (PBL) with B lymphocytes, in particular, expressing relatively high levels of Tlr20. In vitro stimulation of PBL with T. borreli induces an upregulation of tlr20, supportive of a role for Tlr20 in the immune response to protozoan parasites.
Chronic Allopurinol Treatment during the Last Trimester of Pregnancy in Sows: Effects on Low and Normal Birth Weight Offspring
Gieling, E.T. ; Antonides, A. ; Fink-Gremmels, J. ; Haar, K. ter; Kuller, W.I. ; Meijer, E. ; Nordquist, R.E. ; Stouten, J.M. ; Zeinstra, E. ; Staay, F.J. van der - \ 2014
PLoS ONE 9 (2014)1. - ISSN 1932-6203 - 15 p.
intrauterine growth-restriction - for-gestational-age - children born - placental insufficiency - attentional problems - newborn piglets - spleen weight - brain - memory - stress
Low-birth-weight (LBW) children are born with several risk factors for disease, morbidity and neonatal mortality, even if carried to term. Placental insufficiency leading to hypoxemia and reduced nutritional supply is the main cause for LBW. Brain damage and poor neurological outcome can be the consequence. LBW after being carried to term gives better chances for survival, but these children are still at risk for poor health and the development of cognitive impairments. Preventive therapies are not yet available. We studied the risk/efficacy of chronic prenatal treatment with the anti-oxidative drug allopurinol, as putative preventive treatment in piglets. LBW piglets served as a natural model for LBW. A cognitive holeboard test was applied to study the learning and memory abilities of these allopurinol treated piglets after weaning. Preliminary analysis of the plasma concentrations in sows and their piglets suggested that a daily dose of 15 mg.kg-1 resulted in effective plasma concentration of allopurinol in piglets. No adverse effects of chronic allopurinol treatment were found on farrowing, birth weight, open field behavior, learning abilities, relative brain, hippocampus and spleen weights. LBW piglets showed increased anxiety levels in an open field test, but cognitive performance was not affected by allopurinol treatment. LBW animals treated with allopurinol showed the largest postnatal compensatory body weight gain. In contrast to a previous study, no differences in learning abilities were found between LBW and normal-birth-weight piglets. This discrepancy might be attributable to experimental differences. Our results indicate that chronic prenatal allopurinol treatment during the third trimester of pregnancy is safe, as no adverse side effects were observed. Compensatory weight gain of treated piglets is a positive indication for the chronic prenatal use of allopurinol in these animals. Further studies are needed to assess the possible preventive effects of allopurinol on brain functions in LBW piglets.
A closer look at Toll-like receptor 4 (TLR4) and toll-like receptor 20 (TLR20) of common carp (Cyprinus carpio)
Pietretti, D. ; Forlenza, M. ; Fink, I.R. ; Wiegertjes, G.F. - \ 2013
Fish and Shellfish Immunology 34 (2013)6. - ISSN 1050-4648 - p. 1673 - 1673.
Toll-like receptors (TLRs) constitute an important class of pattern-recognition receptors, which recognize a multitude of pathogen-associated molecular patterns (PAMPs). We focused on TLR4 and TLR20 of common carp (Cyprinus carpio), their signaling pathway and their possible functions as activating receptors involved in innate immune responses to various pathogens or immunostimulants. So far the expression of TLR4 and TLR20 genes has been reported only for few fish species of the Cypriniformes (zebrafish, grass carp, common carp) and close relatives Siluriformes (channel catfish). Multiple fish TLR4 genes, created by a (recent) duplication rather than speciation event, seem to exist. It is possible that these paralogs have evolved to express different ligand specificities. In mammals TLR4 recognizes lipopolysaccharides from Gram-negative bacteria. The ligand(s) for fish TLR4 have not been confirmed. Multiple TLR20 genes also seem to exist in zebrafish, but not in carp. Our phylogenetic analysis indicates an ancestral relationship could exist between TLR20 and TLR11/TLR12, two TLRs found in mice but not in humans with ligand specificity for profilin from parasitic Toxoplasma gondii. At present, the ligand of TLR20 is unknown. We have cloned full-length coding sequences of carp TLR4a/b and carp TLR20 to study the function of these molecules. Expression analysis using real-time PCR show both TLR4 and TLR20 are mainly expressed in immune organs such as head kidney, gut and spleen with neutrophilic granulocytes as the primary source. We have also investigated the expression of TLR4 and of TLR20 after infection with protozoan parasites or after infection with spring vireamia of carp virus (SVCV). For TLR4, neither challenge with parasites nor with virus induced major changes in gene expression. However, we find high gene expression of TLR20 after infection with the protozoan parasites Trypanoplasma borreli or Trypanosoma carassii. We used the full-length sequences to create fluorescent protein-tagged TLRs for visualization of localization of these receptors by fluorescence microscopy. Our study suggests both receptors are located mainly in the cytoplasmic region. To define the unknown ligands of TLR4 and TLR20 we overexpressed these receptors in both human cell lines (HEK 293) and fish cell lines (EPC, CLC) stably transfected with a promoter of the transcription factor NF-¿B and a luciferase reporter gene. These studies may help to identify ligands for carp TLR4 a/b and TLR20.
