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.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Biomarkers and mechanisms of natural disease resistance in dairy cows
    Altena, S.E.C. van - \ 2016
    Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Edwin Tijhaar. - Wageningen : Wageningen University - ISBN 9789462578005 - 158
    dairy cows - biomarkers - disease resistance - immunity - antibodies - proteomics - immune response - dendritic cells - immunology - melkkoeien - biomarkers - ziekteresistentie - immuniteit - antilichamen - eiwitexpressieanalyse - immuniteitsreactie - dendritische cellen - immunologie

    The aim of this thesis was to define and test biomarkers for disease resistance in dairy cows and to determine the underlying mechanism in natural disease resistance. The health status of the cows is an important issue in dairy farming. Due to the mandatory reduction in the use of antibiotics, alternatives are required to prevent the development and expression of illness in dairy cows. The identification of biomarkers associated with such disease offers the opportunity to adapt the management of cows at risk, and in this way, prevent them from developing overt disease. Previously, natural antibodies (NAbs) in serum and milk were used as candidate biomarkers for natural disease resistance in cows. In this thesis, we continue on the occurrence and mode of action of NAbs and also focus on their source: the B-1 cells. We performed a literature study on the identification and function of B-1 cells in different species and defined the limitations in the current identification of these cells in pigs, sheep and cows (Chapter 2). B-1 cells were described in cows by using widely accepted cell surface markers CD5 and CD11b. However, in literature several findings suggest that these cell surface markers are not unique markers for B-1 identification. The similarities between mice and veterinary animals in foetal B-cell development and antibody production, implies that B-1 cells are present in cows. In chapter 3, we carefully studied new markers to selectively identify B-1 cells in cows. The combination of B-1 cell markers IgM++ and pSYK++ (indicator constitutive intracellular signalling) identifies a distinct cell population with essential B-1 characteristics such as high CD80 expression. In addition, the development of these B-1 cells in calves before colostrum intake and 3 weeks afterwards shows the same kinetics as the development of NAbs represented by IgM antibodies binding to the well-accepted NAb-antigen phosphatidylcholine (PtC). In calves up to half a year of age, it is shown that the production of such NAbs increases from birth and stabilises from 6 weeks onwards. This implies an endogenous NAb production, which follows the same age-related kinetics as can be expected from B-1 cell development. In contrast, the development of total IgM antibody levels in calves shows a bimodal distribution, which is caused by the uptake and breakdown of maternally-derived IgM and simultaneous endogenous production of specific and natural IgM. Chapter 4 describes the role of such NAbs in bovine immunity. NAbs were represented by the binding of IgM to the naïve antigen keyhole limpet hemocyanin (KLH). Cows with high serum NAb levels were shown to have more IgM and IgG antibodies binding to common microbial structures LPS, LTA and PGN, than cows with low serum NAb levels. In addition, they also have more IgM antibodies binding to intact, fixed E. coli and S. Typhimurium bacteria. However, the killing of live E. coli and S. Typhimurium bacteria via antibody-mediated complement killing does not differ between cows with high and low NAb levels. The antibody-mediated complement killing was determined in a newly developed serum bactericidal test. Cows that performed less in the bactericidal test were more likely to develop mastitis in the future. This association was observed for the killing of E. coli and S. Typhimurium and the development of mastitis within the next one year. For S. Typhimurium the association was still present for the cases of mastitis occurring within four years after testing. Alternative biomarkers for disease resistance in cows were defined in chapter 5 by using a contemporary proteomics approach. Milk samples from high and low disease resistant cows were selected from the “Resilient Cattle” (Weerbaar Vee) biobank. Comparing the spectrum of milk proteins of high and low disease-resistant cows showed potential candidate biomarkers that were elevated in the milk of low-resistant cows. Two candidate marker proteins were validated with ELISA in a new and larger group of high- and low-resistant cows. Lactoferrin (LF) levels were significantly increased in milk of low-resistant cows. In addition, LF levels in milk were associated with clinical manifestations of lameness and had a predictive value for subsequent culling.

    In conclusion, we found that also in cows NAbs are produced by B-1 cells that can be identified based on the combined expression of cell surface IgM and internal pSYK. In addition, the frequency of these B-1 cells after birth follows a similar kinetics as described before in mice. These NAbs can be more precisely identified based on their PtC binding ability and their functional activity in a bactericidal test. However, the true predictive value of B-1 cells and NAbs for the health status and immunocompetence of dairy cattle remains to be established. Proteomics turned out to be a useful approach for identifying potential new biomarkers for health and disease in milk of cows. Application and further development of their predictive capacity is dependent on the availability of robust, sensitive and quantitative assays. This project was part of the “Resilient Cattle” project providing biological samples and essential data on the health status during respective lactation periods of individual dairy cows. The impact of this research now requires translation into management tools and principles for the individual farmer impacting on the overall health status and economic performance of his herd of dairy cattle.

    Effects of early life conditions on immunity in broilers and layers
    Simon, K. - \ 2016
    Wageningen University. Promotor(en): Bas Kemp, co-promotor(en): Aart Lammers. - Wageningen : Wageningen University - ISBN 9789462576711 - 188
    broilers - hens - ontogeny - poultry feeding - chicken housing - immune response - antibiotics - gastrointestinal microbiota - immunology - immunity - vleeskuikens - hennen - ontogenie - pluimveevoeding - huisvesting van kippen - immuniteitsreactie - antibiotica - microbiota van het spijsverteringskanaal - immunologie - immuniteit


    The course for later life immune responses is set early in life during the developmental phase of the immune system and accordingly disturbances of immune development may have long-term consequences for host health. In terms of immune activation and immune development the gut microbiota play an important role and consequently disturbances of early life microbial colonization may affect host immunity later in life. In chickens, disturbances of microbial colonization may be caused by various early life conditions which in turn may affect robustness of the chick in the long term. The aim of this thesis was to assess the effects of several early life factors including time of access to feed post hatch (immediately or 72 hours delayed), housing conditions, antibiotic treatment, and intestinal pathology on the intestinal microbiota composition, immune development, and specific antibody response later in life in chickens. Additionally, possible differences between broilers and layers were taken into account as unintentional co-selection of immunological traits may have taken place during the selection process for different production traits. Delayed access to feed and administration of antibiotics early in life led to a shift in early life microbiota composition, which seemed to be restored quite quickly in both cases. Microbiota composition in response to DSS was not investigated, but based on rodent studies was expected to be influenced. Ileal immune development, which was assessed in terms of relative cytokine and immunoglobulin mRNA expression levels was not affected by feeding strategy post hatch (early vs. delayed), but a downregulation of ileal immunoglobulin expression levels could be observed during DSS treatment. All early life factors investigated affected the specific antibody response towards an immunological challenge later in life. Interestingly, there seemed to be an interaction between immediate access to feed post hatch and immune responsiveness towards the environment, thus early feeding may influence the adaptive capacity of chickens in different environments. Regarding the differences between breeds it is interesting to note that broilers seem to have developed a more humoral oriented immune strategy, while layers seem to react in a more pro-inflammatory way. Taken together, results suggested that early life conditions may influence priming of the immune system during its developmental phase, leading to altered antibody responses later in life. Furthermore, broilers and layers seem to have developed different immune strategies. Early life conditions as well as possible differences between breeds should therefore be taken into account in future immunological studies.

