Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Innate immunity of carp : fishing for receptors
    Fink, Inge - \ 2017
    Wageningen University. Promotor(en): Geert Wiegertjes; Huub Savelkoul, co-promotor(en): Maria Forlenza. - Wageningen : Wageningen University - ISBN 9789463430753 - 240
    carp - cyprinus - immunity - platelets - macrophage activation - receptors - polarization - immunostimulation - immunology - karper - cyprinus - immuniteit - bloedplaatjes - macrofaag activering - receptoren - polarisatie - immunostimulatie - immunologie

    Recent decades have seen a significant intensification of aquaculture leading to increased risk of infections with several pathogenic organisms. On economical and ethical grounds it is more appropriate to improve general welfare conditions and prevent infections rather than treating disease outbreaks once they have occurred. Immunostimulation through feed can provide more efficient and sustainable control of diseases in aquaculture through enhancing the immunocompetence of fish; however, the underlying mechanisms are poorly characterized. The overall aim of this thesis was to perform a molecular and functional characterization of how pathogen-associated molecular patterns (PAMPs), such as β-glucans, affect the innate immune response of carp and which receptors on carp leukocytes are likely candidates to play a role in sensing such PAMPs.

    In chapter 1 we provide a framework for this thesis by introducing different classes of PAMPs, including β-glucans. These molecules were the centrepiece of an intra-European training network called NEMO (Protective immune modulation in warm water fish by feeding glucans), which this PhD project was part of. The scientific aim of the NEMO network was to develop a sustainable and cost-effective use of β-glucans as immunostimulants for aquaculture, using common carp as the model fish species, since on a global scale common carp is the most cultured fish species for food consumption. Our aims within the NEMO project entailed both the characterization of carp leukocytes and the characterization of candidate pattern recognition receptors (PRRs) that could play a role in sensing PAMPs and initiating immune responses. Chapter 1 therefore introduces the thrombocytes and macrophages pertinent to this thesis, as well as important classes of PRRs.

    In our first experimental study, described in chapter 2, we investigated the relevance of thrombocytes for the immune system of carp. We found that thrombocytes from healthy carp express a large number of immune-relevant genes, among which several cytokines and Toll-like receptors (Tlrs). Furthermore, we dissected the role of thrombocytes during infections with two different, albeit related, protozoan parasites, Trypanoplasma borreli and Trypanosoma carassii, and found thrombocytes were massively depleted from blood and spleen of fish infected with T. borreli. The pathology of this infection is associated with elevated levels of tissue nitration, prompting us to investigate, ex vivo, the effect of nitric oxide on thrombocytes. Our studies revealed that nitric oxide can induce a clear and rapid apoptosis of thrombocytes from healthy carp, supporting a role for nitric oxide-mediated control of immune-relevant thrombocytes during infection with T. borreli. Thereby, this particular study provided an excellent example of interplay between pathogen and the innate immune system of carp.

    We reviewed in chapter 3 another cell type central to innate immunity: the macrophage. We focused on the heterogeneity of macrophage activation states as these cells, at least in humans and mice, have the ability to polarize in several directions during an immune response. Based on the signals that lead to activation and the effector functions and cytokine profile as a result thereof, macrophages can be broadly divided into two types: classically activated macrophages induced in a T helper 1 (TH1) cytokine environment, and alternatively activated macrophages, induced in a TH2 cytokine environment. Mirroring the TH1–TH2 dichotomy, classically activated macrophages have also been termed M1, whereas alternatively activated macrophages have been termed M2. Classically activated macrophages are typically induced by stimulation with microbial ligands such as LPS in combination with pro-inflammatory cytokines such as IFNγ, and can be viewed as an extension of innate activated macrophages which are induced by microbial ligands only, thus are independent of cytokines. Alternatively activated macrophages are generated in the presence of IL4 and/or IL13. In addition to M1 and M2, one can distinguish regulatory macrophages, which are associated with the presence of the cytokine IL10. In this chapter, we reviewed the evidence of existence of polarized macrophages in teleost fish, among other things based on observations of the fundamentally different immune responses elicited by the parasites T. borreli and T. carassii.