Molecular cloning and cellular localization of the scavenger receptor SCARF1 in common carp
Østergaard, A.E. ; Fink, I.R. ; Sukinta, A. ; Yixian, L. ; Forlenza, M. ; Wiegertjes, G.F. - \ 2013
Fish and Shellfish Immunology 34 (2013)6. - ISSN 1050-4648 - p. 1727 - 1727.
The recognition of pathogens by the innate immune system relies on a wide range of germ-line encoded pattern recognition receptors of which some belong to the large superfamily of scavenger receptors (SRs). These receptors are expressed on cells surveying potential portals of entry, including macrophages, dendritic cells and endothelial cells and are involved in recognition of both endogenous and pathogen-derived ligands. Here we describe for the first time an evolutionarily conserved member of the SR family, named scavenger receptor class F-member1 (SCARF1), in the common carp (Cyprinus carpio). Orthologs of mammalian SCARF1 have been reported in nematodes and shown to be involved in recognition of e.g. beta-glucans. Also, SCARF1 has been shown to mediate an inflammatory cytokine response upon ligand binding whilst functioning as co-receptor for Toll-like receptors (TLRs). We found two SCARF1 genes in the common carp genome, confirming the hypothesis that carp has undergone an extra genome duplication event compared to zebrafish, where only one SCARF1 gene is found. The two SCARF1 genes in carp are 94% similar and show comparable mRNA expression levels. Sequence analysis of SCARF1 reveals a high level of amino acid conservation compared to the human orthologue. Phylogenetic analysis of carp SCARF1 with other vertebrate SCARF1 genes groups carp and zebrafish together with high bootstrap values. Synteny studies show conserved linkage with several neighbouring genes when comparing genomic regions from different species. Gene expression analysis of SCARF1 shows expression in several organs, whereas gene expression analysis of purified cell populations shows highest expression in endothelial cells, but also macrophages, granulocytes, thrombocytes and thymocytes show SCARF1 gene expression. In contrast, no expression was observed in B cells. Previously, our group has cloned TLR2 from common carp and we are now investigating the possible role of SCARF1 acting as an internalizing receptor that would facilitate TLR activation upon phagocytosis of ligands. The localization of SCARF1 was studied by confocal microscopy of human (HEK293) and fish (EPC) cell lines transfected with a fluorescently tagged receptor. In addition, antibodies directed against a V5-tagged version of SCARF1 were also used to detect SCARF1 localization. These studies showed that carp SCARF1 is expressed in the cytoplasm, but also on the cell membrane, which is comparable to human SCARF1 localization. We are performing phagocytosis studies in order to verify the internalizing ability of SCARF1 and its potential role in TLR signaling.
|Toll-like receptor-1 and -2 in common carp
Fink, I.R. ; Pietretti, D. ; Forlenza, M. ; Wiegertjes, G.F. - \ 2013
Fish and Shellfish Immunology 34 (2013)6. - ISSN 1050-4648 - p. 1706 - 1707.
|Research on herbal products for production animals in the Netherlands
Groot, M.J. ; Asseldonk, A.G.M. ; Fink-Gremmels, J. ; Lourens, S. ; Wagenaar, J.P. ; Kleijer Ligtenberg, G. - \ 2013
In: Proceedings of the 61st Annual Meeting of the Society for Medicinal Plant and Natural Product Research, 1-5 September 2013, Stuttgart, Germany. - Stuttgart : Georg Thieme Verlag KG - p. 1123 - 1123.