    Waves of change: immunomodulation of the innate immune response by low frequency electromagnetic field exposure
    Golbach, L.A. - \ 2015
    Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Lidy van Kemenade. - Wageningen : Wageningen University - ISBN 9789462574090 - 172
    immuniteitsreactie - elektromagnetisch veld - cellen - celbiologie - blootstelling - immune response - electromagnetic field - cells - cellular biology - exposure

    In this thesis we investigated possible modulatory roles of low frequency electromagnetic fields (LF EMFs) exposure on the innate immune system. Recent decades have seen a huge increase in the use of electronic devices that nowadays enable us to communicate with distant family, enjoy music everywhere or order food without leaving the house. However besides the benefits, this evolution has also resulted in increased public concern about the potential adverse health effects of non-ionizing radiation. Every power line or electronic device emits a wide range of electromagnetic waves, which can pass through our bodies or damage our skin, depending on the characteristics of the waves. The symptoms attributed to continuous EMF exposure range from non-specific physical symptoms, such as fatigue 1, headaches 2, and redness of the skin to increased prevalence of childhood leukaemia 3. Although many theories regarding a potential mechanism of induction are put forward, to date no clear mechanism of action has been elucidated. Experimental evidence that could support an association between exposure and health status appears to be insufficient and inconsistent 4,5. We investigated the potential effect of LF EMF exposure on neutrophils, one of the key players of the innate immune response. We tried to elucidate a possible mechanism of interaction between intracellular signalling pathways and LF EMF exposure. We aimed to investigate calcium signalling, actin reorganization, cell migration and antimicrobial activity during exposure with different in vitro approaches.

    Impact of health status on amino acid requirements of growing pigs : towards feeding strategies for farms differing in health status
    Kampman-van de Hoek, E. - \ 2015
    Wageningen University. Promotor(en): Wouter Hendriks, co-promotor(en): Walter Gerrits; Alfons Jansman. - Wageningen : Wageningen University - ISBN 9789462573437 - 184
    varkens - afmesten - diergezondheid - aminozuren - voedingsstoffenbehoeften - voedingseiwit - aminozuurmetabolisme - stikstofretentie - immuniteitsreactie - immuunsysteem - varkensvoeding - diervoeding - pigs - finishing - animal health - amino acids - nutrient requirements - dietary protein - amino acid metabolism - nitrogen retention - immune response - immune system - pig feeding - animal nutrition


    There is large variation in the production performance of commercial growing-finishing pig farms. This variation even exists when pigs have a similar genetic background and fed similar diets. The health status is one of the major factors contributing to this large variation in pig performance, as activation of the immune system can decrease feed intake, body weight gain and increase nutrient utilisation for immune system functioning. As a consequence, amino acids (AA) are repartitioned from skeletal muscle deposition towards utilisation for immune system functioning. Current requirement estimates for growing-finishing pigs are formulated to maximize protein deposition for growth and do not take into account the increased utilization of AA for immune functioning as induced by health challenging conditions. This lack of knowledge hampers the ability of feed manufacturers to optimize diets and improve pig performance. The main objective of the present thesis was to quantify the effect of health status on AA requirements for body protein deposition and for immune system functioning of growing pigs.

    A health status web was developed as a tool to categorize growing-finishing pig farms on the basis of their health status. The health status web can be of use for feed manufacturers to develop targeted strategies to accommodate the nutritional requirements of pigs belonging to particular groups of farms sharing a common health status. A dose-response technique was developed, which is a simple, accu­rate technique to quantitatively estimate changes in AA requirements of individual meal-fed pigs. Nevertheless, a minimum time period of 21 days is required for each individual, which makes the technique inappropriate for studying the effect of immune system activation on AA requirements. The combined measurements of whole body N retention, plasma irreversible loss rate (ILR, i.e. the amount of free AA that disappears per unit of time from the plasma pool for protein synthesis or oxidation), urea entry and appearance of 13C into plasma proteins, provided insight into the consequences of immune system activation on AA metabolism.

    Pigs selected from a farm with a suboptimal health status had greater serum haptoglobin, lower serum albumin concentrations, and greater leukocyte counts in blood at the start of the experiment than pigs selected from a farm with a high health status, indicating a higher level of immune system activation. The occurrence of compensatory gain in pigs from a farm characterized as having a suboptimal health status proves, however, that it is difficult to maintain a contrast in health status, and that pigs can adapt quickly to a change in housing conditions. In the absence of effects on feed intake, health challenging conditions may affect performance due to alterations in post-absorptive AA metabolism, as also indicated by increased urinary N losses, and a tendency for a reduced N retention and a lower utilization of digestible N for N retention in pigs with a systemic inflammation, or by a reduction in faecal nutrient digestibility as indicated for dry matter and N in pigs from a farm with a suboptimal health status. The observed changes in protein and AA metabolism after immune stimulation imply that especially tryptophan may become limiting during immune system activation, whereas lysine becomes excessive. Furthermore, the utilization of methionine, tyrosine, and valine for immune system functioning seems to increase in pigs with a systemic lung inflammation. In addition, the dietary AA or protein supply was able to modulate the acute phase response pre- and post-challenge, stressing the importance of an adequate dietary AA supply for appropriate functioning of the immune system of growing-finishing pigs.

    Before implementing targeted feeding strategies for farms sharing a common health status, future research should be conducted to study the possible beneficial effects of increasing the dietary supply of particularly tryptophan, methionine, tyrosine, and valine relative to lysine for immune system function and for body protein deposition in pigs from farms with a different health status.

    Activation and evasion of the type I Interferon response by infectious bronchitis virus : roles of the accessory proteins
    Kint, J. - \ 2015
    Wageningen University. Promotor(en): Geert Wiegertjes; Huub Savelkoul, co-promotor(en): Maria Forlenza. - Wageningen : Wageningen University - ISBN 9789462573376 - 138
    interferon - coronavirus - infectieus bronchitisvirus - coronaviridae - immuniteitsreactie - kippen - kippenziekten - pluimveeziekten - vaccinontwikkeling - kwantitatieve methoden - eiwit - virale inmenging - interferon - coronavirus - infectious bronchitis virus - coronaviridae - immune response - fowls - fowl diseases - poultry diseases - vaccine development - quantitative methods - protein - viral interference


    Viruses are intracellular parasites that exploit the machinery of the host cell to replicate. To defend themselves against invading viruses, animal cells have evolved an anti-viral mechanism, known as the type I interferon response. Through natural selection viruses have in turn evolved mechanisms to counteract or evade the type I IFN response. Coronaviruses are a large group of positive-stranded RNA viruses that cause a range of human and veterinary diseases. Infectious bronchitis virus (IBV) is a member of the genus Gammacoronavirus and it is the causative agent of a highly contagious respiratory disease of poultry. To date, only few studies have investigated the interaction between IBV and the type I IFN response.

    In this thesis, we describe for the first time the activation of the type I interferon response (IFN response) by the Gammacoronavirus IBV, and the repressive role of accessory proteins therein. In Chapter 1 I provide a general introduction into coronaviruses in general and the Gammacoronavirus IBV in particular. I also introduce the IFN response, and highlight differences between the mammalian and chicken IFN response. Finally, I review current knowledge on the roles of coronavirus accessory proteins in counteraction of the IFN response. In Chapter 2 we describe our studies which demonstrated that activation of the IFN response by IBV is dependent on the intracellular double-stranded RNA sensor MDA5. We show that detection of IBV-infection by MDA5 is delayed with respect to the peak of viral replication, and demonstrate that this delay is not due to inhibition of dsRNA detection by IBV. Using mutant viruses that cannot express accessory proteins (null viruses), we found that accessory proteins 3a and 3b of IBV mediate transcription and translation of Ifnβ mRNA.