    We further investigated the polarization of carp macrophages in chapter 4, where we obtained gene signature profiles of carp macrophages via a transcriptome approach. Independently of cytokines, carp macrophages showed the ability to differentiate into cells with functional characteristics highly comparable to those of mammalian M1 and M2, consistent with a conserved ability of macrophages to polarize into distinct subsets. In addition to obtaining a global view of gene expression, our transcriptome approach identified gene signatures for M1 and M2 macrophages which appear conserved from fish to mammals. We selected a number of these interesting genes that were differentially regulated between M1 and M2 macrophages and discussed in detail five potential M1 markers; il1β, ptx3a, saa, nos2b, and il12a – as well as five potential M2 markers; cyr61, inhba, timp2, tgm2, and arg2. These transcriptome studies may pave the way for future studies of polarized macrophages during immune responses in fish. Furthermore, additional analyses of the datasets described in this chapter will undoubtedly lead to the characterization of more genes relevant to macrophage polarization and recognition of immunostimulants.

    As part of the characterization of candidate PRRs that could play a role in sensing PAMPs and initiating immune responses, we studied the scavenger receptor Cd36 (chapter 5), which in mammals is expressed by many different (immune) cell types and plays a role in highly diverse processes, both homeostatic and pathologic. Among other things, it is often found associated with sensing of β-glucans and also with M2 macrophage activation, sparking our interest in this molecule in fish. We studied Cd36 in common carp as well as in zebrafish, a closely related cyprinid fish species. Whereas a single cd36 gene is present in zebrafish, carp was shown to have two paralogs of cd36. Although all genes show conserved synteny compared to mammalian CD36, unexpectedly we could not detect gene expression of cyprinid cd36 in macrophages or any other immune cell type or immune organ. Yet, because gene expression of cd36 was down-regulated during Mycobacterium marinum infection of zebrafish, and knockdown of cd36 in zebrafish embryos led to higher bacterial burden upon such infection, our data imply a role for Cd36 in immune responses of fish. Future studies are needed to clarify the exact mechanisms involved.

    As characterization of candidate PRRs we also examined the Toll-like receptors Tlr1 and Tlr2 (chapter 6). We identified a full-length, expressed tlr1 gene, a tlr1 pseudogene, and a second tlr2 gene next to the tlr2 which had been described previously. Sequence, phylogenetic and synteny analyses supported the conserved nature of these genes, and three-dimensional modelling showed a good fit with the mammalian TLR1/TLR2 heterodimer including the potential to bind to the prototypical ligand Pam3CSK4. However, we were unable to demonstrate Tlr1/Tlr2-mediated ligand binding in transfected cell lines through NFκB activation, despite showing the expression and co-localization of Tlr1 and Tlr2. This prompted a discussion of methods available for studying ligand-binding properties of fish Tlrs.

    Finally, we discuss in chapter 7 the findings of this thesis in the context of the NEMO project. We present the concept of trained immunity, which could provide the conceptual framework within which the immune-stimulating ability of compounds such as β-glucans could be explained. We discuss recent advances in the field of TLR research as well as that of macrophage polarization, and highlight immunometabolism as a new area of interest which may help to illuminate the molecular events occurring in immune cells during health and disease. In conclusion, we found that carp leukocytes, along with their pattern recognition receptors, are central players of the innate immune system of carp. Our findings contribute to the understanding of mechanisms of immunostimulation, and expect this will enable the valorisation and use of immunostimulants for sustainable aquaculture and improvement of fish health.