Cytotoxicity of surface-functionalized silicon and germanium nanoparticles: the dominant role of surface charges
Bhattacharjee, S. ; Rietjens, I.M.C.M. ; Singh, M.P. ; Atkins, T.M. ; Purkait, T.K. ; Xu, Z. ; Regli, S. ; Shukaliak, A. ; Clark, R.J. ; Mitchell, B.S. ; Alink, G.M. ; Marcelis, A.T.M. ; Fink, M.J. ; Veinot, J.G.C. ; Kauzlarich, S.M. ; Zuilhof, H. - \ 2013
Nanoscale 5 (2013). - ISSN 2040-3364 - p. 4870 - 4883.
block copolymer nanoparticles - cerium oxide nanoparticles - quantum dots - in-vitro - oxidative stress - gold nanoparticles - cellular toxicity - dependent endocytosis - hemolytic-activity - epithelial-cells
Although it is frequently hypothesized that surface (like surface charge) and physical characteristics (like particle size) play important roles in cellular interactions of nanoparticles (NPs), a systematic study probing this issue is missing. Hence, a comparative cytotoxicity study, quantifying nine different cellular endpoints, was performed with a broad series of monodisperse, well characterized silicon (Si) and germanium (Ge) NPs with various surface functionalizations. Human colonic adenocarcinoma Caco-2 and rat alveolar macrophage NR8383 cells were used to clarify the toxicity of this series of NPs. The surface coatings on the NPs appeared to dominate the cytotoxicity: the cationic NPs exhibited cytotoxicity, whereas the carboxylic acid-terminated and hydrophilic PEG- or dextran-terminated NPs did not. Within the cationic Si NPs, smaller Si NPs were more toxic than bigger ones. Manganese-doped (1% Mn) Si NPs did not show any added toxicity, which favors their further development for bioimaging. Iron-doped (1% Fe) Si NPs showed some added toxicity, which may be due to the leaching of Fe3+ ions from the core. A silica coating seemed to impart toxicity, in line with the reported toxicity of silica. Intracellular mitochondria seem to be the target for the toxic NPs since a dose-, surface charge- and size-dependent imbalance of the mitochondrial membrane potential was observed. Such an imbalance led to a series of other cellular events for cationic NPs, like decreased mitochondrial membrane potential (¿¿m) and ATP production, induction of ROS generation, increased cytoplasmic Ca2+ content, production of TNF-a and enhanced caspase-3 activity. Taken together, the results explain the toxicity of Si NPs/Ge NPs largely by their surface characteristics, provide insight into the mode of action underlying the observed cytotoxicity, and give directions on synthesizing biocompatible Si and Ge NPs, as this is crucial for bioimaging and other applications in for example the field of medicine.
|Studies into the formation of heterodimers of Toll-like receptor-1 and -2 in common carp
Fink, I.R. ; Østergaard, A.E. ; Forlenza, M. ; Wiegertjes, G.F. - \ 2012
In: 12th Congress of ISDCI program and abstracts, Fukuoka, 9-13 July 2012. - - p. 51 - 51.
|Pattern recognition receptors
Fink, I.R. - \ 2012
In: Fish Immunology Workshop 2012, Wageningen, 22-26 April 2012. -
Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array
Schuster, M. ; Kilaru, S. ; Fink, G. ; Collemare, J.A.R. ; Roger, Y. ; Steinberg, G. - \ 2011
Molecular Biology of the Cell 22 (2011)19. - ISSN 1059-1524 - p. 3645 - 3657.
fungus ustilago-maydis - tug-of-war - lipid-droplet transport - cytoplasmic dynein - molecular motors - intracellular-transport - vesicle transport - cargo transport - caenorhabditis-elegans - polarity orientation
The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to the growing cell tip (anterograde). Occasionally, EEs run up to 90 mu m in one direction. The underlying MT array consists of unipolar MTs at both cell ends and antipolar bundles in the middle region of the cell. Cytoplasmic MT-organizing centers, labeled with gamma-tubulin ring complex protein, are distributed along the antipolar MTs but are absent from the unipolar regions. Dynein colocalizes with EEs for 10-20 mu m after they have left the cell tip. Inactivation of temperature-sensitive dynein abolishes EE motility within the unipolar MT array, whereas long-range motility is not impaired. In contrast, kinesin-3 is continuously present, and its inactivation stops long-range EE motility. This indicates that both motors participate in EE motility, with dynein transporting the organelles through the unipolar MT array near the cell ends, and kinesin-3 taking over at the beginning of the medial antipolar MT array. The cooperation of both motors mediates EE movements over the length of the entire cell.