    The observation that IBV delays the activation of the IFN response, prompted us to investigate the sensitivity of IBV to IFN treatment in Chapter 3. Here we show that IBV is relatively resistant to treatment with type I IFN, as relatively high doses of type I IFN are required to decrease propagation of the virus. Next, we studied which viral protein(s) contribute to resistance of IBV to type I IFN and found that absence of accessory proteins 3a and 3b increased sensitivity of IBV to type I IFN, via a presently unknown mechanism. In addition, we observed that independent of accessory proteins 3a and 3b, IBV blocks signaling of IFN by inhibiting phosphorylation and translocation of the transcription factor STAT1. To explain the delayed kinetics of IFN production observed in Chapter 2, we investigated whether delayed protein production was restricted to IFN, or whether IBV, like Alpha- and Betacoronaviruses, inhibits general translation of host proteins (i.e. induces host shutoff). In Chapter 4 we demonstrate that IBV-induced transcription of Ifnβ mRNA leads to the production of relatively little IFN protein. We discovered that limited production of IFN protein by IBV-infected cells is the result of general inhibition of host translation, confirming that IBV induces a shutoff of host-protein production. This finding indicates that evasion of the innate immune system by Gammacoronaviruses may be more similar to that of Alpha- and Betacoronaviruses than previously thought. Using accessory protein null viruses we discovered that accessory protein 5b of IBV is essential for the inhibition of host-protein synthesis by IBV. In Chapter 5 and Chapter 6 we describe the methods used in this thesis to quantify the number of infectious virus particles of IBV as well as methods used to quantify the activation of the type I IFN response in chicken cells. Although the studies described in this thesis have answered several questions about the interaction of IBV with the type I IFN response of its host, they have also raised new questions to be addressed in future research. In the final Chapter of this thesis (Chapter 7), I discuss a number of remaining questions and future perspectives regarding evasion of the IFN response by IBV. Finally, I explore the possible implications of our findings on the in vivo pathogenicity of IBV and on the rational design of attenuated IBV vaccines.

    In conclusion, the work described in this thesis demonstrates for the first time how IBV evades, activates, and antagonises the IFN response. Also, this thesis comprises the first study that describes a function for the accessory proteins of IBV and shows that these poorly understood proteins play an important role in antagonism of the type I IFN response.

    Mucosal immunity : barriers, bugs, and balance
    Neerven, R.J.J. van - \ 2014
    Wageningen : Wageningen University, Wageningen UR - ISBN 9789462571952 - 24
    immuniteit - immuniteitsreactie - immuunsysteem - immunologie - infectieziekten - ontsteking - orale vaccinatie - voeding - immunity - immune response - immune system - immunology - infectious diseases - inflammation - oral vaccination - nutrition
    'Je moeder leert je wie je vijanden zijn'
    Makkink, C. ; Parmentier, H.K. - \ 2014
    De Molenaar 2014 (2014)7. - ISSN 0165-4284 - p. 26 - 27.
    vleeskuikenouderdieren - immuunsysteem - immuniteitsreactie - epigenetica - diervoeding - broiler breeders - immune system - immune response - epigenetics - animal nutrition
    Via de voeding van ouderdieren kan de werking van het immuunsysteem van de nakomelingen worden beïnvloed. Het is een uitdaging om interventies te vinden die het immuunsysteem van landbouwhuisdieren optimaliseren, zo luidde de conclusie tijdens de Feed4Foodure-themamiddag.
    Stimulation of the innate immune system of carp: role of Toll-like receptors
    Pietretti, D. - \ 2013
    Wageningen University. Promotor(en): Geert Wiegertjes; Huub Savelkoul, co-promotor(en): Maria Forlenza. - Wageningen : Wageningen University - ISBN 9789461737878 - 213
    karper - cyprinus - immuunsysteem - verdedigingsmechanismen - immuniteitsreactie - immunostimulatie - bèta-glucaan - macrofagen - receptoren - immunologie - visteelt - aquacultuur - carp - cyprinus - immune system - defence mechanisms - immune response - immunostimulation - beta-glucan - macrophages - receptors - immunology - fish culture - aquaculture

    Toll-like receptors (TLRs), named after the Toll gene identified in fruit flies, are a family of evolutionary conserved proteins that play a key role in the innate immune system. TLRs are found inside or on the surface of immune cells of virtually all-living animals and recognize integral parts of microbes. Thereby, they are excellent candidate receptors for controlled stimulation of the innate immune system of, for example, fish in aquaculture. β-glucans are microbial compounds routinely added to fish feed for their health-promoting effects. They regulate innate immunity by stimulating fish cells to produce more oxygen and nitrogen radicals but are not recognized by TLRs.Instead, TLRs of cyprinid fish (zebrafish, carp) are stimulated by viral and/or parasitic infection. Although immunostimulation by β-glucans occurs via yet undefined receptors certainly, addition of integral but harmless parts of microbes to fish feed may help controlfish diseases in aquaculture.

    Verkenning van mogelijkheden voor plantweerbaarheid tegen bladluis in paprika
    Messelink, G.J. ; Bloemhard, C.M.J. ; Kok, L.W. - \ 2013
    Bleiswijk : Wageningen UR Glastuinbouw (Rapporten WUR GTB 1242) - 22
    biologische bestrijding - myzus persicae - reductie - paprika's - capsicum annuum - onderdrukking - insectenplagen - populatiegroei - immuniteitsreactie - glastuinbouw - nederland - biological control - myzus persicae - reduction - sweet peppers - capsicum annuum - suppression - insect pests - population growth - immune response - greenhouse horticulture - netherlands
    Het doel van dit onderzoek was om maatregelen te vinden waarmee bladluis in paprika via de plant geremd kan worden. Hoewel er in de literatuur duidelijke aanwijzingen zijn dat de plantweerbaarheid verhoogd kan worden, is dit niet naar voren gekomen in dit onderzoek. Een verhoogde kaliumgift, wormenhumus en plantversterkers op basis van huminezuren of salicylzuur konden géén significante remming geven van bladluis. Wel is het omgekeerde gevonden: wanneer veen voor 20 procent werd gemengd met wormenhumus resulteerde dit in 35 procent meer populatiegroei van rode perzikluis ten opzichte van onbehandelde planten op alleen veen. Dit onderzoek heeft verder laten zien dat de reactie van bladluis op de plantbehandeling zelfs binnen een soort kan variëren. Het groene fenotype van perzikluis reageerde anders dan het rode fenotype, waarschijnlijk omdat de positie die deze bladluizen innemen op de plant ook anders is.
    Relevance of IgA in allergy: regulation by mucosal factors
    Hartog, C.G. den - \ 2013
    Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Joost van Neerven. - S.l. : s.n. - ISBN 9789461734495 - 170
    iga - immunoglobulinen - allergieën - slijmvlies - immuunsysteem - immuniteitsreactie - immunologie - iga - immunoglobulins - allergies - mucosa - immune system - immune response - immunology

    Allergy is mediated by allergen-specific antibodies of various classes of which immunoglobulin E (IgE) produced by B cells upon stimulation by T helper type 2 cells is of crucial importance. Immunoglobulins have varying antigen and allergen specificity (introduced in chapter 1). IgE has to be specific for an allergen (sensitization) and bind simultaneously to several epitopes on the allergen to cause cross-linking of IgE immobilized on high affinity IgE receptors expressed on mast cells and basophils to induce degranulation (Chapter 2). IgE molecules can be specific for a protein of one species or bind to similar regions on allergens from different species, thereby causing cross-reactivity as discussed for shrimp and mussel tropomyosin in chapter 2 and fig and ficus in chapter 3. In most cases of cross-reactivity the primary sensitizing allergen can be identifying by inhibition assays (Chapter 3).

    The presence of allergen-specific IgE in serum of allergic patients does not necessarily cause clinically relevant symptoms of allergy when blocking immunoglobulins of non-IgE isotypes are present, like IgG4, which is induced by specific immunotherapy. Until now, serum-derived IgA has not been shown to be able to block IgE-allergen interactions and the potential protective role of allergen-specific IgA is still debated. The aim of this thesis was to study the potential protective role of the mucosal immune system and especially IgA production by B cells against allergy.