    Effects of genetic origin and social environment on behavioral response to manual restraint and monoamine functioning in laying hens
    Uitdehaag, K.A. ; Rodenburg, T.B. ; Reenen, C.G. van; Koopmanschap, R.E. ; Vries Reilingh, G. de; Engel, B. ; Buist, W.G. ; Komen, J. ; Bolhuis, J.E. - \ 2011
    Poultry Science 90 (2011)8. - ISSN 0032-5791 - p. 1629 - 1636.
    feather pecking behavior - open-field - dopamine turnover - tonic immobility - group selection - serotonin - chicks - corticosterone - platelets - lines
    Purebred laying hen lines of White Leghorn (WL) origin have been found to be more flighty and to show more feather pecking than lines of Rhode Island Red (RIR) origin. It has been found, however, that when RIR birds were housed together with WL birds, RIR birds became more flighty and those mixed groups developed more feather damage than pure-line cage-housed groups. It is unknown, however, whether this effect of social environment is accompanied by changes in stress-related behavior and neurophysiological activity, which are assumed to be associated with increased feather damage. The objective of this study was therefore to investigate the effects of genetic origin (WL or RIR) and social environment (mixed or pure groups) on behavioral response to manual restraint and monoamine functioning. Monoamine functioning was measured by brain serotonin (5-HT) and dopamine turnover. Furthermore, correlations between 5-HT turnover in the brain and peripheral measures of 5-HT in the blood were calculated. Experimental birds, housed either with other birds from the same genetic origin (pure groups) or with both RIR and WL birds (mixed groups) from hatching onward, were subjected to a manual restraint test at 47 wk of age. The WL birds struggled less during restraint and had higher dopamine and 5-HT turnover levels after restraint than did RIR birds. The WL birds also showed higher levels of platelet 5-HT uptake than did RIR birds. No effects of social environment were found. Blood and brain 5-HT measures were found to be correlated, with correlations ranging from 0.34 to 0.57, which seems to offer opportunities for less invasive peripheral indicators of 5-HT activity. In conclusion, genetic origin, but not social environment, affected the behavioral response to manual restraint and monoamine functioning in laying hens
    A novel activating chicken IgY FcR is related to leukocyte receptor complex (LRC)
    Viertlboeck, B.C. ; Schmitt, R. ; Hanczaruk, M.A. ; Crooijmans, R.P.M.A. ; Groenen, M.A.M. ; Gobel, T.W. - \ 2009
    The Journal of Immunology 182 (2009)3. - ISSN 0022-1767 - p. 1533 - 1540.
    family - thrombocytes - diversification - expression - prediction - clearance - evolution - platelets - system - cells
    FcRs have multifaceted roles in the immune system. Chicken FcRs were demonstrated on macrophages decades ago; however, only recently the chicken Ig-like receptor AB1, encoded in the leukocyte receptor complex, was molecularly identified as a high-affinity FcR. The present study was initiated to identify additional receptors with the capability to bind chicken immunoglobulins. Based on database searches, we cloned a novel chicken FcR, designated gallus gallus FcR (ggFcR), which was shown to bind selectively chicken IgY. The receptor consists of four extracellular C2-set Ig domains, followed by a transmembrane region containing arginine as a positively charged amino acid and a short cytoplasmic tail. ggFcR associates with the common -chain, indicative for an activating receptor, and real-time RT-PCR revealed high expression on PBMC, thrombocytes, and macrophages. The genomic organization is similar to most Ig-like receptor genes, where each Ig domain is encoded by a separate exon. Additionally, the ggFcR signal peptide is encoded by two exons, the second of which is 36 bp, a hallmark for genes encoded in the leukocyte receptor complex. Phylogenetic analysis also showed a relationship to genes encoded in the leukocyte receptor complex. Surprisingly, ggFcR is not encoded in the leukocyte receptor complex, but it is located as a single isolated gene at the extremity of chicken chromosome 20
    Crystal structure of fervidolysin from Fervidobacterium pennivorans, a keratinolytic enzyme related to subtilisin
    Kim, J.S. ; Kluskens, L.D. ; Vos, W.M. de; Huber, R. ; Oost, J. van der - \ 2004
    Journal of Molecular Biology 335 (2004)3. - ISSN 0022-2836 - p. 787 - 797.
    extracellular keratinase - microsporum-canis - serine-protease - fibronectin - complex - purification - platelets - binding - site - metalloprotease
    Structure-forming fibrous proteins like keratins, gelatins and collagens are degraded only by a few proteases as their tight packing limits access to the potential cleavage sites. To understand the keratin degradation in detail, we describe the first crystal structure of a keratin-degrading enzyme (keratinase), fervidolysin, from Fervidobacterium pennivorans as an immature form with propeptide (PD)-bound. The 1.7 Angstrom resolution crystal structure shows that the protease is composed of four domains: a catalytic domain (CD), two P-sandwich domains (SDs), and the PD domain. A structural alignment shows a distant relationship between the PD-CD substructure of fervidolysin and pro-subtilisin E. Tight binding of PD to the remaining part of the protease is mediated by hydrogen bonds along the domain surfaces and around the active cleft, and by the clamps to SD1 and SD2. The crystal structure of this multi-domain protein fervidolysin provides insights into proenzyme activation and the role of non-catalytic domains, suggesting a functional relationship to the fibronectin (FN)-like domains of the human promatrix metalloprotease-2 (proMMP-2) that degrades the fibrous polymeric substrate gelatin. (C) 2003 Elsevier Ltd. All rights reserved.
    Characterisation of fish leucocytes : an immunocytochemical and functional study in carp (Cyprinus carpio L.)
    Koumans - van Diepen, J.C.E. - \ 1993
    Agricultural University. Promotor(en): W.B. van Muiswinkel, co-promotor(en): J.H.W.M. Rombout. - S.l. : Koumans-van Diepen - ISBN 9789054851110 - 167
    cyprinidae - karper - bloedserum - fibrine - bloedplaatjes - bloed - erytrocyten - leukocyten - bloedplasma - reticulo-endotheliaal systeem - antilichamen - immunoglobulinen - immunocytochemie - cyprinidae - carp - blood serum - fibrin - platelets - blood - erythrocytes - leukocytes - blood plasma - reticuloendothelial system - antibodies - immunoglobulins - immunocytochemistry