|Molecular and functional characterization of toll-like receptor 4 in common carp (Cyprinus carpio)
Pietretti, D. ; Fink, I.R. ; Forlenza, M. ; Wiegertjes, G.F. - \ 2011
In: 15th international conference on disease of fish and shellfish Split, Croatia, 12 - 16 September, 2011. - - p. 72, O - 060.
|Molecular and functional characterization of toll-like receptor-1 in common carp (Cyprinus carpio)
Fink, I.R. ; Forlenza, M. ; Wiegertjes, G.F. - \ 2011
In: 15th international conference on disease of fish and shellfish, Split Croatia, 12 - 16 September, 2011. - - p. 70, O - 058.
|Heterogeneity of macrophage activation during parasite infections
Wiegertjes, G.F. ; Fink, I.R. ; Forlenza, M. - \ 2011
In: The 15th International Conference on Diseases of Fish and Shellfish organized by the EAFP, Split, Croatia, 12 - 16 September, 2011. - - p. 17 (O - 008).
|Molecular and functional characterization of scavenger receptor class F member 1 (SCARF1) and CD36 in common carp (Cyprinus carpio)
Østergaard, A.E. ; Fink, I.R. ; Sukinta, A. ; Wiegertjes, G.F. - \ 2011
In: Abstract book of the 15th International Conference on Disease of Fish and Shellfish, Split, Croatia, 12-16 September 2011. - - p. 71, O - 059.
|Functional evidence for toll-like receptors in modern bony fish
Wiegertjes, G.F. ; Ribeiro, C.M.S. ; Pietretti, D. ; Fink, I.R. ; Østergaard, A.E. ; Forlenza, M. ; Scheer, M.H. ; Savelkoul, H.F.J. - \ 2011
In: Proceedings of the 25th Annual EMDS Meeting, clinical and fundamental aspects of monocyte macrophage and DC plasticity, Brussels, Belgium, 22-24 september 2011. - - p. 21, B10 - 21, B10.
Heterogeneity of macrophage activation in fish
Forlenza, M. ; Fink, I.R. ; Raes, G. ; Wiegertjes, G.F. - \ 2011
Developmental and Comparative Immunology 35 (2011)12. - ISSN 0145-305X - p. 1246 - 1255.
tumor-necrosis-factor - cyprinus-carpio l. - trout oncorhynchus-mykiss - nitric-oxide synthase - toll-like receptors - interferon-gamma genes - carassius-auratus l. - expression analysis - rainbow-trout - factor-alpha
In this review, we focus on four different activation states of fish macrophages. In vitro, stimulation with microbial ligands induces the development of innate activated macrophages whereas classically activated macrophages can be induced by stimulation with LPS in combination with (recombinant) IFN¿. Both types of macrophages show elevated phagocytic activity, expression of pro-inflammatory cytokine genes and radical production. Alternatively activated macrophages require the cytokines IL-4/IL-13 for induction of, among others, arginase activity. Until in vitro studies identify the effects of putative IL-4 and IL-13 homologues on fish macrophages, arginase enzyme activity remains the most reliable marker for the presence of alternatively activated macrophages in fish. The best evidence for the existence of regulatory macrophages, associated with the presence of IL-10, comes from in vivo studies, for example during parasitic infections of carp. Altogether, differentially activated macrophages in fish largely resemble the phenotypes of mammalian macrophages. However, the presence of fish-specific ligand recognition by TLRs and of duplicated genes coding for proteins with particular activities, poses additional challenges for the characterization of phenotype-specific gene signatures and cell surface markers.
Adaptive braking by Ase1 prevents overlapping microtubules from sliding completely apart.