    The mechanisms underlying differential regulation of the two IgA subclasses IgA1 and IgA2 production characteristic for humans are largely unknown. Most likely factors in the local mucosal tissue are involved in differentiating between IgA1 and IgA2 production. Evidence for the role of dendritic cells (DC) in IgA production is increasing. We generated and characterized two DC types not previously described in literature by using the mucosal factors retinoic acid (RA) and transforming growth factor (TGF)-ß (Chapter 5). Those RA- and TGF-ß-derived dendritic cells have tolerogenic characteristics, as shown by reduced inflammatory cytokine production (IL-12 and TNF-α), but non-significant reduction in IL-10 production, and reduced expression of activation marker CD86 and maturation marker CD83 after stimulation with bacterial ligands.

    To study the role of epithelial cell- and DC-derived molecules in induction of production of IgA by B cells and plasma cells, the mucosa-related cytokines APRIL, BAFF, IL-10, TGF-ß, and vitamin A-derived RA were added to B cells. Addition of RA resulted in differentiation of B cells into plasma cells (CD38+) and enhanced secretion of IgA1 and IgA2 when also IL-10 and APRIL or BAFF was present. Our data indicate that production of IgA1 is increased in the presence of BAFF (probably by plasma cells), whereas IgA2 production is increased in the presence of APRIL which is produced by either DCs or epithelial cells in vivo (probably by de-novo induction). When RA and IL-10 were added, B cells upregulated homing integrin ß7 on the membrane, which is believed to preferably direct homing to the small intestine. In literature, increased levels of APRIL have been associated with decreased prevalence of eczema, whereas increased levels of TSLP have been associated with presence of eczema. In chapter 6, it is shown that APRIL is involved in induction of IgA2, and that TSLP potently inhibits IgA1, and to a lesser extent also IgA2 production, which is consistent with reports showing that increased levels of IgA correlate with less eczema. Also in chapter 4, an association was found between reduced levels of allergen-specific IgA2 in serum and presence of eczema and asthma. Also the tissue (systemic, e.g. serum versus mucosal, e.g. saliva) where IgA is determined is differentially linked to the clinical status in house dust mite-allergic patients. In addition, allergen-specific levels of IgA2 rather than IgA1 appeared to be associated with absence of allergy. Within house dust mite-allergic patients, the ratio between serum-IgE and saliva-IgA2 was associated with severity of local (mucosal) clinical symptoms.

    IgA production is diminished in the absence of intestinal bacteria and colonization of the gut induces production of IgA. The ontogeny of the bacteria-specific repertoire of intestinal IgA in relation to composition of the intestinal microbiota has not been studied before. In chickens we analysed the CDR3-repertoire development in the first ten weeks post hatch. No association between bacterial composition and IgA CDR3 repertoire was found, indicating that bacteria may induce IgA production but not cause extensive modification in the specificity of IgA (Chapter 7). Transitional changes in the composition of the microbiota were restored once IgA production was initiated, suggesting that IgA is directly involved in regulation of the intestinal microbiota composition.

    Diet-derived RA is a key regulator of tolerance and IgA production in the mucosa. In addition, dietary ingredients can directly interact with cells of the intestinal immune system. In chapter 8 we show that bovine IL-10 binds to the human IL-10 receptor and thereby modulates bacterial ligand induced activation of monocytes and DCs. Bovine milk also contains immunoglobulins that are specific for bacterial ligands and potential allergens that are also encountered by the human mucosal immune system. Bovine IgG efficiently binds to human IgG receptors and can modulate myeloid cell activation by LPS (Chapter 9). Immunoglobulin from bovine milk may therefore provide potent opportunities to either induce tolerance or antigen-specific immune responses resulting in the production of protective IgA, thereby assisting in providing protection against pathogenic infections. This protection may be mediated by bovine IgG alone, or in cooperation with IL-10, TGF-ß and vitamin A-derived RA.

    The findings from chapters 2-9 are discussed and applied for the field of allergy, both for the clinic and experimental research. The finding that human IgA1 and IgA2 are differentially regulated by innate factors and differentially correlated with the presence or absence of allergy and severity of clinical symptoms is a promising finding. This finding may provide new opportunities for allergen-specific immunotherapy. In addition, efficacy of other mucosal vaccination strategies could be affected by the innate mucosal mechanisms of regulation of IgA production.

    Adaptive capacity of rearing hens : effects of early life conditions
    Walstra, I. - \ 2011
    Wageningen University. Promotor(en): Bas Kemp, co-promotor(en): Henry van den Brand; Jan ten Napel. - [S.l.] : S.n. - ISBN 9789461731265 - 147
    hennen - opfoktechnieken - broeden - uitbroeden - embryogenese - experimentele infectie - warmtestress - immuniteitsreactie - immunologie - adaptatiefysiologie - hens - rearing techniques - incubation - hatching - embryogenesis - experimental infection - heat stress - immune response - immunology - adaptation physiology

    The traditional strategy to deal with pathogens in the layer industry is based on monitoring and control methods, primarily aimed at minimizing the risk of infection with the pathogen. The aim of this thesis was to investigate whether the adaptive capacity of layers could be influenced by early life conditions as they may occur in layer practice, as an alternative strategy for improving layer health and disease resistance. The first study investigated whether suboptimal versus optimal incubation, hatch and early rearing conditions could influence the adaptive capacity during infectious challenges with Eimeria and Infectious Bronchitis (IB). The second study investigated effects of prenatal high temperature manipulation on postnatal temperature preference and adaptive response of layers to heat stress. The third study investigated effects of suboptimal and optimal incubation temperature on the adaptive response to Eimeria under normal circumstances or following exposure to a high (35oC) environmental temperature. The fourth study investigated effects of feed provision immediately after hatch (early feeding) and suppression of gram negative intestinal bacteria (by use of the antibiotic Colistin) for 21 d post hatch on microbial composition of the intestines, layer development and response to a mix challenge with lipopolysaccharide (LPS) and humane serum albumin (HuSA). Finally, effects of early feeding and Colistin treatment on organ weights and response to an infectious challenge with Eimeria were investigated. Results demonstrated that optimized incubation, hatch and rearing resulted in a better adaptive response to Eimeria and IB, as was shown by a higher feed intake and reduced weight loss. Optimal incubation as a single early life condition also had a positive influence on the adaptive response of layers toEimeria, as demonstrated by tendencies to higher feed intake and BW gain, less duodenal lesions and a lower oocyst production. Early feeding resulted in higher body and organ weights, a changed microbiota composition in the intestines, and a changed response to E. acervulina and LPS/HuSA. Colistin treatment resulted in a changed microbiota composition of the intestines and a changed response to E. acervulina and LPS/HuSA. These results confirmed the hypothesis that early life conditions can be used to influence the adaptive capacity to infectious challenges. In conclusion, improving the adaptive capacity with the use of particular early life conditions may be the first step towards an alternative method to maintain or improve layer health and disease resistance.