    A panel of monoclonal antibodies (MAbs) against carp serum immunoglobulin (Ig), WCIs or carp thymocytes (T), WCTs were used for the characterisation of carp leucocytes. Unfortunately, all WCTs and some WCIs react with common carbohydrate determinants present on all leucocytes and Ig. Most WCIs react specific with protein determinants at the heavy chain of Ig. Consequently, B lymphocyte (sub) populations, plasma cells and Ig-binding cells could be studied. Ig molecules are found in clusters at the cell membrane of B cells and plasma cells, and in contrast to mammalian plasma cells, most carp plasma cells still have Ig at their surface membrane. Mainly the dull surface Ig-positive (sIg +) cells were stimulated by the mammalian B cell mitogen LPS and not by PHA (T cell mitogen) in vitro , whereas the sIg-negative (sIg -) cells were stimulated by PHA and not by LPS. The percentages of B cells and plasma cells showed an increase during ontogeny and reached a plateau at about 3 months and 8 months of age respectively. It is suggested that full development of the carp (humoral) immune system needs at least 8 months (at 21-22 °C). Three different subpopulations of B cells and plasma cells and at least two Ig isotypes can be distinguished based upon their reactivity with WCI 4 arid WCI 12. The distribution of the three B cell subpopulations appeared to be organ and age dependent which indicates functional differences between the Ig isotypes. Fc-like receptors were mainly demonstrated on gut macrophages while pronephros macrophages and neutrophilic granulocytes did not show Ig binding. Consequently, other forms of antigen opsonisation (e.g. complement) may play a role in phagocytosis by these non Ig-binding cells. Several procedures were tested for obtaining MAbs specific for Ig -lymphoid cells. It is concluded that the presence of immunodominant carbohydrate determinants is the major problem for obtaining specific MAbs. Tolerisation of mice against these determinants or the use of isolated membrane lysates from (sIg -) PBL appeared promising but till now only specific thrombocyte markers have been obtained. The use of more purified antigen is recommended in further attempts. The data presented in this thesis can be used for fundamental studies on cell interactions in the immune response, but also for more applied investigations on fish health control.