Braun, M. ; Lansky, Z. ; Fink, G. ; Ruhnow, F. ; Diez, S. ; Janson, M.E. - \ 2011
Nature Cell Biology 13 (2011). - ISSN 1465-7392 - p. 1259 - 1264.
fission yeast - spindle midzone - cross-linkers - protein - kinesin-14 - binding - motors - prc1 - organization - cytokinesis
Short regions of overlap between ends of antiparallel microtubules are central elements within bipolar microtubule arrays. Although their formation requires motors1, recent in vitro studies demonstrated that stable overlaps cannot be generated by molecular motors alone. Motors either slide microtubules along each other until complete separation2, 3, 4 or, in the presence of opposing motors, generate oscillatory movements5, 6, 7. Here, we show that Ase1, a member of the conserved MAP65/PRC1 family of microtubule-bundling proteins, enables the formation of stable antiparallel overlaps through adaptive braking of Kinesin-14-driven microtubule–microtubule sliding. As overlapping microtubules start to slide apart, Ase1 molecules become compacted in the shrinking overlap and the sliding velocity gradually decreases in a dose-dependent manner. Compaction is driven by moving microtubule ends that act as barriers to Ase1 diffusion. Quantitative modelling showed that the molecular off-rate of Ase1 is sufficiently low to enable persistent overlap stabilization over tens of minutes. The finding of adaptive braking demonstrates that sliding can be slowed down locally to stabilize overlaps at the centre of bipolar arrays, whereas sliding proceeds elsewhere to enable network self-organization.
Molecular characterization of Toll-like and scavenger receptors activated by B-glucans in common carp (Cyprinus carpio)
Fink, I.R. ; Oestergaard, A.E. ; Wiegertjes, G.F. - \ 2010
|Molecular characterization of TOLL-like receptor 4B in common carp
Pietretti, D. ; Fink, I.R. ; Oestergaard, A.E. ; Savelkoul, H.F.J. ; Wiegertjes, G.F. - \ 2010
In: Abstracts First EOFFI Symposium European Organisation of Fish Immunology, Viterbo, Italy, 23-27 May 2010. - - p. 85 - 85.
Biofilm formation and genotyping of Staphylococcus aureus bovine mastitis isolates: Evidence for lack of penicillin-resistance in Agr-type II strains
Melchior, M.B. ; Osch, M.H.J. ; Graat, R. ; Duijkeren, E. van; Mevius, D.J. ; Nielen, M. ; Gaastra, W. ; Fink, J. - \ 2009
Veterinary Microbiology 137 (2009)1-2. - ISSN 0378-1135 - p. 83 - 89.
polysaccharide intercellular adhesin - quaternary ammonium-compounds - sequence element is256 - somatic-cell count - antimicrobial susceptibility - genetic-variability - epidermidis - milk - infections - protein
The increasing evidence for a role of biofilm formation in bovine mastitis caused by Staphylococcus aureus led to further investigations on biofilm formation by S. aureus isolates from mastitis in two growth media (TSBg and bovine milk serum). The ability of 99 S. aureus strains that were recently isolated or obtained from a culture collection (historical strains) to form biofilm, in both growth media as well as the correlation of biofilm formation with the presence of the ica-, bap-, and IS257 genes are described. These genes have been correlated with biofilm formation by human S. aureus isolates. All strains were also genotyped with respect to their Agr-type and -subtype, and for the presence of the antibiotic resistance genes blaZ and smr by PCR. The prevalence of the Agr-types and the investigated genes and their correlation with biofilm formation were statistically evaluated. The Agr-type of a strain had a marked effect on the biofilm formation, by that strain, however in contrast to human isolates no significant effect of ica- and IS257 genes on biofilm formation was observed. The bap gene was not found in any of the investigated strains. The presence of biofilm related genes showed a high correlation with the Agr-type of the strains. The data give evidence for a very strong correlation of Agr-type I strains and penicillin-resistance in the bovine S. aureus mastitis strains; none of the Agr-type II strains was found to harbor penicillin-resistance genes. These data indicate that the most prevalent Agr-types in S. aureus bovine mastitis, Agr-type I and II, can be regarded as different subspecies, with different abilities for the formation of biofilm in bovine milk serum. The very high correlation between Agr-type II and penicillin-susceptibility strongly suggests that these strains are not able to accommodate blaZ genes