    Innate immune receptors in carp: recognition of protozoan parasites
    Ribeiro, C.M.S. - \ 2010
    Wageningen University. Promotor(en): Huub Savelkoul, co-promotor(en): Geert Wiegertjes. - [S.l. : S.n. - ISBN 9789085857747 - 217
    karper - immuniteitsreactie - receptoren - protozoa - parasieten - immuunsysteem - vaccins - hulpstoffen - immunologie - immuniteit - carp - immune response - receptors - protozoa - parasites - immune system - vaccines - adjuvants - immunology - immunity
    This PhD thesis reports on pattern recognition receptors involved in the immune responses of common carp (Cyprinus carpio) to two protozoan parasites Trypanoplasma borreli and Trypanosoma carassii. The immune responses of carp are fundamentally different when comparing these two extracellular blood parasites. T. borreli induces a characteristically high production of nitric oxide by macrophages, whereas T. carassii parasites seem to preferentially induce an alternative state of macrophage activation. These differences could be driven by differences in the initial engagement of pattern recognition receptors on carp macrophages with either of the two types of parasites. Based on known host-parasite interactions in mammalian vertebrates, Toll-like receptor 2 (TLR2) and TLR9 were selected as candidate receptors for parasite recognition by receptors carp macrophages. Transfection of human cell cultures with carp TLR2 and overexpression of TLR2 in carp macrophages, corroborated the ability of this receptor to bind peptidoglycan from Gram-positive bacteria and glycosylphosphatidylinositol anchors from protozoan parasites. The parasite T. carassii, in particular, induced a TLR2-mediated formation of the cytokine IL-23, leading to a Th17-like immune response in fish infected with T. carassii. Transfection of human cell cultures with carp TLR9 indicated this receptor recognizes bacterial DNA, but not protozoan DNA, and studies in carp macrophages indicated this recognition to be protease-dependent. A novel pattern recognition receptor of carp, named Soluble Immune-Type Receptor (SITR), was identified by investigating an enriched cDNA repertoire from macrophages stimulated by T. borreli. SITR is abundantly expressed in carp macrophages and seems to be secreted upon stimulation with T. borreli. Overexpression of SITR in mouse macrophages and knock-down of SITR in carp macrophages provided evidence for the involvement of this receptor in T. borreli-induced production of nitric oxide. The results presented in this PhD thesis have shed light on the evolution of innate immune receptors involved in the recognition of pathogens.
    Genetic analysis of production, immunity and behaviour in laying hens
    Biscarini, F. - \ 2010
    Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Jan van der Poel; Henk Bovenhuis. - [S.l. : S.n. - ISBN 9789085857860 - 132
    genetische analyse - hennen - eierproductie - immuniteit - diergedrag - dierveredeling - fokwaarde - heritability - immuniteitsreactie - single nucleotide polymorphism - genetic analysis - hens - egg production - immunity - animal behaviour - animal breeding - breeding value - heritability - immune response - single nucleotide polymorphism
    The new regulations about the husbandry of laying hens and the so-called genomic revolution offer both opportunities and challenges for the breeding of layers. Hens are currently housed mainly in battery cages of 4 individuals each. Following recent developments of the communitarian legislation, many countries will soon adopt furnished cages or non-cage systems, which will lead to larger groups of hens. Also, beak-trimming will be prohibited in EU countries in the near future. Advancements in sequencing technology are making an always greater number of genetic markers available at increasingly cheaper prices, making genome-wide studies possible and helping geneticists to start unraveling the mystery of the genetic make-up of animals, which until a few years ago was considered a black-box. This thesis touches upon the impact of such innovations on the breeding of laying hens.

    Use of pooled data in the genetic evaluation of laying hens
    Hens are usually housed in cages and therefore pooled instead of individual egg records are often available: a pooled egg record is the total production of a cage, when the egg production of the individual hens is unknown. Current selection schemes are carried out in nucleus herds where hens are housed individually, so that egg production of individual birds can be recorded and used for genetic evaluations. Based on this information sires and dams are selected. Such a selection scheme based on individually housed hens introduces a discrepancy between the environment where hens are selected and the environment in which hens are kept for commercial egg production (group housing). Selecting animals in one environment and using them in a different environment might lead to genotype x environment interaction (Besbes and Ducroq, 2003), thereby reducing the realized response to selection. Future husbandry conditions, with larger groups of hens or hens housed in furnished cages might make this problem even worse. A method to use pooled data in the genetic evaluation of laying hens would therefore be of interest. In Chapters 2 and 3 of this thesis it is described how to use pooled records for the estimation of heritability and breeding values. In chapter 2 the use of individual and pooled observations is compared. Individual body weights of hens at different ages were available: these were then pooled by cage in order to create pooled records. Heritabilities estimated from pooled and individual data correlated well: the standard error of estimates based on pooled records was however about twice that of estimates based on individual records. The accuracy of EBVs from pooled data is lower than the accuracy of EBVs from individual data; in the case of sires with at least 10 offspring the reduction in accuracy was about 23%. This loss of precision in estimating genetic parameters and breeding values is understandable considering that pooled records are a less detailed of information. However, this lower accuracy should be interpreted in the context of direct vs indirect selection. The breeding goal is the trait under commercial conditions (group housing), and if testing is under individual housing, the genetic correlation between group and individual housing is relevant. The ratio of the selection response for direct and indirect selection is a function of the accuracies for both situations, the standard deviations of the traits and the genetic correlation between the traits (Falconer, 1989). Similarly, the ratio between accuracies based on pooled and individual data provides a threshold for the genetic correlation between individual and group housing below which pooled data would result in a greater selection response. In practical breeding also the costs of individual housing relative to the costs of group housing are relevant. Since group housing is cheaper than individual housing, more selection candidates could be tested for the same level of costs. This would in turn result in higher selection intensity and larger response to selection.
    In chapter 3 the method of analyzing pooled data developed in chapter 2 was compared with an approximation consisting in assigning cage means to each hen in a cage, then treating them as individual observations. Cross-validation was used to compare the two methods: the method developed in Chapter 2 performed consistently better than the approximate method in terms of predicting ability.
    In the general discussion, finally, it was described how to estimate genetic and phenotypic correlations from pooled data.

    Across-line association studies for immune response and feather pecking behaviour
    The great number of genetic markers available at increasingly lower prices has been fostering developments in genomic research. Association studies between genetic markers and phenotypes are typically conducted within populations (breeds, or lines): the amount of LD conserved in a population is exploited using high marker density, such as SNP chips, and markers relatively close to QTLs are expected to show significant effects in association studies. In this thesis we propose to take it one step further and perform association studies across lines. This requires higher marker density but increases the resolution. The amount of LD conserved across lines is expected to be lower than within lines and the phase of the marker-phenotype association might be different in the different lines. On the other hand markers that happen to show significant effects in an across-line association study are likely to be close to the QTL. These issues in conducting marker-phenotype association studies across populations were addressed in Chapters 4 and 5 of this thesis, where it was shown how to deal with multiple populations when analyzing hens from 9 different genetic lines of White Leghorn and Rhode Island Red origin genotyped for a panel of 1536 SNP (Single Nucleotide Polymorphism) markers.
    The traits analysed were immunological parameters and plumage damage due to feather pecking behaviour, two classes of traits for which, given that they have relatively low heritability and are difficult and expensive to measure, genomic information may be particularly valuable. Immunological parameters might be used in selection programmes aimed at improving disease resistance of laying hens, while information on the genetic background of feather pecking behaviour can be useful in reducing problems due to this behavioural disorder of layers. Under future husbandry conditions susceptibility to infectious diseases and feather pecking are expected to become more serious problems: both aspects of layer production are in fact related to the number of individuals that interact with each other, which will increase as a result of the application of the EU directive 1999/74/EC. In addition, the ban of beak-trimming will make it more difficult to control the consequences of feather pecking (plumage damage, cannibalism, mortality). Genetic selection might represent an appealing addition to the current control measures. The association studies identified several regions of interest. The gene for interleukin 17 (IL17), on chromosome 3, was found to be associated with natural and acquired antibody titres, and with the classical and alternative pathways of complement activation. The major histocompatibility complex (MHC) genes on chromosome 16 showed significant association with natural and acquired antibody titres and classical complement activity. The interleukin 12B gene (IL12B) on chromosome 13 was associated with natural antibody titres. As for feather pecking behaviour, a role of the gene for the serotonin receptor 2C (HTR2C) on chromosome 4 was found. This supports existing evidence of a prominent involvement of the serotonergic system in the modulation of this behavioural disorder in laying hens. The genes for IL9, IL4, CCL4 and NFKB were found to be associated to plumage condition, revealing relationships between the immune system and behaviour.