    Influence of wet vs. dry by-product ingredients and addition of branched-chain volatile fatty acids and valerate to dairy diets. 3. Kinetics of rumen ingesta turnover, whole tract digestibility, and some plasma hormone concentrations.
    Robinson, P.H. ; Tamminga, S. ; Vuuren, A.M. van - \ 1987
    Netherlands Journal of Agricultural Science 35 (1987)4. - ISSN 0028-2928 - p. 459 - 471.
    blood - blood plasma - blood serum - carboxylic acids - dairy cattle - dairy farming - endocrinology - erythrocytes - fatty acids - fibrin - hormones - leukocytes - platelets - rumen - rumen digestion - rumination
    Vergelijking in de handel verkrijgbare runderbloedplaten
    Peters-Groenen, A.A.M. ; Winnubst, L. ; Broex, N.J.G. ; Hartog, J.M.P. de - \ 1987
    Wageningen : RIKILT (Rapport / RIKILT 87.03) - 5
    rundvee - vleesvee - slachtdieren - bloedplaatjes - vleeskeuring - cattle - beef cattle - meat animals - platelets - meat inspection
    Vergelijkend onderzoek naar de toepasbaarheid van "kant en klare" runderbloedplaten voor het bacteriologisch onderzoek van slachtdieren.
    Genetical and some environmental influences affecting the level of leucocyte counts in the milk of cows
    Afifi, Y.A. - \ 1967
    Wageningen University. Promotor(en): T. Stegenga. - Wageningen : Veenman - 81
    rundvee - rauwe melk - diergeneeskunde - melkklieren - melksecretie - lactatie - dierlijke producten - vervalsing - besmetting - verouderen - gebreken - achteruitgang (deterioration) - bloedserum - fibrine - bloedplaatjes - bloed - erytrocyten - leukocyten - bloedplasma - mastitis - cattle - raw milk - veterinary science - mammary glands - milk secretion - lactation - animal products - adulteration - contamination - aging - defects - deterioration - blood serum - fibrin - platelets - blood - erythrocytes - leukocytes - blood plasma - mastitis
    The progeny groups of different sires varied widely in white-cell count in milk, even after exclusion of all cows which had suffered from mastitis. The sire had a demonstrable effect on white-cell count in milk, especially during the second half of lactation. Heritability estimates of white-cell count in milk showed that values for the fourth lactation were higher than those for heifers. But at the end of lactation heritability values for 4th lactation cows and heifers were nearly equal (about 0.40). The daughter groups with high average white-cell counts mostly showed frequent mastitis. There was a high phenotypic and genetic correlation between clinical mastitis and white-cell count. Within seasons for cows which, so far known, had never mastitis, very high and very low producers had higher white-cell counts than other cows. White-cell counts increased remarkably with advancing lactation. A relation between white-cell count and ease of milking could not be demonstrated.

    Increasing milking vacuum over 40 cm mercury pressure, especially at the end of lactation, or increasing pulsation to over 50 per min. tended to increase white-cells. Milking routine (man/machine ratio) affected white-cell count in the end of lactation. More cows per milker increased the number of white-cells.

    Verspreiding van bloedgroepen in het Nederlandse zwartbonte rundvee : een onderzoek naar de frequenties van bloedgroepen en naar enige factoren, die de frequenties beinvloeden
    Kraaij, G.J. - \ 1967
    Wageningen University. Promotor(en): T. Stegenga. - Wageningen : Veenman - 127
    bloedserum - fibrine - bloedplaatjes - bloed - erytrocyten - leukocyten - bloedplasma - rundvee - melkveerassen - nederland - genetica - heritability - genetische variatie - blood serum - fibrin - platelets - blood - erythrocytes - leukocytes - blood plasma - cattle - dairy breeds - netherlands - genetics - heritability - genetic variation
    Blood groups are genetically determined components of the red blood cells. In cattle there were 13 loci known to determine blood groups and some of these loci had large series of alleles. There were also 14 other loci known to determine proteins and enzymes in blood and milk of cattle.

    The author examined how the distribution of blood groups in the Dutch Friesian population had been influenced by the restrictions of breeders. He found that parent cattle were paired independently of blood group. In offspring there was no selection for a certain phenotype until the end of the first year.

    There were clear differences in the frequency of some genes between adult bulls and cows. The gene for blood group A was less frequent in bulls and that for blood group F was less frequent in cows. This occurred in some foreign breeds of cattle.

    The distribution of blood groups over the population was not even. There were differences in gene frequency between breeding areas and in the breeding areas there were large differences between artificial insemination stations. Differences within farms and within breeding pedigrees were even greater. These differences could be ascribed largely to the use of one or only a few sires.

    Het kopergehalte van lever en bloedserum bij het Fries-Hollandse rund
    Grift, J. van der - \ 1955
    's-Gravenhage : Staatsdrukkerij (Verslagen van landbouwkundige onderzoekingen 61.10) - 61
    melkveerassen - rundvee - bloedserum - fibrine - bloedplaatjes - bloed - erytrocyten - leukocyten - bloedplasma - dairy breeds - cattle - blood serum - fibrin - platelets - blood - erythrocytes - leukocytes - blood plasma
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