    Nutritional zinc deficiency, immune capacity and malaria : a study on mediators of immunity to malaria caused by Plasmodium falciparum in African children
    Mbugi, E.V. - \ 2009
    Wageningen University. Promotor(en): Huub Savelkoul; J.F. Shao, co-promotor(en): Hans Verhoef. - [S.l. : S.n. - ISBN 9789085855316 - 174
    malaria - plasmodium falciparum - immuniteit - zink - kinderen - geneesmiddelresistentie - immuniteitsreactie - mineraaltekorten - voedingsstoffentekorten - cytokinen - antilichamen - endotheel - anemie - tropische ziekten - tanzania - malaria - plasmodium falciparum - immunity - zinc - children - drug resistance - immune response - mineral deficiencies - nutrient deficiencies - cytokines - antibodies - endothelium - anaemia - tropical diseases - tanzania
    This thesis aimed at investigating the role of genetic and nutritional factors that affect the immune response to malaria in Tanzanian children. The introductory chapter (Chapter 1) reviews the importance of nutritional deficiencies, particularly of zinc, and presents the hypothesis that such deficiencies lead to impaired immunity and contribute to the burden of malaria. The chapter also describes current efforts to prevent malaria through intermittent preventive treatment, both in infants (IPTi) and pregnant women (IPTp). Sulfadoxinepyrimethamine is still used for first-line treatment of uncomplicated malaria, or, in many countries, to prevent malaria and anaemia in pregnancy. In malaria endemic areas, development of resistance to previously valuable antimalarial drugs has been continuously reported for decades. Thus our initial longitudinal study aimed at measuring the prevalence of resistance-associated mutations on dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes (dhfr and dhps) that confer parasite resistance to sulphadoxinepyrimethamine (SP) that was used as an interim antimalarial drug after chloroquine resistance. Although SP resistance-associated point mutations were highly prevalent, we observed an adequate parasite response to SP (Chapter 2). We speculated that the impact of the dhfr and dhps mutations on SP resistance may be dependent at least in part on the protective immunity that has developed in response to frequent exposure to infection and may be weighed down by host immunity in endemic areas and thus impacts in the continued use of the drug for treatment of malaria. The impact of other drugs with similar mechanisms of action used as antibiotics in selecting mutations responsible for SP resistance needs therefore to be studied for their modulating activity of the immune response. These findings underscore the relevance to further study the crucial involvement of the immune system in the development of protection against malaria but also affecting the efficacy of treatment modalities of malaria in various African conditions.
    In the subsequent cross-sectional studies, we assessed the effect of deficiencies of zinc and magnesium as well as iron deficiency anaemia on malaria-specific cytokine responses indicative of innate immunity to Plasmodium falciparum (Chapter 3). In this study, we used Plasmodium falciparum-parasitised red blood cells (pRBCs) as antigens for in vitro stimulation of peripheral blood mononuclear cells (PBMCs). Cytokines were measured in the supernatant of cultured PBMCs after 24 hours of stimulation. Zinc deficiency was associated with a marked increase in monocyte-derived TNF-α concentration in children with malarial infection but not in their uninfected peers. In children with malarial infection, iron deficiency anaemia was associated with elevated concentrations of TNF-α, whereas magnesium deficiency in children without malaria seemed to be associated with increased concentrations of IL-10. Our findings reflected plasticity in cytokine profiles of monocytes reacting to malaria infection under conditions of different nutrient deficiencies. Following the observation of the variable impact of micronutrients on innate cytokines, we evaluated the profile of both type I and type II cytokines and whether they were influenced by nutritional and malaria status (Chapter 4). The cytokine measurements were performed at day 7 of stimulation anticipating that this timing was optimal for measuring effects on these cytokines mainly derived from activated T-cells. The results indicated a variable influence of nutrient deficiencies on increased cytokine response with zinc deficiency and iron deficiency anemia having greater impact on type I and magnesium deficiency on type II cytokines. The subsequent study evaluated the plasma levels of naturally acquired antimalarial antibodies of variousIgG subclasses plus the total IgG and IgM levels and whether they were associated with zinc deficiency based on preceding chapters (Chapter 5). The results indicated a high variability in antibody levels with zinc deficiency, varying with age of the affected child. IgG3 appeared to be predominant across all age subgroups within < 5 yrs aged children providing clues that IgG3 might confer immune protection to malaria under conditions of zinc deficiency. Chapter 6 explored the association between CD36 deficiency, P. falciparum in vitro adherence on purified CD36 and anemia in children. CD36 is a receptor that occurs on the surface of activated immune cells and vascular endothelial cells and participates in phagocytosis and lipid metabolism. We hypothesized that it could play a fundamental role in cytoadherence of erythrocytes that are parasitized by Plasmodium. Our results showed that CD36 deficiency was associated with protection against the development of malarial anemia in children and that it may be mediated through reduced cytoadherence of infected red blood cells to vascular endothelium.
    These studies demonstrate that despite antimalarial drug resistance, there is a potential for optimizing the immunological protective capacity in the population to confer parasite clearance that can be variably influenced by micronutrient status. Improving nutritional status in this population could be rewarding not only to increase protection to malaria but possibly also to other infections.
    Immune responses of carp : a molecular and cellular approach to infections
    Forlenza, M. - \ 2009
    Wageningen University. Promotor(en): Huub Savelkoul; Geert Wiegertjes. - S.l. : s.n. - ISBN 9789085855125 - 212
    karper - immuniteitsreactie - immuunsysteem - experimentele infecties - moleculaire biologie - celbiologie - immuniteit - immunologie - diermodellen - vergelijkend onderzoek - carp - immune response - immune system - experimental infections - molecular biology - cellular biology - immunity - immunology - animal models - comparative research - cum laude
    cum laude graduation (with distinction)
    Immuuninterventie : 'Drukken op de juiste knoppen van het afweersysteem'
    Schijns, V.E.J.C. - \ 2009
    Wageningen : Wageningen Universiteit - ISBN 9789085852773 - 32
    immunologie - immuniteitsreactie - immuniteit - immuunsysteem - immunisatie - vaccins - vaccinatie - immunology - immune response - immunity - immune system - immunization - vaccines - vaccination
    Intratype heterologous vaccination in cattle can confer antibody response in presence of maternally derived antibodies
    Dekker, A. ; Eblé, P.L. ; Stockhofe, N. ; Selman, P.J. ; Chénard, G. - \ 2008
    vaccinatie - kalveren - immuniteitsreactie - vaccination - calves - immune response
    Poster about intratype heterologous vaccination in cattle. It can confer antibody response in presence of maternally derived antibodies
    Major histocompatibility (MH) polymorphism of common carp : link with disease resistance
    Rakus, K.L. - \ 2008
    Wageningen University. Promotor(en): Huub Savelkoul; A. Pilarczyk, co-promotor(en): Geert Wiegertjes; I. Irnazarow. - [S.l.] : S.n. - ISBN 9789085852445 - 169
    karper - major histocompatibility complex - polymorfisme - ziekteresistentie - genen - immuniteitsreactie - immunologie - visteelt - aquacultuur - carp - major histocompatibility complex - polymorphism - disease resistance - genes - immune response - immunology - fish culture - aquaculture
    The impact of diseases caused by a wide range of pathogens (viruses, bacteria
    and parasites) is the most important problem in aquaculture of common carp (Cyprinus
    carpio L.). Genetic selection aimed at obtaining population of more resistant common
    carp is potential and sustainable approach to disease control in semi-intensive carp pond
    farming. Genes of the major histocompatibility complex (MHC) are candidate marker
    genes for studies on association with disease resistance. The MHC contains some of the
    most polymorphic genes known to date and are considered crucial to adaptive
    immunity. MHC molecules bind both self and foreign peptides and present them to T
    lymphocytes (T cells). MHC class I molecules present endogenously derived peptides to
    CD8+ T cells, while MHC class II molecules present exogenously derived peptides to
    CD4+ T cells. Each MHC molecule has the ability to bind and present different groups
    of peptides in more or less successful ways. This can influence the immune response of
    an organism since the peptides derived from a certain pathogen may either not be
    presented by specific MHC molecules, which can result in higher susceptibility or, may
    be bound with a high affinity by specific MHC molecules which could lead to increased
    resistance to the pathogenic organism.
    In teleosts, unlike to humans, tetrapods and cartilaginous fish, class I and class II
    genes are not linked and segregate independently, which allows for association studies
    of only class I or only class II MH genes with disease resistance. MHC class II
    molecules generally have a broader spectrum of action in the immune system than the
    MHC class I genes. There are also observations that suggest a more intense selection
    pressure and a more rapid evolution of MH class II than class I alleles in fish. Although
    the expression of both MH class II chains is equivalent, the beta chain generally has a
    higher degree of polymorphism than the alpha chain. This thesis addressed possible
    implementations of MH class II B genes for selection aimed at improving of a common
    carp resistance in semi-intensive pond farming.
    In common carp there are two paralogous groups of MH class II B genes, Cyca-
    DAB1-like and Cyca-DAB3-like genes. In a preliminary study, we examined the
    polymorphism for the Cyca-DAB1-like and Cyca-DAB3-like genes in different
    European common carp lines (chapter 2). These carp lines were of various
    geographical origins and part of carp live gene bank, which is maintained at the Institute
    of Ichthyobiology and Aquaculture in Gołysz. Previous observations over a period of at least 15 years revealed significant differences between lines in survival rate and parasite
    load under natural conditions. Also, differences in resistance to atypical Aeromonas
    salmonicida in laboratory based challenge tests was observed, suggesting genetic
    differences in resistance between the carp lines. Analysis of polymorphism of MH class
    II B genes in selected carp lines revealed a ubiquitous presence and high polymorphism
    of Cyca-DAB1-like but not Cyca-DAB3-like genes. The observed allelic polymorphism
    for Cyca-DAB1-like genes rather than Cyca-DAB3-like genes stimulated further studies
    into the association of Cyca-DAB1-like allelic polymorphism and disease resistance of
    common carp.
    In order to study association between Cyca-DAB1-like gene polymorphism and
    resistance of common carp we optimized a technique designated polymerase chain
    reaction -restriction fragments- single strand conformation polymorphism (PCR-RFSSCP)
    to be able to screen and type large numbers of individual carp (chapter 3). The
    advantages of this technique are simplicity, high sensitivity and low costs. PCR-RFSSCP
    analysis of n = 79 carp individuals from 8 lines challenged with Aeromonas
    hydrophila revealed the presence of different genotypes consisting of unique
    combinations of Cyca-DAB1 and Cyca-DAB2 sequences. We found four alleles for the
    Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02). We noted that the
    Cyca-DAB2 gene was either homozygous or absent. The degree of heterozygosity of the
    Cyca-DAB1 and Cyca-DAB2 genes clearly correlated with the number of SSCP bands.
    Thus, we proved that PCR-RF-SSCP is a reliable technique that can be used for
    screening large number of individuals for investigating the Cyca-DAB1 and Cyca-DAB2
    genes polymorphism in common carp.
    Previously, we performed a long-term divergent selection of common carp for
    antibody production, which successfully resulted in carp lines with a different immune
    response. We studied the segregation of Cyca-DAB genes with the DNP-specific
    antibody response and we showed that the presence of Cyca-DAB1-like, but not Cyca-
    DAB3-like genes, preferentially leads to a high DNP-specific antibody response in carp
    (chapter 4). Background genes other than Cyca-DAB genes also influenced the level of
    antibody response. We also studied the transcription of both Cyca-DAB1-like and Cyca-
    DAB3-like genes in different organs of carp. The constitutive transcription for both
    Cyca-DAB1-like and Cyca-DAB3-like genes was high, although Cyca-DAB1-like genes consistently showed slightly higher mRNA transcription than Cyca-DAB3-like genes in
    some immunological relevant organs. Sequence information, constitutive transcription
    levels and co-segregation data indicated that both paralogous Cyca-DAB1-like and
    Cyca-DAB3-like groups represent functional MH class II B genes.
    We then proceeded to study association of Cyca-DAB1-like genotypes with
    resistance to four different pathogens; the bacterium Aeromonas hydrophila, the
    ectoparasite Argulus japonicus, and the blood parasite Trypanoplasma borreli (chapter
    5) and the viral pathogen Cyprinid herpesvirus-3 (CyHV-3) (chapter 6). We used a
    large number of individuals of different carp lines and revealed, using PCR-RF-SSCP,
    the presence of n = 9 unique Cyca-DAB1-like genotypes, of which three genotypes (B,
    D, and E) were most common (chapter 5). In general, Cyca-DAB2 was often
    homozygous or absent while allelic polymorphism was detected in Cyca-DAB1 gene.
    We could detect significant associations between genotype E and abundance of
    A. japonicus and between genotype D and higher level of parasitaemia after T. borreli
    infection. We also observed a significant association between Cyca-DAB1
    heterozygosity and lower level of parasitaemia after T. borreli infection. In chapter 6,
    we showed a strong association between Cyca-DAB1-like genotypes and resistance or
    susceptibility to CyHV-3. One genotype (E) performed significantly better, resulting in
    carp more resistant to CyHV-3, while three other genotypes (B, H and J) could be
    linked to higher susceptibility to the virus. Subsequent analysis of the alleles that
    compose the Cyca-DAB1-like genotypes linked one particular allele (Cyca-DAB1*05)
    to significantly increased, and two alleles (Cyca-DAB1*02 and Cyca-DAB1*06) to
    significantly decreased resistance to CyHV-3. The resistant genotype E did not
    comprise the Cyca-DAB2 gene and consisted of a homozygous Cyca-DAB1*05 allele.
    Phylogenetic analysis of all Cyca-DAB1 alleles showed that the Cyca-DAB1*05 allele
    represents the oldest allele in our study (chapter 7). We discussed the possibility for
    using Cyca-DAB1 allelic polymorphism as a potential genetic marker in future breeding
    programs of common carp (chapter 7). We expect that selection of carp for particular
    MH class II B genotypes or alleles could allow for an increased survival upon challenge
    with selected pathogens and possibly, increased survival rate under pond conditions.
    A multidisciplinary study of allergy : mouse models, immune modulation and lifestyle
    Jeurink, P.V. - \ 2008
    Wageningen University. Promotor(en): Huub Savelkoul; Gerrit Antonides, co-promotor(en): Johan van Ophem; Harry Wichers. - S.l. : S.n. - ISBN 9789085049616 - 275
    allergieën - immunotherapie - levensstijl - diermodellen - antigenen - t cellen - cytokinen - immuniteitsreactie - immuniteit - immunologie - allergies - immunotherapy - lifestyle - animal models - antigens - t lymphocytes - cytokines - immune response - immunity - immunology
    Allergic diseases affect a substantial part of the global population. Although extensive studies have elucidated the allergic mechanism, no conclusive answer has yet been found that will prevent the onset of an allergic disease. Literature suggests that no single factor like a gene mutation, environmental factor or lifestyle component can be hold accountable for the allergic cascade. Therefore, the main goal of this research project was to use a multidiscipli¬nary approach to allergies, by combining information on the genetic components, lifestyles and in vivo and in vitro assessment of the immune cells involved in allergy.
    The accomplishment of this multidisciplinary goal required knowledge on the genetic factors involved in the immunopathology of allergic diseases, but also immunological and cell biological knowledge. In addition, we investigated the influence of environmental factors on the allergic response which also required knowledge on sociology and economics to assess involved lifestyle factors. Within the multidisciplinary research areas, allergic responses are studied at multiple levels. For example, the genetic differences can be studied by means of mice with different genetic backgrounds (BALB/c, STS/A, C57BL/6) or in knock-out (CD4 or CD8 knock-out) or transgenic (IL-5 transgenic) mice. These differences in the genetic background on their turn have an effect on the expression of the allergic disease characteristics. Examples of the assessed allergic characteristics comprise antigen-presentation by specialized antigen-presenting cells, the presence of T helper 2 cells, the involvement of Th2 produced cytokines like IL-4 and IL-5, and the isotype switching of B cells towards allergen-specific IgE. Alterations of the allergic characteristics were studied by the use of in vitro cultures of human peripheral blood mononuclear cells (PBMC) that display all above mentioned characteristics when they are properly isolated, cryopreserved and cultured. These alterations were elicited by the use of heat-treatment of allergens or the exposure to fungal derived proteins or polysaccharides. Taken together, these different levels within the allergic individual can on their turn be influenced by environmental and nutritional factors. To study this, lifestyle factors have been assessed by the use of a personalized internet-based questionnaire and these data were thereafter linked to allergen-specific immunoglobulin levels. To further stress the importance of multilevel research within a multidisciplinary study, a more detailed description of the separate chapters within this thesis are combined with their major results, and finalized with future perspectives.
    A differential role for corticosteroid receptors in neuroendocrine-immune interactions in carp (Cyprinus carpio L.)
    Stolte, H.H. - \ 2008
    Wageningen University. Promotor(en): Huub Savelkoul; G. Flik, co-promotor(en): Lidy van Kemenade. - [S.l.] : S.n. - ISBN 9789085851998 - 231
    karper - corticoïden - hormoonreceptoren - immunologie - immuniteit - immuniteitsreactie - interacties - stress - cytokinen - visteelt - neuroendocrinologie - carp - corticoids - hormone receptors - immunology - immunity - immune response - interactions - stress - cytokines - fish culture - neuroendocrinology
    In this thesis we investigated the involvement of the receptors for the stress hormone cortisol in stress and immune regulation. We set out to characterise the pro-inflammatory cytokine interferon gamma (IFN-γ). Furthermore, we used a genome wide screen (microarray) to search for additional genes that might be involved in regulation of the stress or the immune response.

    In teleostean fishes cortisol can be bound by different receptors encoded by at least three different genes. An ancestral corticosteroid receptor (AncCR) is assumed to have been an effective receptor for cortisol in the ancestors of fishes. An early genomic duplication in the fish lineage, over 450 million years ago, led to separate glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) genes, both of which retained the ability to bind cortisol. A second major genomic duplication event took place only in teleostean fishes (not in other vertebrates), and gave rise to duplicate GR genes (GR1 and GR2). Even more variants developed as a result of alternative splicing of the GR1 gene which introduces a nine amino acid insert in the DNA-binding domain of GR1a, GR1b does not have this insert.

    To investigate how one ligand can regulate many and very diverse functions using multiple receptors, we describe the expression of GR1 (a and b), GR2 and MR and their sensitivity for cortisol in chapters 3 and 4. The three receptors are expressed in tissues that make up the neuroendocrine stress-axis (brain, hypothalamus and pituitary) and in cells that produce corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). Decreased mRNA expression in brain after prolonged stress suggests an involvement in regulation of hypothalamo-pituitary-interrenal (HPI)-axis activity. In cells of the immune system MR expression is very low compared to GR expression and GR2 is preferentially expressed in lymphocytes. Transactivation assays shows that GR1 is a relatively ‘insensitive’ or ‘stress’ receptor, which can only become activated at stress levels of, whereas GR2 is a ‘sensitive’ receptor that will already be activated at basal levels of cortisol such as occur in non-stressed fish. MR sensitivity for cortisol is intermediate. We predict by tertiary protein modelling and confirmed by transfection assays, that the transactivation capacity of both splice variants (GR1a and GR1b) is similar. Based on the very low expression level in immune cells and the moderate transactivation capacity of MR we concluded that GRs rather then the MR primarily convey stress signals to the immune system. Next, we determined the expression profile of the duplicated GR genes in the immune system in chapters 4 and 5 to investigate the regulation of stress-induced immune modulation. Simultaneously we investigated the expression profile of (among others) heat shock protein 70 (Hsp70). This protein is required for binding of cortisol to the GR, but also has intrinsic immune modulatory functions, as it was shown to downregulate LPS-induced pro-inflammatory cytokine expression in vitro and in vivo. In head kidney phagocytes we found that only physiological stress levels of cortisol could reduce LPS-induced expression of pro-inflammatory cytokines, a response that appears mediated by the ‘stress’ receptor GR1. Moreover, we found that Hsp70 and GR1 (a and b) expression is increased after an immune stimulus in vitro and in vivo, whereas 24 hr restraint stress or 100nM cortisol-treatment hardly increases Hsp70 and GR1 expression levels. This suggests that an immune stimulus rather than increased cortisol levels increases the sensitivity for glucocorticoid regulation and thereby of the cytokine profiles in immune cells.

    To find additional genes involved in bidirectional neuroendocrine-immune communication we applied a genome wide screen of 9000 randomly picked cDNA clones. This has the advantage of an unprejudiced overview of regulated genes, but the sensitivity of the technique is limited. In chapter 6 we describe a microarray experiment in which we compared mild acute stress, to prolonged severe immune stimulation. We show that an immune response after parasite infection appears tightly regulated and comparable between individuals, whereas a mild acute stressor allows for more variable gene expression profiles. We found LOC406744 of the DUF727 protein family and nephrosin as new interesting candidate genes that may be involved in neuroendocrine-immune communication.

    The key pro-inflammatory cytokine IFN-γ, which is hypothesised to affect neurotransmitter and hormone release, had not been investigated in carp. In chapter 7 we show that carp have duplicate IFN-γ genes that are expressed in immune cells. IFN-γ-2 shows structural and functional characteristics simlar to those in other vertebrate IFN-γ genes and appears to be involved in T-lymphocyte function, whereas IFN-γ-1 is expressed in stimulated B-lymphocytes. Currently recombinant proteins are being produced which will enable us to further elucidate the role of both IFN-γ gene products in the immune system as well as in mediating the neuroendocrine stress response.

    Interestingly, as explained in chapter 8, both the glucocorticoid receptor and the IFN-γ genes are duplicated. The duplication-degeneration-complementation (DCC) model has been proposed as an explanation for the high retention of duplicate genes in fishes. The hypothesis assumes that following gene duplication, the two gene copies degenerate over time by random mutation to perform complementary functions that jointly match that of the single ancestral gene, termed ‘subfunctionalisation’. Indeed it appears that the duplicate GR genes have divided the general and ‘stress-related’ functions, reflected by their different sensitivity for cortisol. The duplicate IFN-γ genes appear to have divided B- and T-lymphocyte functions as targets suggested by their gene expression profiles upon selective stimulation.

    An important conclusion of this thesis is that duplicated glucocorticoid receptors and heat shock proteins are an integral part of the immune system. Immune stimuli rather than increased cortisol levels control GR and Hsp70 expression in immune cells. The differentially regulated expression of GR genes is at the basis of a balanced pro- and anti-inflammatory
    cytokine profile, immune cell viability and thus at the basis of the success of the fishes. This thesis illustrates the importance of extensive and effective bidirectional communication between the neuroendocrine and immune systems, which are at the basis of the successful evolution of the vertebrates.
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